Multidrug resistant

Rinsho Ketsueki, 1996 Feb, 37(2), 109 - 15
{Multidrug resistant P-glycoprotein expression on acute nonlymphocytic leukemia cells at diagnosis}; Kaya H et al.; In performing cancer chemotherapy, it is essential to know the expression of multidrug resistant (MDR) P glycoprotein (p-gp) on cancer cells . In the present study, in order to clarify the relationship between MDR of leukemic cells and cytologic, immunological and clinical features of acute nonlymphocytic leukemia (ANLL), leukemic cells in peripheral blood and/or bone marrows obtained from 28 ANLL patients were examined . Each smear was stained with C219 monoclonal antibody against P-gp by the APAAP method, and then 1,000 ANLL cells in each smear were observed . Among the FAB subtypes, M4 showed the highest proportion of leukemic cells expressing P-gp . Concerning the response to chemotherapy, five of seven patients (71%) having 1.0% or more of P-gp positive leukemic cells and 11 of 19 patients (58%) having less than 1.0% of those cells achieved complete remission . However, there was no significant correlation between P-gp expression and clinical outcome . There was also no significant correlation between P-gp expression and CD7 or CD34 . Furthermore, no significant correlation between chromosome 7 abnormality and P-gp expression was observed . From these results, if we can clarify the mechanism of MDR and the relationship between MDR and cytogenetic or clinical features of ANLL with further study, P-gp expression may become a useful marker for predicting the outcome of ANLL.

Leuk Lymphoma, 1996 Feb, 20(5-6), 381 - 7
Multidrug resistance-associated protein (MRP) in haematological malignancies; Nooter K et al.; The presence of multidrug resistant cells, either acquired or de novo, severely limits treatment outcome in haematological malignancies . Although expression of the Mr 170,000 P-glycoprotein drug pump is likely to play a role in multidrug resistance (MDR) in haematological malignancies, it is now evident that other MDR mechanisms may be operational as well in leukaemias, lymphomas, and multiple myeloma . We determined the expression of a newly recognised drug resistance gene, the Multidrug Resistance-associated Protein (MRP) gene, in peripheral blood cells from healthy volunteers and from patients with haematological malignancies . Expression of MRP mRNA and its Mr 190,000 glycoprotein were estimated by RNase protection assay and immunocytochemistry, respectively . MRP appeared to be ubiquitously expressed at low levels in all nonmalignant haemopoietic cell types . However, some leukaemias showed elevated levels of MRP, probably due to transcriptional activation or increased mRNA stability . High to very high MRP expression levels were frequently found in chronic lymphocytic leukaemia and prolymphocytic leukaemia . Acute myelocytic leukemia often exhibited low but occasionally high MRP expression levels, while in the other acute and chronic leukaemias, lymphomas, and multiple myeloma, predominantly low, basal levels of MRP were found . We conclude that hyperexpression of MRP is observed in leukaemias, and that further studies are needed to assess the clinical relevance of MRP.

Leuk Lymphoma, 1996 Feb, 20(5-6), 357 - 64
The biological significance of the multidrug resistance gene MRP in inversion 16 leukemias; Kuss BJ et al.; Multidrug resistance represents an important mechanism by which leukaemic and solid tumour cells escape cell death after exposure to anthracyclines and other natural products . Acute myeloid leukaemia (AML) associated with the inversion chromosome 16: inv(16)(p13q22) has a favourable prognosis and is known to be chemosensitive . The inversion chromosome is seen in a number of FAB subclasses but is most commonly associated with acute myelomonocytic leukaemia with abnormal eosinophils, M4Eo . It results in the creation of a fusion between the myosin heavy chain gene (MYH11) on the short arm and the gene for a transcription factor, core binding factor beta (CBFB) on the long arm . In a subset of these inv(16) AML patients, inversion also results in loss of the gene for the multidrug resistance protein (MRP) at the short arm breakpoint . This gene maps to 16p13.13, centromeric to the primary short arm breakpoint, separated from MYH11 by a distance of approximately 150kb . Deletion of the MRP gene has been demonstrated by in situ hybridisation, gene dosage studies and by loss of heterozygosity of a flanking microsatellite marker (D16S405) . Twenty two patients with inv(16) leukaemia were analysed for deletion of the MRP gene . Deletion of the gene was detected in seven patients, fourteen patients showed retention of the gene and in one case the findings were indeterminate . Clinical data from 13 of these patients were analysed revealing deletion of the MRP gene to be significantly associated with longer time from diagnosis until failure (death or relapse from complete remission) in these patients (p = 0.007) . From this work and the growing literature concerning MRP, it appears likely that the deletion of an MRP allele, may favourably affect the biology of inv(16) AML and may have important prognostic implications.

Zhonghua Fu Chan Ke Za Zhi, 1996 Feb, 31(2), 75 - 8
{Experimental study on the mechanism of cisplatin resistance and its reversion in human ovarian cancer}; Liang Z et al.; OBJECTIVE: To explore the mechanism of cisplatin resistance and its reversion in human ovarian cancer . METHODS: A xenografted cisplatin resistant mice model of human ovarian cancer, SKOV3/cp, was developed by the microencapsulated technique . The multiple changes of bio-chemical markers in the model were determined, and various modulators for reversion were tested . RESULTS: Intracellular platinum accumulation in SKOV3 was 5.1 times, Pt-DNA adducts 2.4 times and interstrand cross link of DNA (ISC) 4.8 times of those in cisplatin-resistant cell line, SKOV3/cp . These changes in SKOV3/cp could not be reversed by verapamil . Amphotercin B (AmB) and Novobiocin (NVB) could raise the concentrations of platinum and Pt-DNA adducts in SKOV3/cp, resulting in complete or partial reversion of cisplatin-resistance of SKOV3/cp . There were no differences in total glutathione (GSH) level and in sensitivity to CdCl2 between SKOV3 and SKOV3/cp . CONCLUSIONS: It is suggested that the primary factor causing SKOV3/cp resistance to cisplatin is the reduction of intracellular platinum accumulation and the augmentation of the ability to remove Pt-DNA adducts . The resistance is not considered to be associated with the multidrug resistant, GSH, metallothionein systems . AmB and NVB can overcome cisplatin resistance of SKOV3/cp in vitro and in vivo.

Jpn J Physiol, 1996 Feb, 46(1), 33 - 41
Quantitative characterization of P-glycoprotein-mediated transport in mdr1-gene-transfected lymphoma cells; Oonishi T et al.; We have established a quantitative flow cytometry system to elucidate the causal role of P-glycoprotein in the phenomenon of multidrug resistance . We have used this method to analyze the accumulation and release of adriamycin (ADM) in intact L5178Y and L5178Y/VMDR/C.06 (L5178Y/R) cells, by determining the effect of sodium orthovanadate (Na3VO4), verapamil, bovine serum albumin (BSA) and physiologically operative materials on the cells . Based on the experiments, we prepared a standard solution that contained NaCl, D-glucose, L-cysteine, HCO3- and BSA, which was sufficient to perform transport experiments . In particular, BSA caused a decrease in ADM accumulation and a facilitation of the rate of ADM release in both L5178Y and L5178Y/R cells, probably due to its relatively high affinity for ADM as compared to the cell membrane . In multidrug-resistant L5178Y/R cells, sodium orthovanadate, a strong ATP-binding inhibitor, caused a marked increase in the accumulation of ADM, whereas vanadate-treated drug-sensitive L5178Y cells showed little increase in ADM accumulation . In a release (0-trans exit) experiment, vanadate-treated L5178Y/R cells exhibited an apparent decrease in ADM release (increase in ADM retention), to a level which was almost the same as L5178Y cells . We thus confirmed that the P-glycoprotein-mediated efflux system is coupled with P-glycoprotein-associated ATP-hydrolysis . Further, verapamil, a potent inhibitor of P-glycoprotein-mediated transport, facilitated the ADM accumulation in L5178Y/R cells up to the level of L5178Y and vanadate-treated L5178Y/R cells . A more important finding is that, in the release experiment, verapamil-treated L5178Y/R cells exhibited a much greater ADM retention than drug-sensitive L5178Y and vanadate-treated L5178Y/R cells . These findings, in particular the potent effect of verapamil on drug-resistant cells, may afford new insight into the pathophysiology of the phenomenon of multidrug resistance and the mechanism of action of the multidrug transporter.

Cytometry, 1996 Feb 1, 23(2), 120 - 5
Quantitative determination of the MDR-related P-glycoprotein, Pgp 170, by a rapid flow cytometric technique; Ferrand VL et al.; Pgp 170 is the main integral membrane protein involved in acquired or de novo multidrug resistance (MDR), frequently implicated in chemotherapeutic failure . Because there is at present no method for quantitating Pgp 170 levels, a new and convenient assay, using flow cytometry with a standard fluorescence curve and MRK 16, a mAb recognizing an external epitope of human Pgp 170, was developed . Assuming a 1:1 stoichiometry, we calculated for the first time the apparent number of Pgp 170 molecules per cell . The method was applied successfully to cells in suspension or grown as monolayers and their mixtures . All quality criteria were checked and proved the suitability of the method for quantifying Pgp 170.

Anticancer Drugs, 1996 Feb, 7(2), 182 - 8
Paclitaxel sensitizes multidrug resistant cells to radiation; Mote PA et al.; The unique action of paclitaxel, to stabilize microtubules and block cells at the radiosensitive G2M phase of the cell cycle, suggests, it may sensitize tumors to radiotherapy . We have investigated the potential of this interaction to overcome multidrug resistance in vitro using the HL60 cell line and its P-glycoprotein expressing, multidrug resistant H/E8 subline . HL60 cells showed a modest 1.4-fold (p < 0.01) increase in sensitivity to 2 Gy radiation given 24 h after a 1 h treatment with paclitaxel . The H/E8 subline, which has increased radiation resistance and expresses an extended multidrug resistance phenotype, showed significant sensitization to radiation (up to 2.3-fold sensitization; p < 0.01) even with doses of paclitaxel which had no effect on cell viability or were associated with any G2/M block in the cell cycle . In the presence of verapamil, an inhibitor of P-glycoprotein mediated efflux, drug resistant cells could be sensitized to 2 Gy radiation by similar paclitaxel doses as the parental cell (> or = 30 nM; p < 0.01) . These results indicate a therapeutic advantage may be possible in the treatment of resistant tumors by the combined use of paclitaxel with radiation.

J Ind Microbiol, 1996 Feb, 16(2), 134 - 43
Cyclic peptides and depsipeptides from cyanobacteria: a review; Moore RE; An elaborate array of structurally-novel and biologically-active cyclic peptides and depsipeptides are found in blue-green algae (cyanobacteria) . Several of these compounds possess structures that are similar to those of natural products from marine invertebrates . Most of these cyclic peptides and depsipeptides, such as the microcystins and the lyngbyatoxins, will probably only be useful as biochemical research tools . A few, however, have the potential for development into useful commercial products . For example, cryptophycin-1, a novel inhibitor of microtubule assembly from Nostoc sp GSV 224, shows impressive activity against a broad spectrum of solid tumors implanted in mice, including multidrug-resistant ones, and majusculamide C, a microfilament-depolymerizing agent from Lyngbya majuscula, shows potent fungicidal activity and may have use in the treatment of resistant fungal-induced diseases of domestic plants and agricultural crops.

J Nucl Med, 1996 Feb, 37(2), 312 - 4
Evaluation of carbon-14-colchicine biodistribution with whole-body quantitative autoradiography in colchicine-sensitive and -resistant xenografts; Mehta BM et al.; Quantitative autoradiography (QAR) with radiolabeled monoclonal antibodies in xenografted animals has been extensively described in the past, either on individual tissues or on the whole body . We applied whole-body QAR to identify multidrug resistant tumors using 14C-colchicine (14C-CHC) . METHODS: Two groups of five animals each were xenografted with CHC-sensitive and CHC-resistant human neuroblastoma cells . Animals were injected intravenously with 4 microCi/0.11 mumole 14C-CHC per gram of body weight and sacrificed after 60 min . Whole-body QAR was carried out using 25-microns thick sections . RESULTS: Fusion images allowed direct comparison of 14C-CHC uptake in tumor and nontumor tissues . Mean 14C-CHC distribution in sensitive and resistant tumors was 882.0 +/- 43.6 and 399.6 +/- 157.7 nCi/g corresponding to 24.5 +/- 1.21 and 11.1 +/- 4.38 nmole/g, respectively (p < 0.001), with normal tissue distribution in both groups being similar . Three-dimensional QAR showed that the uptake of 14C-CHC was in the cellular zones of the tumor . This method has potential in biodistribution studies of novel radiopharmaceuticals such as 14C-CHC . CONCLUSION: These studies further suggest that PET imaging of 11C-CHC is feasible to distinguish between sensitive and resistant tumor deposits in vivo.

J Nucl Med, 1996 Feb, 37(2), 286 - 9
Technetium-99m-sestamibi uptake by human benign and malignant breast tumor cells: correlation with mdr gene expression; Cordobes MD et al.; Early diagnosis of multidrug-resistance (MDR) development is extremely important for the judicious choice of treatment protocols in breast cancer chemotherapy . In this study, the mechanism of 99mTc-sestamibi uptake by nine human breast tumor cell lines was analyzed as a function of P-glycoprotein (PgP) expression . METHODS: Technetium-99m-sestamibi radioactivity incorporation into the cells was determined after different times of incubation at 37 degrees C . We analyzed the mechanism of 99mTc-sestamibi uptake as follows: (a) effect of temperature (4 degrees C); (b) influence of extracellular 99mTc-sestamibi concentration; and (c) competitive inhibition of cell uptake with cold 99mTc-sestamibi . Technetium-99m-sestamibi uptake was compared to the level of PgP determined by Western blotting . The PgP reversing effect of verapamil was evaluated at different drug concentrations (50, 200, 500 microM) . RESULTS: Technetium-99m-sestamibi uptake plateaued at 60 min, which was 14 times lower at 4 degrees C than at 37 degrees C and was directly proportional to the extracellular concentration between 0.3 and 10 nM . Technetium-99m-sestamibi percentage uptake by cells expressing nonimmunodetectable levels of PgP was significantly higher (7.3% +/- 0.6% (s.d.) to 14.9% +/- 1.9%) than that by cells expressing high PgP levels (0.7% +/- 0.4%, p < 0.001) . In the presence of verapamil, a known reverser of PgP functions, 99mTc-sestamibi uptake was increased by a factor of 2 in cells expressing no detectable levels of PgP and by a factor of 12 in cells with high PgP levels . CONCLUSION: Technetium-99m-sestamibi uptake by these breast tumor cells is energy-dependent but not specific . These data suggest that 99mTc-sestamibi imaging may be used as a noninvasive technique to diagnose the presence of MDR in breast tumors in vivo.

J Arthroplasty, 1996 Feb, 11(2), 217 - 22
Periprosthetic infections due to Mycobacterium tuberculosis in patients with no prior history of tuberculosis; Spinner RJ et al.; Although uncommon, infection of prostheses with Mycobacterium tuberculosis can be managed successfully if it is diagnosed early and treated correctly . A case of M . tuberculosis infection of a prosthetic knee first diagnosed 4.5 years after initial arthroplasty is described . This case and a review of the literature led to the conclusion that there are two distinct patterns of M . tuberculosis infection following joint implant surgery in patients without a history of tuberculosis . (1) Mycobacterium tuberculosis infection may be an unexpected finding at the time of arthroplasty . These patients generally have favorable outcomes using standard antituberculous chemotherapy, without implant removal . (2) Late-onset M . tuberculosis joint infection may be identified in patients with painful, clinically infected, or malfunctioning prostheses . In these cases, medical treatment alone is usually unsuccessful; prosthesis removal is often required . With recent increases in the incidence of tuberculosis in the United States and the emergence of multidrug-resistant strains of M . tuberculosis, periprosthetic tuberculous infection is likely to become more common.

J Cell Biol, 1996 Feb, 132(4), 701 - 16
Human multidrug resistance 3-P-glycoprotein expression in transgenic mice induces lens membrane alterations leading to cataract; Dunia I et al.; We have generated mice transgenic for a human multidrug resistance (MDR)3 mini-gene driven by a hamster vimentin promoter . The MDR3 gene encodes a P-Glycoprotein that resembles the mouse multidrug resistance 2 P-Glycoprotein shown to be involved in the translocation of the phospholipid phosphatidylcholine through the hepatocyte canalicular membrane (Smit et al., 1993 . Cell . 75:451-462) . The vimentin promoter drives expression of the MDR3 transgene in mesenchymal tissues and in the eye lens . We show here that the presence of human multidrug resistance 3 P-Glycoprotein in the lens results in a severe lenticular pathology . Lens structural abnormalities initiate at a late embryonic stage and increase during postnatal lens development . Differentiation of the primary fibers is affected, and the terminal differentiation of the lens epithelium into secondary fibers is also perturbed . The ultrastructural alterations, particularly of the lens plasma membranes, resemble those identified in congenital mouse osmotic cataract.

J Cell Biol, 1996 Feb, 132(4), 549 - 63
The PAL1 gene product is a peroxisomal ATP-binding cassette transporter in the yeast Saccharomyces cerevisiae; Swartzman EE et al.; The PAL1 gene was isolated using PCR and degenerate oligonucleotide primers corresponding to highly conserved amino acid sequence motifs diagnostic of the ATP-binding cassette domain of the superfamily of membrane-bound transport proteins typified by mammalian multidrug resistance transporter 1 and Saccharomyces cerevisiae Ste6 . The deduced PAL1 gene product is similar in length to, has the same predicted topology as, and shares the highest degree of amino acid sequence identity with two human proteins, adrenoleukodystrophy protein and peroxisomal membrane protein (70 kD), which are both presumptive ATP-binding cassette transporters thought to be constituents of the peroxisomal membrane . As judged by hybridization of a PAL1 probe to isolated RNA and by expression of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the biogenesis of functional peroxisomes for its metabolism . A pal1delta mutant grew normally on either glucose- or glycerol-containing media; however, unlike PAL1+ cells (or the pal1delta mutant carrying the PAL1 gene on a plasmid), pal1delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carbon source . Antibodies raised against a chimeric protein in which the COOH-terminal domain of Pal1 was fused to glutathione S-transferase specifically recognized a protein in extracts from wild-type cells only when grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1delta cells and more abundant in pal1delta cells expressing PAL1 from a multicopy plasmid . The Pal1 polypeptide was highly enriched in the organellar pellet fraction prepared from wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component of the peroxisomal matrix, e-oxoacyl-CoA thiolase . As judged by both subcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 was present, absent, or overexpressed . These findings demonstrate that Pal1 is a peroxisome-specific protein, that it is required for peroxisome function, but that it is not necessary for the biogenesis of peroxisomes or for the import of 3-oxoacyl-CoA thiolase (and at least two other peroxisomal matrix proteins).

Oncogene, 1996 Feb 1, 12(3), 651 - 8
Gene-specific DNA repair and steady state transcription of the MDR1 gene in human tumor cell lines; Evans MK et al.; We have explored the relationship between DNA repair and transcription in vivo . A gene-specific repair assay has been employed to study removal of ultraviolet light-induced cyclobutane pyrimidine dimers in the MDR1 gene at different levels of MDR1 mRNA expression . The parental human adenocarcinoma cell line, KB-3-1, has very low levels of MDR1 mRNA expression, but its multidrug resistant derivatives KB-8-5 and KB-C1 have 42-fold and 3800-fold increases in MDR1 mRNA expression, respectively . In the KB-3-1 cell line that has a low level of MDR1 mRNA expression, we find a low level of MDR1 gene-specific repair and inefficient repair of the transcribed strand of the gene . In the KB-8-5 cell line that has a modest increase in MDR1 mRNA expression, we find only a minor increase in dimer repair in the MDR1 gene . Here, the repair in the transcribed strand is not significantly higher than that in the KB-3-1 cell line . However, in the KB-C1 derivative, where there is a 3800-fold increase in the level of MDR1 mRNA expression, we find a substantial increase in the level of dimer repair in the MDR1 gene . In addition, the MDR1 transcribed strand repair is markedly more efficient than the repair in the nontranscribed strand . Our data suggest that the rate of transcription in the MDR1 gene must be substantially increased before there is any measurable effect on DNA repair . Repair in the housekeeping gene, dihydrofolate reductase (DHFR), was similar in all three tumor cell lines . Repair in its transcribed strand was markedly lower than previously reported in normal human fibroblasts . We suspect that these human HeLa-derived tumor cell lines have deficient gene-specific DNA repair . This may be an important aspect of their malignant phenotype.

J Clin Oncol, 1996 Feb, 14(2), 610 - 8
Phase I study of etoposide with SDZ PSC 833 as a modulator of multidrug resistance in patients with cancer; Boote DJ et al.; PURPOSE: To determine the maximum-tolerated dose (MTD) and toxicity of PSC 833 infusion administered with etoposide for 5 days in patients with cancer, and to determine the effect of PSC 833 on etoposide pharmacokinetics . PATIENTS AND METHODS: Thirty-five patients were entered onto the study, one of whom was ineligible . Etoposide was delivered from day 1 as a 2-hour infusion over 5 consecutive days at a dose of 75 to 100 mg/m2/d . PSC 833 was administered from day 2 as a 2-hour loading dose and as a 5-day continuous infusion . Doses were escalated from 1 to 2 mg/kg (loading dose) and 1 to 15 mg/kg/d (continuous infusion) . RESULTS: Thirty-four patients were treated with 53 cycles of PSC 833 and etoposide . Steady-state blood PSC 833 levels more than 1,000 ng/mL were achieved in all patients treated at PSC 833 doses > or = 6.6 mg/kg/d by continuous infusion . Myelosuppression was the most common toxicity . The major dose-related toxicity of PSC 833 was reversible hyperbilirubinemia, which occurred in 83% of cycles . The dose-limiting toxicity of PSC 833 was severe ataxia, which occurred in two of nine patients treated at 12 mg/kg/d and in both of the single patients treated at 13.5 and 15 mg/kg/d . PSC 833 concentrations more than 2,000 ng/mL resulted in an increase in etoposide area under the curve (AUC) of 89%, a decrease in etoposide clearance (Cl) of 45%, a decrease in volume of steady-state distribution (Vss) of 41%, and an insignificant increase in alpha half-life (t 1/2 alpha) and significant increase of beta half-life (t 1/2 beta) of 19% and 77%, respectively . CONCLUSION: PSC 833 can be administered in combination with etoposide with acceptable toxicity . The recommended continuous infusion dose of PSC 833 for this schedule is 10 mg/kg/d over 5 days . PSC 833 results in an increase in etoposide exposure and etoposide doses should be reduced in patients receiving PSC 833.

DNA Cell Biol, 1996 Feb, 15(2), 105 - 11
Evidence that SP1 modulates transcriptional activity of the multidrug resistance-associated protein gene; Zhu Q et al.; In previous studies, we have cloned and sequenced a 5'-end region of the multidrug resistance-associated protein (MRP) gene that contains promoter activity as assessed through transient transfections of constructs contained in a pCAT basic reporter plasmid . In the present study, using a series of deletion mutants, evidence was obtained that the SP1 binding sites contained in the promoter are essential for optimal MRP transcriptional activity . These results were supported by the finding that introduction of site-specific mutations into the wild-type SP1 sequence produced a major reduction in CAT activity . DNase I protection assays also demonstrated that SP1 sites are protected from hydrolysis with proteins from nuclei of a variety of cell lines . Gel mobility-shift assays with proteins extracted from CHO, HeLa, HL60, or HL60/ADR demonstrated the presence of a protein that bound to the wild-type SP1 sequence but not to an SP1 sequence containing site-specific mutations . The mobility shift with nuclear extracts was closely similar to that occurring after incubating purified SP1 protein with wild-type SP1 sequence . DNA supershift experiments with antibody to SP1 strongly suggest that the complexes formed with nuclear extracts contain the SP1 protein.

Semin Oncol, 1996 Feb, 23(1 Suppl 3), 11 - 20
Preclinical evaluation of CPT-11 and its active metabolite SN-38; Lavelle F et al.; CPT-11 (irinotecan) is a water-soluble analogue of camptothecin (CPT), an antitumor drug extracted from the Chinese tree Camptotheca acuminata . SN-38 is an active metabolite of CPT-11 that contributes significantly to its activity . The antitumor effects of CPT-11 and SN-38 are exerted through a novel mechanism of action; inhibition of DNA topoisomerase I . CPT-11 and its metabolite have demonstrated potent inhibitory activity against a variety of cancer cell lines in vitro and against several murine and human tumors grafted in mice in vivo, including those that express multidrug resistance . CPT-11 has also shown synergistic activity in combination with 5-fluorouracil and cisplatin in vitro . No irreversible or unusual toxicities were observed with CPT-11 in animal toxicity studies . In summary, the preclinical profile of CPT-11 confirmed this drug to be an attractive candidate for clinical development.

Leuk Res, 1996 Feb, 20(2), 101 - 7
bcl-2 protein downregulation is not required for differentiation of multidrug resistant HL60 leukemia cells; Blagosklonny MV et al.; Parental and multidrug resistant HL60 leukemia cell lines were used to study coupling of expression of apoptotic/cytostatic (bcl-2, bax, bclxL, p21/Waf1, and c-myc) genes during differentiation . The multidrug resistant HL60 cell line, HL60/ADR, was less sensitive than parental cells to cytostatic activity of low (0.4-2 ng/ml) doses of PMA . However, during treatment with standard differentiating doses of PMA (10 ng/ml), no difference between the two cell lines in cytostasis and differentiation was found . Downregulation of c-myc and upregulation of p21/Waf1 proteins showed the same time-course in both cell lines . The bcl-2 mRNA was rapidly downregulated while bax and bclxL gene expression was not altered in both differentiating HL60 and HL60/ADR cells . Significant downregulation of bcl-2 protein occurred only in parental HL60 cells . In HL60/ADR, despite rapid cessation of bcl-2 protein synthesis, almost no change in steady-state bcl-2 protein level was found . The lack of bcl-2 protein downregulation was a result of the prolonged half-life of this protein in HL60/ADR cells . Thus, although downregulation of bcl-2 mRNA is coupled to differentiation, actual loss of bcl-2 protein is not required for accomplishment of the differentiation program.

Am J Trop Med Hyg, 1996 Feb, 54(2), 210 - 3
Comparative clinical trial of artesunate followed by mefloquine in the treatment of acute uncomplicated falciparum malaria: two- and three-day regimens; Looareesuwan S et al.; The difficulties in treating drug-resistant falciparum malaria in Thailand are compounded by the necessity of giving antimalarials over long periods of time . The resultant decrease in patient compliance not only lowers cure rates but also predisposes to the further spread of drug resistance . We compared the efficacy of two sequential treatment regimens given over two and three days in 111 patients with acute uncomplicated falciparum malaria . Sixty-seven patients received two 400-mg doses of artesunate (total dose = 800 mg) followed by two doses of mefloquine (750 mg given immediately and 500 mg 12 hr later; total dose = 1,250 mg) in Group 1 . Forty-four patients (Group II) received four 200-mg doses of artesunate (total dose = 800 mg) given 12 hr apart followed by a mefloquine regimen similar to that for Group I . All patients were admitted to hospital in Bangkok for 28 days to preclude reinfection . Ninety-six patients completed the study . Cure rates for the two groups were 84% (49 of 58) for Group I and 100% (38 of 38) for Group II . The mean parasite clearance time and fever clearance time were significantly shorter in Group II (P < 0.02) . There were no serious adverse reactions . All nine of the treatment failures in Group I were of the RI types . The results indicate that the sequential treatment with artesunate followed by mefloquine given over three days is effective and well-tolerated in patients with acute, uncomplicated falciparum malaria and suitable as an alternative treatment for multidrug-resistant falciparum malaria.

Am J Trop Med Hyg, 1996 Feb, 54(2), 205 - 9
Clinical study of pyronaridine for the treatment of acute uncomplicated falciparum malaria in Thailand; Looareesuwan S et al.; One hundred one adult patients with acute uncomplicated falciparum malaria were treated with pyronaridine . All patients were admitted to the Bangkok Hospital for Tropical Diseases for 28 days to exclude reinfection . Sixty-nine patients (Group I) received pyronaridine 1,200 mg over a three-day period and 32 patients (Group II) received 1,800 mg pyronaridine over a five-day period . Cure rates for the two groups were 63% (38 of 60) for Group I and 88% (23 of 26) for Group II (P<0.05) . No RII or RIII type response was seen . Mean fever and parasite clearance times were not significantly different in the two groups . The drug was well-tolerated . In vitro drug sensitivity tests of the paired parasite isolates obtained prior to treatment and after recrudescence indicated that the Plasmodium falciparum isolates of the successfully treated patients had a lower mean concentration for 50% inhibition of growth (IC50) and a much narrower range of the individual IC50 values (15.69 +or- 3.82 ng per ml (mean +or- SD)) as compared with those from the recrudescence cases (22.98 +or- 12.05 ng per ml) . Nevertheless, there was no evidence of an increase of the IC50 and IC95 values after recrudescence . The results of the study show that pyronaridine alone at a total dose of 1,800 mg given over five days is well-tolerated in patients suffering from acute uncomplicated malaria and has evident activity against multidrug-resistant falciparum malaria . However, it cannot be recommended for use in Thailand as long as the recrudescence rate is as high as 12% . Further studies of its combinations with other antimalarial drugs are needed.

Jpn J Cancer Res, 1996 Feb, 87(2), 184 - 93
Modulation of multidrug resistance by SDZ PSC 833 in leukemic and solid-tumor-bearing mouse models; Watanabe T et al.; P-Glycoprotein inhibitors, including the nonimmunosuppressive cyclosporin D analog SDZ PSC 833 (PSC 833), have been developed to circumvent multidrug resistance . In the present study, the potential of PSC 833 in reversing multidrug resistance was evaluated in various systemic treatment models with leukemic and solid-tumor-bearing mice . Having a relatively wide therapeutic window of daily p.o . doses from 12.5 to 75 mg/kg, PSC 833 significantly improved the antileukemic activity of the anticancer drugs adriamycin (ADM), vincristine (VCR) and etoposide (VP-16) given i.p . or i.v . against i.p.-inoculated vincristine-resistant P388 tumor (P388/VCR) . PSC 833 in combination with i.p.-injected anticancer drugs in optimal schedule and dosage induced apparent cures in some leukemic mice, whereas no cures were obtained with the cyclosporin A/anticancer drug combinations . PSC 833 combined with i.v.-injected anticancer drugs was highly active, but not curative, against P388/VCR and parental P388 tumors (maximum T/C>175%) PSC 833 in combination with intravenous treatment with ADM showed prominent anti-solid-tumor activity against s.c.-inoculated colon adenocarcinoma 26 and human colorectal adenocarcinoma HCT-15 . Against colon adenocarcinoma 26, the PSC 833/ADM combinations induced cure in two or three of six mice . PSC 833/ADM combinations significantly inhibited the growth of the tumor with maximum percent inhibitions of 83 and 73% in the early and advanced stages of the HCT-15 tumor models, respectively . The present study demonstrated that PSC 833 is highly active in potentiating the antitumor activity of systemically administered ADM, VCR and VP-16 against four murine and human tumors with a relatively wide therapeutic window of daily p.o . dose range of 12.5-100 mg/kg.

Semin Oncol, 1996 Feb, 23(1), 46 - 65
Transfer of drug resistance genes into hematopoietic progenitors to improve chemotherapy tolerance; Koc ON et al.; A number of drug resistance genes have been identified that may be useful in gene therapy approaches to ameliorate chemotherapy toxicity . Hematopoietic tissue is the most suitable target for drug resistance gene therapy because myelosuppression is the dose-limiting toxicity of the many chemotherapeutic agents . Recent studies have shown that murine and human hematopoietic progenitors can be transduced ex vivo using retroviral vectors to overexpress P-glycoprotein, dihydrofolate reductase, and O6-alkylguanine DNA alkyltransferase . In all instances, gene transfer results in significant drug resistance in hematopoietic progenitors both in vitro and in vivo . Clinical trials are underway to evaluate the role of MDR-1 gene therapy in amelioration of chemotherapy induced myelosuppression . Other genes being examined for their potential to transfer drug resistance to hematopoietic cells include genes encoding aldehyde dehydrogenase, nucleotide excision repair proteins, multidrug resistant protein, and superoxide dismutase . As a group these proteins could confer significant levels of chemotherapy drug resistance to bone marrow cells . When compared with other somatic gene therapy approaches, drug resistance gene therapy has the aim of protecting normal cells and preventing toxicity . In addition many of these genes could be used to select for cells carrying the drug resistance gene as well as cotransduced therapeutic gene . Thus, gene transfer of drug resistance genes will have broad applications in the field of gene therapy as well as in protecting hematopoietic cells from chemotherapy toxicity.

Cancer Genet Cytogenet, 1996 Feb, 86(2), 116 - 9
Acquisition of doxorubicin resistance in human leukemia HL-60 cells is reproducibly associated with 7q21 chromosomal anomalies; Ganapathi R et al.; Tumor cell resistance to doxorubicin (DOX) is usually associated with the overexpression of P-glycoprotein (PGP) in model systems . We have characterized the karyotypic changes in two sublines of HL-60 cells which differ in the induction of differentiation by retinoic acid . The parental sublines, designated HL-60A/S and HL-60Y/S, were selected in increasing concentrations of 0.025-0.1 micrograms/mL DOX . Monosomy 8 in HL-60Y/S was the only karyotypic difference prior to DOX exposure . Both sublines acquired 7q+ markers upon exposure to DOX . In HL-60Y/S, and add(7)(q21) replaced one homologue at 0.025 micrograms/mL DOX, and an add(7)(q32) appeared which replaced the other normal 7 at 0.05 micrograms/mL DOX . The HL-60A/S cells acquired an add(7)(q21) at 0.025 micrograms/mL DOX . The 7q+ abnormalities involved breakpoints in the midregion of 7q . The overexpression of phosphorylated PGP in immunoprecipitates with C-219 antibody was identified in both sublines of DOX-resistant HL-60 cells with 7q+ abnormalities, and this is consistent with the location of mdr-1 sequences to 7q21-21.1 . Also, analysis of RNA from parental-sensitive and DOX-resistant sublines by reverse transcriptase-polymerase chain reaction revealed: a) comparable expression of multidrug resistance related protein (MPR) in sensitive and resistant sublines; and b) overexpression of mdr-1 only in the DOX-resistant sublines . Thus, the selection of DOX resistance in two sublines of HL-60 cells which differ in their response to retinoic acid-induced myeloid differentiation is reproducibly associated with overexpression of mdr-1 versus MRP.

Cancer Res, 1996 Feb 1, 56(3), 574 - 81
Reversal of multidrug resistance in vivo by dietary administration of the phytochemical indole-3-carbinol; Christensen JG et al.; A major obstacle to successful chemotherapy is the development of multidrug resistance (MDR) by cancer cells . MDR is characterized by enhanced cellular efflux of many structurally and functionally diverse compounds, including many anticancer drugs, due to overexpression of the MDR-1 gene product, P-glycoprotein . We hypothesized that the phytochemical, indole-3-carbinol (I3C), and some of its acid-condensation derivatives may inhibit P-glycoprotein-mediated transport due to their aromatic and nitrogen components, thus increasing the accumulation and efficacy of anticancer drugs and acting as a dietary adjuvant to conventional chemotherapy . I3C was subjected to acid conditions similar to those occurring in the stomach following ingestion and three acid-condensation products; a dimer, a noncyclic trimer, and a cyclic trimer were isolated and purified by high-performance liquid chromatography . The ability of I3C and its acid-condensation derivatives to reverse MDR was investigated using murine B16 melanoma cells that were transfected with the human MDR-1 gene (B16/hMDR-1 cells) and were cross-resistant to vinblastine and doxorubicin . The I3C acid-condensation product mixture, but not I3C, sensitized B16/hMDR-1 transfectants to the toxicity of vinblastine and doxorubicin . All three I3C acid-condensation products also increased the accumulation of the P-glycoprotein substrate, doxorubicin, in B16/hMDR-1 transfectants to levels comparable to parental B16 cells . The I3C acid-condensation product mixture competed with azidopine for binding to P-glycoprotein, suggesting that the observed MDR-reversing effect of the acid-condensation products was due to direct interaction with P-glycoprotein . The ability of p.o . administered I3C to reverse MDR was also tested in vivo . The resistance of B16/hMDR-1 transfectants to vinblastine and doxorubicin was preserved after i.p . injection and growth in nude mice . Tumor mass in mice that were provided with 333 or 500 mg/kg mouse/day I3C in their diet and injected s.c . with the anticancer drugs doxorubicin or vinblastine was significantly reduced as compared to tumor mass in mice provided with standard diet and injected with these anticancer drugs or mice provided with 500 mg/kg mouse/day I3C and not injected with anticancer compound . The concentrations of I3C used had no effect on survival or the general appearance and behavior of the mice . Collectively, these results indicate that ingestion of the common dietary constituent I3C results in its conversion to acid-condensation derivatives that sensitized MDR tumors to chemotherapeutic drugs without eliciting direct toxicity to the host.

Br J Cancer, 1996 Feb, 73(3), 307 - 15
Regulation of P-glycoprotein 1 and 2 gene expression and protein activity in two MCF-7/Dox cell line subclones; Davies R et al.; The MCF-7 doxorubicin-resistant cell line MCF-7/Dox has been used extensively for studies of the multidrug resistance phenomenon . Using fluorescence-activated cell sorting (FACS), these cells were separated into two populations on the basis of rhodamine 123 (R123) accumulation . We designated these as low P-glycoprotein (LP-gp) and high P-gp (HP-gp) cells on the basis of their P-gp content . Using the reverse transcriptase polymerase chain reaction technique controlled by homologous internal standards, we analysed levels of MDR1 and MDR2 mRNA in each cell type . LP-gp and HP-gp cells had MDR1 mRNA levels of 2.17 +/- 0.17 and 6.65 +/- 2.29 amol ng-1 total RNA respectively, compared with 0.00088 +/- 0.00005 amol ng-1 in wild-type MCF-7 cells (MCF-7/WT) . MCF-7/WT cells additionally contained 0.023 +/- 0.016 amol ng-1 of MDR2 mRNA, which was unchanged in LP-gp cells, but lower than in HP-gp cells, which contained 0.42 +/- 0.08 amol ng-1 . Both LP-gp and HP-gp cells contained increased copies of the MDR1 gene . However, the degree of gene amplification did not correlate with the changes in MDR1 mRNA levels, indicating further regulatory levels of gene expression . The level of P-gp detected by MRK 16 correlated with R123 accumulation . HP-gp cells expressed a 10-fold higher level of P-gp1 than LP-gp cells . However, there was only a 3-fold increase in MDR1 mRNA level in HP-gp cells compared with LP-gp cells . These data suggest that some regulation of P-gp1 expression also occurred at the post-translational level . Phosphorylation of P-gp by protein kinase C (PKC)-alpha is necessary for its activity . Our analysis of PKC-alpha, 0 and epsilon isozyme levels, and subcellular distribution, shows a co-regulation of expression with P-gp, suggesting a necessary role for PKC in P-gp regulation.

J Virol, 1996 Feb, 70(2), 1086 - 90
Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy; Iversen AK et al.; Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI) . Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis . Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine . The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors . Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)) . Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period . The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.

Biochim Biophys Acta, 1996 Jan 31, 1278(2), 213 - 22
Saturable P-glycoprotein kinetics assayed by fluorescence studies of drug efflux from suspended human KB8-5 cells; Ghauharali RI et al.; This article describes a new and rapid method to determine the pumping rate of P-glycoprotein (P-gp) in intact cells . Multidrug resistant (MDR) human epidermoid carcinoma KB8-5 cells (containing P-gp) were loaded with daunorubicin (DNR) in the absence or in the presence of verapamil, sufficient to inhibit DNR pumping by P-gp . In either case, the cells were resuspended in medium devoid of DNR and the subsequent increase of the DNR fluorescence intensity was measured as a function of time . For cells loaded with the same amount of drug, the free cytosolic drug concentration (Ci(t)) was a unique function of the DNR medium concentration (Co(t)) . The cellular drug content in the presence of verapamil decreased nonlinearly with decreasing extracellular drug concentration, indicating that the intracellular drug apparent distribution volume increased with decreasing cellular drug content . At each fluorescence intensity, we calculated the P-gp mediated (verapamil-inhibitable) DNR transport rate from the rate of increase of the DNR fluorescence intensity in the absence of verapamil minus the rate of increase of the DNR fluorescence intensity in the presence of verapamil . When plotted against the intracellular free drug concentration (as calculated from the total cellular drug content and a separately determined relation between the total cellular drug content and the intracellular free drug concentration: the apparent distribution volume), this P-gp mediated DNR transport rate showed saturation of P-gp at higher DNR concentrations . The results imply that P-gp mediated DNR transport is saturable (the value of Km is in the order of 1 microM).

Biochem Pharmacol, 1996 Jan 26, 51(2), 117 - 23
Induction of daunorubicin carbonyl reducing enzymes by daunorubicin in sensitive and resistant pancreas carcinoma cells; Soldan M et al.; Daunorubicin (DRC) and other anthracyclines are valuable cytotoxic agents in the clinical treatment of certain malignancies . However, as is the case with virtually all anticancer drugs, tumor cell resistance to these agents is one of the major obstacles to successful chemotherapy . In addition to an increased energy-dependent efflux of chemotherapeutic agents, enzymatic drug-inactivating mechanisms also contribute to multidrug resistance of tumor cells . In the case of DRC, carbonyl reduction leads to 13-hydroxydaunorubicinol (DRCOL), the major metabolite of DRC with a significantly lower antineoplastic potency compared to the parent drug . In the present study, we compared two pancreas carcinoma cell lines (a DRC-sensitive parental line and its DRC-resistant subline) with respect to their capacity of DRC inactivation via carbonyl reduction . In addition, we cultured the two cell lines in the presence of increasing sublethal concentrations of DRC . Evidence is presented that DRC treatment itself leads to a concentration-dependent induction of DRC carbonyl reduction in subcellular fractions of both the sensitive and resistant pancreas carcinoma cells, resulting, surprisingly, in different susceptibilities to DRC . The principal difference between the two cell lines becomes most apparent at high-dose DRC supplementation (1 microgram/mL), at which DRC resistant cells exhibited higher inducibility of DRC-inactivating enzymes, whereas respective sensitive cells already showed an impairment of cellular viability . The use of the diagnostic model substrates metyrapone and p-nitrobenzaldehyde reveals that this adaptive enhancement of DRC inactivation can be attributed to the induction of DRC carbonyl reductases different from known aldehyde and carbonyl reductases . In conclusion, these findings suggest that inactivation of anthracyclines by carbonyl reduction is inducible by the substrate itself, a fact that might be considered as one of the enzymatic mechanisms that contribute to the acquired resistance to these drugs.

Int J Cancer, 1996 Jan 26, 65(3), 351 - 9
SDZ 281-977: a modified partial structure of lavendustin A that exerts potent and selective antiproliferative activities in vitro and in vivo; Cammisuli S et al.; The chemical derivatization of biologically active microbial metabolites continues to be a promising approach to the identification of new drugs . We recently synthesized the novel antiproliferative compound SDZ 281-977, 5-{2-(2,5-dimethoxy-phenyl)ethyl}-2-hydroxy-benzoic acid methylester, a derivative of the EGF receptor tyrosine kinase inhibitor lavendustin A . Here we report on our studies of the anticancer efficacy and the mode of action of SDZ 281-977 . The growth of both the human pancreatic tumor cells MIA PaCa-2 and the human vulvar carcinoma cells A431 was inhibited in the low micromolar range . Tumors from these cells were induced in nude mice and were shown to respond to orally or intravenously administered SDZ 281-977 . In contrast, no antitumor effect was detected in rats bearing dimethylbenzanthracene-induced mammary tumors . Studies in mice indicated that SDZ 281-977 was neither immunosuppressive nor hematosuppressive at doses effectively inhibiting tumor growth . Surprisingly, the mode of action of SDZ 281-977 apparently does not involve inhibition of EGF receptor tryosine kinase, because, in contrast to lavendustin A, SDZ 281-977 failed to inhibit this enzyme in a cell-free assay . The mechanism of the antiproliferative effect can be explained on a cellular level by the ability of the compound to arrest cells in mitosis . SDZ 281-977 is thus the first example of an antimitotic agent derived from the potent tyrosine kinase inhibitor lavendustin A . The therapeutic potential of SDZ 281-977 is enhanced by the fact that it is not subject to multidrug resistance, because tumor cells expressing the multidrug resistance phenotype were as sensitive to SDZ 281-977 as their nonresistant counterparts . In conclusion, SDZ 281-977 represents a novel lavendustin A derivative with potent antiproliferative properties in vitro and in vivo that may be explained on the basis of its antimitotic effects . SDZ 281-977 may be a candidate drug for the treatment of selected cancers, including those expressing the multidrug resistance phenotype.

J Biol Chem, 1996 Jan 26, 271(4), 2102 - 11
Partial reversal of multidrug resistance in human breast cancer cells by an N-myristoylated protein kinase C-alpha pseudosubstrate peptide; Gupta KP et al.; The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein . Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein . Evidence has been presented that the isozyme PKC-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation . Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo . In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha . P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 kinase, and P-glycoprotein . A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC-alpha substrates . The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive . P1 did not affect P-glycoprotein expression . MCF7-MDR cells were not cross-resistant to P1, which suggest that the peptide was not transported by P-glycoprotein . Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit {3H}azidopine photoaffinity labeling of P-glycoprotein . P1 actually increased {3H} azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump . P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-alpha-catalyzed P-glycoprotein phosphorylation.

J Biol Chem, 1996 Jan 26, 271(4), 1877 - 83
Altered drug-stimulated ATPase activity in mutants of the human multidrug resistance protein; Muller M et al.; The characteristics of P-glycoprotein (MDR1), an ATP-dependent drug extrusion pump responsible for the multidrug resistance of human cancer, were investigated in an in vitro expression system . The wild-type and several mutants of the human MDR1 cDNA were engineered into recombinant baculoviruses and the mutant proteins were expressed in Sf9 insect cells . In isolated cell membrane preparations of the virus-infected cells the MDR1-dependent drug-stimulated ATPase activity, and 8-azido-ATP binding to the MDR1 protein were studied . We found that when lysines 433 and/or 1076 were replaced by methionines in the ATP-binding domains, all these mutations abolished drug-stimulated ATPase activity independent of the MgATP concentrations applied . Photoaffinity labeling with 8-azido-ATP showed that the double lysine mutant had a decreased ATP-binding affinity . In the MDR1 mutant containing a Gly185 to Val replacement we found no significant alteration in the maximum activity of the MDR1-ATPase or in its activation by verapamil and vinblastine, and this mutation did not modify the MgATP affinity or the 8-azido-ATP binding of the transporter either . However, the Gly185 to Val mutation significantly increased the stimulation of the MDR1-ATPase by colchicine and etoposide, while slightly decreasing its stimulation by vincristine . These shifts closely correspond to the effects of this mutation on the drug-resistance profile, as observed in tumor cells . These data indicate that the Sf9-baculovirus expression system for MDR1 provides an efficient tool for examining structure-function relationships and molecular characteristics of this clinically important enzyme.

N Engl J Med, 1996 Jan 25, 334(4), 231 - 8
Expression of the gene for multidrug-resistance-associated protein and outcome in patients with neuroblastoma; Norris MD et al.; BACKGROUND . Overexpression of the gene for the multidrug-resistance-associated protein (MRP) has been linked with resistance to chemotherapeutic agents (multidrug resistance) in vitro . The expression of MRP by neuroblastoma cells correlates with N-myc oncogene amplification, a well-established prognostic indicator in patients with neuroblastoma . METHODS . To relate MRP gene expression to established prognostic markers and the clinical outcome of neuroblastoma, we analyzed MRP expression in specimens of primary tumors from 60 patients with neuroblastoma . RESULTS . Levels of MRP gene expression were significantly higher in tumors with N-myc amplification than in tumors without such amplification (P < 0.001) . High levels of MRP expression were strongly associated with reductions in both survival and event-free survival (P < 0.001) in the overall study population and in subgroups of patients without N-myc amplification and patients with localized disease . For the overall study population, the five-year cumulative survival rates in the groups with high and low levels of MRP expression were 57 percent (95 percent confidence interval, 37 to 78 percent) and 94 percent (95 percent confidence interval, 86 to 100 percent), respectively . In contrast, expression of the MDR1 multi-drug-resistance gene was not predictive of survival or event-free survival . After adjustment by multivariate analysis for the effects of N-myc amplification and other prognostic indicators, high levels of MRP expression retained significant prognostic value for poor survival (relative hazard, 14.9; P = 0.01) and poor event-free survival (relative hazard, 9.7; P = 0.004), whereas N-myc amplification had no prognostic value . CONCLUSIONS . High levels of MRP gene expression in patients with neuroblastoma correlate strongly with poor outcome . The findings suggest that expression of this multidrug-resistance gene accounts for the association between N-myc amplification and reduced survival.

J Biol Chem, 1996 Jan 19, 271(3), 1708 - 16
Characterization of phosphorylation-defective mutants of human P-glycoprotein expressed in mammalian cells; Germann UA et al.; To assess the role of phosphorylation of the human multidrug resistance MDR1 gene product P-glycoprotein for its drug transport activity, phosphorylation sites within its linker region were subjected to mutational analysis . We constructed a 5A mutant, in which serines at positions 661, 667, 671, 675, and 683 were replaced by nonphosphorylatable alanine residues, and a 5D mutant carrying aspartic acid residues at the respective positions to mimic permanently phosphorylated serine residues . Transfection studies revealed that both mutants were targeted properly to the cell surface and conferred multidrug resistance by diminishing drug accumulation . In contrast to wild-type P-glycoprotein, the overexpressed 5A and the 5D mutants exhibited no detectable levels of phosphorylation, either in vivo following metabolic labeling of cells with {32P}orthophosphate or in vitro in phosphorylation assays with protein kinase C, cAMP-dependent protein kinase, or a P-glyco-protein-specific protein kinase purified from multidrug-resistant KB-V1 cells . These results reconfirm that the major P-glycoprotein phosphorylation sites are located within the linker region . Furthermore, the first direct evidence is provided that phosphorylation/dephosphorylation mechanisms do not play an essential role in the establishment of the multidrug resistance phenotype mediated by human P-glycoprotein.

Cancer Lett, 1996 Jan 19, 99(1), 109 - 14
Chemosensitization and drug accumulation assays as complementary methods for the screening of multidrug resistance reversal agents; Quesada AR et al.; Two methods based on the reversion of adriamycin-resistance o the increase of Rhodamine 123 accumulation in a multidrug resistant (MDR) cell line have been simplified and adapted for the screening of MDR reversal agents . Both methods are carried out in microtiter plates, are highly sensitive and can be easily automated . In both assays verapamil and the cyclosporine derivative PSC 833 could be detected at concentrations lower than 1 and 0.05 microM, respectively . Depending on the MDR cell line used, drugs exhibiting the collateral sensitivity phenomenon can be selected in the cytotoxicity assay, while interferences due to sample toxicity are easily avoided in the dye accumulation assay.

Int J Cancer, 1996 Jan 17, 65(2), 230 - 7
Overlapping phenotypes of multidrug resistance among panels of human cancer-cell lines; Izquierdo MA et al.; In addition to P-glycoprotein (Pgp), 2 proteins related to multidrug resistance (MDR) have recently been described . The Multidrug-Resistance-associated protein (MRP) is one of the ATP-binding-cassette (ABC) transporters . The Lung-Resistance Protein (LRP) is the major component of human vaults, which are newly described cellular organelles and thought to mediate intracellular transport processes . Using immunocytochemical methods, we have examined the expression of MRP and LRP among panels of human cancer-cell lines not selected for drug resistance which have been previously characterized for expression of Pgp, and in vitro response to a variety of anti-cancer drugs . Expression of MRP and LRP was observed in 47/55 (87%) and 46/59 (78%) cell lines, respectively . Statistically significant correlations were observed between expression of each of these 3 proteins and in vitro sensitivity to at least one drug classically associated with MDR . LRP showed the greatest individual predictive value, which also applied to several non-classical MDR drugs . Co-expression of 2-3 MDR-related proteins was observed in 64% of the lines and was, in general, associated with high relative levels of drug resistance . Previously identified "classic" MDR lines as well as "pan-resistant" lines concurrently expressed all 3 MDR-related proteins . Some highly drug-resistant cell lines without detectable MDRI/Pgp were found to express relatively high levels of MRP and LRP . The high prevalence of MRP and LRP expression observed in this large set of cell lines, which have not been subjected to laboratory drug selection, suggests that MDR mechanisms associated with these proteins may be widespread in human malignancies . Moreover, the overlapping of these more recently recognized MDR phenotypes with Pgp-type MDR results in a complex phenotype, the understanding of which may be of importance in the development of new drugs and design of clinical treatment protocols, particularly those seeking to employ strategies to reverse the MDR phenotype.

Cancer, 1996 Jan 15, 77(2), 292 - 300
Sequential assessment of multidrug resistance phenotype and measurement of S-phase fraction as predictive markers of breast cancer response to neoadjuvant chemotherapy; Chevillard S et al.; BACKGROUND . The authors examined the relevance of S-phase fraction (SPF) and multidrug resistance (MDR) phenotype as predictive tests of breast cancer response in a series of patients treated by conventional doses of neoadjuvant chemotherapy with (FAC) or without (FTC) doxorubicin . METHODS . Fine needle samplings of tumors were used to measure SPF by flow cytometry before treatment (Day 0), and to assess the MDR phenotype using semiquantified reverse transcriptase polymerase chain reaction and immunocytochemistry, before and after (Days 8 and 28) the first cycle of chemotherapy . RESULTS . Measurement of SPF before treatment was significantly associated with clinical response, but sequential assessment of MDR phenotype identified three groups of tumors with distinct outcomes: (1) tumors with a positive and constant expression of MDR1, in which prediction of resistance was restricted to patients treated by FAC; (2) tumors without any detectable expression, in which resistance to FAC or FTC treatments was rarely observed; and (3) tumors with an early (Day 8) acquired or increased MDR1 gene expression, which were always resistant to therapy to both treatment regimens . These results were confirmed at the protein level . CONCLUSIONS . Sequential assessment of MDR phenotype is a relevant tool for monitoring breast cancer response in neoadjuvant chemotherapy.

Med J Aust, 1996 Jan 15, 164(2), 121 - 4
Multidrug-resistant tuberculosis: prevention is better than cure; Gilbert GL; The incidence of tuberculosis, in decline in Western countries for years, is increasing again . Inadequate therapy leads to the emergence of multidrug-resistant tuberculosis, which is difficult to treat and for which there is no effective chemoprophylaxis . Prevention is the most important strategy.

Blood, 1996 Jan 15, 87(2), 725 - 33
Mechanisms of retinoid resistance in leukemic cells: possible role of cytochrome P450 and P-glycoprotein; Kizaki M et al.; Retinoic acid (RA) regulates the differentiation and proliferation of a wide variety of different cell types and all-trans RA induces complete remission in a high proportion of patients with acute promyelocytic leukemia (APL) . However, clinical resistance to retinoids may develop and poses a serious problem for differentiation-inducing therapy . We studied the effects of RA in combination with a cytochrome P450 inhibitor (clotrimazole) and a P-glycoprotein antagonist (verapamil) on cell growth and differentiation of RA-resistant HL-60 cells and fresh RA-resistant leukemic cells from two APL patients . RA-resistant HL-60 cells and APL cells differentiated to mature granulocytes when cultured with all-trans RA and either clotrimazole and verapamil but not with either of the agents alone . These findings were confirmed in these cells by their increased expression of CD11b antigen and migration-inhibitory factor-related protein-8/14 mRNAs and decreased levels of c-myc mRNA . These combinations also markedly decreased the number of viable cells and inhibited cellular proliferation . After isolation of microsomes, measurements showed that levels of cytochrome P450 activities in both wild-type and RA-resistant HL-60 cells were almost comparable . Moreover, expression of the CYP1A1-type cytochrome P450 gene could not be detected in either cell type . However, RA-resistant HL-60 cells and APL cells, but not RA-sensitive HL-60 cells and APL cells, expressed multidrug-resistance-1 gene transcripts . Taken together, acquired resistance to RA may be explained in part by drug metabolism in leukemic cells . Possible mechanisms for accelerated clearance of RA include the induction of non-CYP1A1 cytochrome P450 enzymes and P-glycoprotein.

Eur J Pharmacol, 1996 Jan 11, 295(2-3), 253 - 60
Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on P-glycoprotein; Boer R et al.; The interaction of cytostatics and chemosensitizers with the dexniguldipine binding site of P-glycoprotein was investigated in photoaffinity labeling experiments . A tritiated azidoderivative of the chemosensitizer dexniguldipine with dihydropyridine structure, {3H}B9209-005, was used to irreversibly label P-glycoprotein . The apparent affinity of cytostatics and chemosensitizers to this binding site was estimated from labeling experiments in the presence of increasing concentrations of compounds . From the cytostatics tested, the vinca alkaloids and taxol showed the highest affinity, anthracyclins possessed moderate affinity while methotrexate, ara C and camptothecin, cytostatics not involved in P-glycoprotein-mediated multidrug resistance, were almost inactive . The chemosensitizers GF 120918, cyclosporin A and SDZ PSC-833 inhibited photoincorporation with the highest potency . Steep dose-inhibition curves were obtained with the cyclic peptides and S9788, indicating that these compounds may bind allosterically to a separate binding site . Compounds with dihydropyridine structure with or without chemosensitizing potency were also tested and some structure-activity relationships could be derived from the data . Our data show that inhibition of photoaffinity labeling by {3H}B9209-005 is a valuable and reliable system for measuring the interaction with and potency of chemosensitizing compounds at P-glycoprotein . Furthermore, data obtained in this test system are well suited to investigate structure-activity relationships for chemosensitizers at P-glycoprotein . In addition cytostatics underlying P-glycoprotein-mediated multidrug resistance can be identified.

Lancet, 1996 Jan 6, 347(8993), 2 - 3
Pyronaridine: a promising drug for Africa?
Winstanley P.
PIP: There are more than 1 million malaria deaths annually in Africa, approximately 90% of such deaths globally . Parasite resistance is such that chloroquine can no longer be relied upon to cure deadly Plasmodium falciparum infections . Pyrimethamine-sulfadoxine is almost as cheap as chloroquine and easier to use, but resistance to this drug is also likely to become widespread across Africa within the next five years . Practical alternatives to Fansidar must be identified now . The author describes pyronaridine, a drug which in Cameroon achieved 100% cure of uncomplicated falciparum malaria . It is a synthetic Chinese drug currently being assessed by the WHO Committee on Drugs for Malaria . Although pyronaridine's mode of action and disposition are poorly understood, it is nonetheless known to be effective against multidrug-resistant P . falciparum; that it does not seem to exhibit much cross-resistance with chloroquine, quinine, or mefloquine; that it has proved clinically effective in trials in China; and that it seems to be well-tolerated . Much remains to be seen, however, before pyronaridine can be recommended for routine use in Africa . Dose schedules are empirical and pharmacokinetic data are urgently needed; current formulations have low oral bioavailability; the geographic variation in parasite chemosensitivity, expected potential for resistance, and toxicity must be investigated; pyronaridine's toxicological profile has not been established to standards acceptable in the US or the European Union; and its clinical risk-benefit profile is poorly understood . Little clinical work has been conducted outside of China . Furthermore, Plasmodium species can develop resistance to the drug in the laboratory, which necessitates careful attention when used in areas of high transmission, and it remains unclear whether pyronaridine will be well tolerated in humans . Assembling the necessary data for regulatory approval will be costly . If pyronaridine is to be developed, attention must be given to further costs; treating an adult currently costs about $3, too expensive for Africa .

Cancer Lett, 1996 Jan 2, 98(2), 199 - 205
Effect of the activated Raf protein kinase on the human multidrug resistance 1 (MDR1) gene promoter; Kim SH et al.; Revealing the regulatory mechanism of the multidrug resistance 1 (MDR1) gene is important to gain understanding of MDR in tumor cells . Using MDR1 deletion constructs and the 22W mutant of c-Raf in which the NH2-terminal half has been deleted, we examined the effect of the activated Raf on human MDR1 promoter activity in transient expression assay and stable transfectants of GHE-L cells . A DNA sequence exhibiting strong activation of MDR1 promoter by 22W was located between -197 and -136 containing the upstream heat shock element (HSE) motifs without other regulatory elements, whereas the MDR1 deletion construct containing downstream HSE motif showed a relatively weaker activation by 22W . We observed that the activated Raf significantly potentiated the induction of MDRCAT activity in GHE-L cells by sodium arsenite or heat shock, which stimulates heat shock factor (HSF) binding to HSE . In addition, protein kinase A inhibitor (H-87) blocked the activation of the MDR1 promoter by 22W in GHE-L cells in a dose-dependent manner . From these results, we propose the possibility that Raf- and protein kinase A-dependent pathways control the transcription of MDR1 gene via a mechanism involving the modulation of HSF activity.

Yao Xue Xue Bao, 1996, 31(5), 346 - 51
Chemosensitizing effect of verapamil on Swiss-3T3 cells transfected with human MDR1 gene; Wang N et al.; To further understand the characteristics of drug resistance reversal of verapamil, the relationship between the level of doxorubicin resistance and the magnitude of modulation by verapamil was examined in multidrug resistant Swiss-3T3 cells transfected with human MDR1 gene . In independently isolated transfectants doxorubicin cytotoxicity decreased markedly compared with parent cells . Potentiation of doxorubicin toxicity by a noncytotoxic concentration of 3 mumol.L-1 of verapamil was much greater in transfectants than in parent cells, while the magnitude of reversal was inversely dependent on the level of resistance . Southern blot hybridization indicated the MDR1 cDNA integration in genomics of each transfectant . Defect in cellular accumulation of doxorubicin was restored by verapamil in transfected cells . The saturation of active drug transport that may involve the magnitude changes of potentiation by verapamil, and the mode of interaction between P-glycoprotein and drugs, were discussed.

Int J Clin Pharmacol Res, 1996, 16(4-5), 89 - 97
Prediction of blood cyclosporine concentrations in haematological patients with multidrug resistance by means of total, lean and different adipose factors dosing body weight using Bayesian and non-linear least squares methods; Wu G et al.; In the present work, we have studied the prediction of blood cyclosporine (CsA) concentrations in haematological patients with multidrug resistance by means of total body weight, 25, 50 and 75 adipose factor dosing body weight, and lean body weight using the Bayesian method (BM) and non-linear least squares method (NLLSM) during the second course of CsA treatment . The results showed that both BM and NLLSM can minimize the prediction difference among different dosing body weight parameter types . The results also slightly favour the predictions with lean body weight.

Pneumonol Alergol Pol, 1996, 64(11-12), 740 - 9
{Drug resistance among patients with tuberculosis}; Szczuka I; Detailed questionnaire has been received from 365 patients with drug resistant tuberculosis . They represented 75% of all such patients in Poland . 360 of them had pulmonary tuberculosis . 76% were men . Among 365 analysed patients 55% were below the age of 50 (of whom 61% had initial drug resistance and 47% had acquired resistance) . Among these patients 52% had initial and 48% acquired drug resistance . In 193 patients (i.e . 54%) resistance to one drug (in 97 patients to isoniazid-H, in 80 to streptomycin-S and in 5 to rifampicin-R was observed . Resistance to two drugs was observed in 25% of patients and among them a majority (57%) was resistant to H and S and 23% to H and R . In 11% of patients, resistance to three drugs was observed, in 8% to four drugs, and in 4% to five or more drugs . Multidrug resistance (at least H and R) was observed in 97 patients (25% of the total number) and in 76 patients resistance to other than H and R drugs was observed . Among the total number of analysed patients in 263 (72%) resistance to H was observed, in 208-to S, and in 102 to R . Among patients with initial drug resistance, the majority was resistant to one drug and among those with acquired resistance the majority was resistant to two or more drugs . On the basis of this analysis the estimated total initial drug resistance-2 . 8% is 6-8 times lower than 30-40 years ago and have remained at this low level for at least 20 recent years . It is concluded therefore that drug resistance in Poland does not present any danger to the effectiveness of tuberculosis programme . Monitoring, however, should continue.

Invest New Drugs, 1996, 14(4), 341 - 7
Cellular transport of CI-980; Hook KE et al.; CI-980, originally synthesized as a potential folate antagonist, is a tubulin-binding mitotic inhibitor currently in pediatric phase I and adult phase II clinical trials . Because of its extensive tissue distribution in animals and its favorable activity against multidrug resistant (MDR)-cells compared with other mitotic inhibitors, such as vincristine, we examined the membrane transport properties of CI-980 . CI-980 accumulated rapidly in L1210 and CHO/K1 cells, reaching intracellular levels 40- and 8-fold higher, respectively, than those in the extracellular medium . Efflux was also quite rapid, but a small fraction of drug remained associated with the cells in drug-free medium . The uptake of CI-980 was not temperature or energy dependent, nor was it saturable up to an extracellular concentration of 100 microM . Inhibitors of nucleoside transport had no effect on CI-980 uptake . A cell line deficient in the transport of reduced folate was not resistant to CI-980, nor did it exhibit reduced CI-980 uptake . A 100-fold excess of the R-enantiomer inhibited CI-980 uptake by only 50% . These results are consistent with a model of CI-980 uptake involving passive diffusion followed by significant but largely reversible binding to intracellular or membrane components.

Mol Membr Biol, 1996 Jan-Mar, 13(1), 33 - 40
Membrane insertion, processing, and topology of cystic fibrosis transmembrane conductance regulator (CFTR) in microsomal membranes; Chen M et al.; Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated C1(-) channel . Malfunction of CFTR causes cystic fibrosis (CF) . CFTR belongs to an ATP-binding cassette (ABC) transporter superfamily which includes P-glycoprotein (Pgp), the molecule that is responsible for multidrug resistance in cancer cells . P-glycoprotein molecules have been suggested to have more than one topology and function . In this study, we analysed the early stages of membrane insertion, processing, and topology of human CFTR using rabbit reticulocyte lysate and wheat germ extract translation systems supplemented with canine pancreatic microsomal membranes . Our results suggest that CFTR contains an uncleavable signal sequence and its membrane targeting and insertion may depend on the signal recognition particle (SRP) and SRP receptor . The topology of CFTR in microsomal membranes is the same as the one predicted based on hydropathy plot analysis . These results, together with our previous findings on Pgp, indicate that (1) the topologies of mammalian ABC transporters can be dissected and studied using protein fusion chimeras in a cell-tree system; and (2) the membrane targeting and insertion of CFTR and Pgp may take the same pathway, i.e., the SRP-dependent pathway, but the membrane folding mechanism of these two proteins in microsomal membranes is probably different.

Ann Oncol, 1996 Jan, 7(1), 75 - 81
Expression of the multidrug resistance-associated protein (MRP) gene in primary non-small-cell lung cancer; Nooter K et al.; BACKGROUND: One of the major problems in the cure of advanced non-small-cell lung cancer (NSCLC) is its lack of response to cytotoxic drug treatment, and the mechanisms underlying this intrinsic drug resistance are unclear . PATIENTS AND METHODS: We determined the expression of a newly recognised drug resistance gene, the Multidrug Resistance-associated Protein (MRP) gene, in normal lung tissue and in tumour biopsies from 35 surgically resected NSCLCs (11 adenocarcinomas, 24 squamous cell carcinomas) . MRP mRNA levels were quantitated by RNase protection assay and expression of the MRP Mr 190,000 glycoprotein was estimated by immunohistochemistry . RESULTS: Using the MRP-specific monoclonal antibody MRPr1, MRP expression was detected by immunohistochemistry in epithelial cells lining the bronchi in normal lung . In NSCLC approximately 35% of the samples showed elevated MRP mRNA levels . Based on MRP-specific immunohistochemical staining the tumours were divided into 4 groups: 12% were scored as negative (-), 14% showed weak cytoplasmic staining of the tumour cells (+/-), 40% had a clear cytoplasmic staining (+), and in 34% a strong cytoplasmic as well as membranous staining was observed (++) . MRP expression, as estimated by immunohistochemistry, correlated with the MRP mRNA levels quantitated by RNase protection assay (correlation coefficient = 0.745, p = 0.0009), with MRP mRNA levels (mean +/- SD) of 3.0 +/- 1.0 U, 3.5 +/- 0.7 U, 7.5 +/- 5.9 U, and 19.3 +/- 10.7 U, in the (-), (+/-), (+), and (++) immunohistochemistry expression groups, respectively . Among the squamous cell carcinomas a correlation was observed between MRP staining and tumour cell differentiation: the strongest MRP staining was predominantly found in the well differentiated tumours . CONCLUSIONS: Hyperexpression of MRP is frequently observed in primary NSCLC, especially in the well differentiated squamous cell carcinomas . Further studies are needed to assess the role of MRP in the mechanism of clinical drug resistance in NSCLC.

Bull World Health Organ, 1996, 74(6), 591 - 7
A clinical trial with halofantrine on patients with falciparum malaria in Colombia; Restrepo M et al.; A total of 120 semi-immune adult male malaria patients from an area of multidrug-resistant Plasmodium falciparum malaria were hospitalized for 42 days in Medellin, Colombia (an area of no malaria transmission), and treated with halofantrine in a double-blind, randomized, prospective clinical trial according to five different treatment schedules . Each patient was assigned to one of the following halofantrine schedules: I, one dose of 1000 mg; II, three doses of 500 mg; III, two doses of 500 mg; IV, three doses of 250 mg; and V, one dose of 750 mg . Best results (75% cure rate) were obtained with schedule II, although there was no statistically significant difference compared with the other schedules . A total of 46 patients experienced recrudescent malaria . Drug levels in plasma 72 hours after beginning treatment showed no statistically significant difference between relapsing and cured patients . Side-effects (mainly gastrointestinal) were uncommon and mild . Cardiotoxicity was studied by electrocardiogram . A mean prolongation of 28.5 ms (6.6 +/- 6.3% increase from baseline) was observed in the Q-Tc interval on day 1 of the trial.

Drugs Exp Clin Res, 1996, 22(6), 295 - 300
Verapamil increases the bacteriostatic and bactericidal effects of adriamycin on Escherichia coli; Abramov Y et al.; The purpose of this study was to evaluate the effect of verapamil on adriamycin-resistant and -sensitive Escherichia coli bacterial strains . Two E . coli strains: B-SR9 and K12-KL16 were incubated with adriamycin in various concentrations in the presence or absence of verapamil . Growth and killing rates were measured using optical densities and colonogenic assays . Transmembrane transport capacity was evaluated by measuring radioactively labelled leucine uptake and intracellular potassium concentrations . While adriamycin (ADR) showed both bacteriostatic and bactericidal effects upon the two bacterial strains, the K12 strain was significantly more resistant to the drug than its peer . Subtoxic concentrations of verapamil augmented these effects in both strains . Verapamil affected bacterial transmembrane transport activity and caused potassium leakage through the cell membrane . Simultaneous exposure to adriamycin and verapamil resulted in rapid, massive damage to membrane functions, indicating accelerated killing rate . The authors concluded that verapamil acts as a potentiator of adriamycin's cytotoxicity in E . coli bacteria in a manner similar to that in multidrug resistant mammalian tumour cells . This observation suggests that the mechanisms of resistance to the drug may be similar in both species.

Invasion Metastasis, 1996, 16(2), 65 - 72
Exposure to vinblastine modulates beta 1 integrin expression and in vitro binding to extracellular matrix molecules in a human renal carcinoma cell line; Duensing S et al.; Solitary stroma-invading tumor cells expressing the ATP-binding cassette transporter P-glycoprotein have been reported to be associated with a significantly higher incidence of vessel invasion and lymph node metastases . In contrast to P-gp-mediated multidrug resistance (MDR) which has become well characterized over the last decade, little is known about further morphological and functional alterations in drug-resistant tumor cells . Binding of malignant cells to components of the extracellular matrix mediated by beta 1 integrins has been suggested to play a substantial role in the metastatic cascade . We studied alterations of beta 1 integrin expression and in vitro adhesiveness to extracellular matrix proteins of the human renal carcinoma line Caki-1 in comparison to the vinblastine resistant sublines Caki-1/V1 and Caki-1/V10 (cultured in the presence of 1 ng/ml and 10 ng/ml vinblastine, respectively) . Both VLA-1 and VLA-2 receptors were acquired by the Caki-1/V10 subline, whereas untreated and Caki-1/VI cells lacked surface expression of these antigens . VLA-6 was found to be decreased in the vinblastine-resistant sublines . Attachment of drug-resistant Caki-1/V1 and Caki-1/V10 cells to collagen type I was significantly increased when compared to parental cells (p < or = 0.005) . Significant differences in the attachment to type IV collagen were observed between Caki-1/V10 and untreated cells (p < or = 0.045) . Both Caki-1/V1 and Caki-1/ V10 cells exhibited increased adhesion to fibronectin when compared to cells of the untreated line (p < or = 0.04) . Whether an aberrant expression of beta 1 integrin receptors in resistant cells in combination with altered tumor cell adhesiveness is caused by MDR induction or whether it is an epiphenomenon of cytotoxic stress is unknown . Future studies will be needed to characterize the clinical relevance of MDR-associated changes in tumor cells.

Tsitologiia, 1996, 38(6), 611 - 5
{The nuclease activity of the cell nuclei in 2 lines of murine myeloma sp2/0 differing in their resistance to cytostatics in adriamycin-induced apoptosis}; Meliksetian MB et al.; The endonuclease activity of two drug-sensitive and drug-resistant mouse myeloma cell lines during cytotoxic drug-induced apoptosis was studied . It was shown that internucleosomal fragmentation of DNA in drug-sensitive line sp2/0, undergoing apoptosis in the presence of adriamycin and colchicine, was not dependent on intracellular calcium content and was associated with activation of both Ca(2+)-Mg(2+)-dependent and acidic cation-independent endonucleases . In contrast, in multidrug resistant spEBR-5 cells, treated with the same drugs, only Ca(2+)-Mg(2+)-dependent endonuclease activity was detected . These data suggest that the differences in the pattern of endonuclease activity revealed in these cells are linked to drug-resistant phenotype and do not depend on the apoptosis-inducing agent used.

Neoplasma, 1996, 43(6), 389 - 95
Cell surface phenotype and increased penetration of human multidrug-resistant ovarian carcinoma cells into in vitro collagen-fibroblasts matrix; Sedlak J et al.; Human multidrug resistant ovarian carcinoma cells (A2780/ADR) exhibited increased in vitro penetration into the collagen-normal human fibroblasts matrix, increased cell surface expression of alpha 6 integrin (CD49f antigen) and slightly increased expression of alpha 2 (CD49b) integrin compared with that of parental drug-sensitive A2780 cells . Both, multidrug-resistant and parental, drug-sensitive, cell lines did not express the 67 kDa non-integrin high affinity laminin receptor on their cell surfaces . As there were no marked differences between metalloproteinase activity of both A2780 cell sublines (with similar intensity of 72 kDa and 92 kDa lysis bands in zymograms), the increased penetration of the drug-resistant subline into the collagen-fibroblast gel matrix might be associated with the increased expression of adhesion proteins (including collagen-binding alpha 2 integrin), or cell surface-associated collagenase-stimulating protein(s) . This multidrug resistant ovarian carcinoma cell line might serve as an in vitro model of neoplastic cells with increased biological aggressiveness, molecular mechanisms of which require further analysis.

Neoplasma, 1996, 43(5), 291 - 5
Stimulation of 1-(beta-D-arabinofuranosyl)cytosine (AraC)-induced apoptosis in the multidrug resistant human promyelocytic leukemia cell lines with protein kinase inhibitors; Hunakova L et al.; Stimulation of apoptosis induced by 1-(beta-D-arabinofuranosyl)cytosine (AraC) with protein kinase inhibitors (i.e . staurosporine, CGP 41251-a protein kinase C (PKC)-selective staurosporine derivative and protein tyrosine kinase (PKT) inhibitor genistein) was examined in two human multidrug-resistant promyelocytic leukemia (HL-60) cell lines with different cell membrane drug resistance-associated glycoproteins (i.e . HL-60/VCR:MDR1 gene coded Pgp/p170 and HL-60/ADR: MRP gene coded non-Pgp/p190) . Staurosporine stimulated AraC-induced apoptosis in the parental drug-sensitive HL-60 cells and both examined multidrug resistant HL-60 sublines . The stimulation of AraC-induced apoptosis by PKC selective inhibitor CGP 412251 and PTK-inhibitor genistein was approximately equal to that of staurosporine in HL-60/ADR cell line . In both parental drug sensitive HL-60 cells and Pgp/p170 positive (MDR1) HL-60/VCR, staurosporine-stimulated AraC-induced apoptosis was higher than that stimulated by the PKC selective CGP 41251 inhibitor, or PTK-inhibitor genistein . These data suggest that the molecular pathway(s) for AraC-induced apoptosis can be activated and stimulated by PKC- and PTK-inhibitors in both examined drug-resistant HL-60 cell lines . Furthermore, these data suggest that although both PKC- and PTK-dependent mechanisms are involved in AraC-induced apoptosis, in the drug-sensitive HL-60 cells and multidrug-resistant HL-60/VCR (Pgp/p170) cells this process is mediated at least partially, also by PKC- and PTK-independent mechanisms, activated by staurosporine.

Cancer Chemother Pharmacol, 1996, 39(1-2), 157 - 61
Comparative activity of idarubicin and idarubicinol in combination with cyclosporin A in multidrug-resistant leukemia cells; Tolomeo M et al.; 4-Demethoxydaunorubicin (idarubicin, IDA) is an anthracycline that has shown good cytotoxic activity in vitro against tumor cell lines displaying the multidrug-resistant (MDR) phenotype . IDA is converted in the liver into idarubicinol (2HIDA) and, in this form, seems to exert its antitumoral activity in vivo . Recent studies have shown that 2HIDA has tumoricidal activity similar to that of the parent drug when tested in vitro in sensitive neoplastic cells . In this work we compared in vitro the effects of IDA and 2HIDA used alone and in combination with 2 microM cyclosporin A (CyA) in the MDR leukemic cell lines FLCR and K562R and in their sensitive parent cell lines FLC and K562 . IDA and 2HIDA showed the same cytotoxic activity in sensitive cells . After 1 h of exposure of cells to each anthracycline, we observed that the cellular uptake of IDA and 2HIDA was also similar . In resistant cells, 2HIDA was 3-4 times less active than IDA . We observed that the intracellular uptake of 2HIDA was lower than that of IDA, and this may be correlated with a greater ability of P-glycoprotein to expel 2HIDA as opposed to IDA . Indeed, when MDR cells were exposed to IDA and 2HIDA in combination with 2 microM CyA, the cytotoxic effect of these anthracyclines was the same, and it was similar to that observed in sensitive cells . These data confirm the utility of the combination of IDA and an MDR-reversing agent in hematological malignancies displaying the MDR phenotype.

Mol Biochem Parasitol, 1996 Jan, 75(2), 145 - 57
Molecular characterization of a P-glycoprotein-related tcpgp2 gene in Trypanosoma cruzi; Dallagiovanna B et al.; We have cloned, sequenced and characterized a gene from Trypanosoma cruzi (Y strain), termed tcpgp2, which encodes a member of the ABC (ATP-binding cassette) superfamily of evolutionarily conserved transport proteins . The nucleotide sequence of the tcpgp2 gene was determined . It presents a 4602-bp open reading frame, coding for a 1534-amino acid protein, with a predicted molecular mass of 169,470 Da . The deduced amino acid sequence of tcpgp2 exhibited a remarkable homology with the P-glycoprotein-related genes of Leishmania tarentolae, the yeast cadmium factor (YCF1) and the human multidrug resistance-associated protein (MRP) . Southern blot analysis using a specific probe indicated that the Tcpgp2 P-glycoprotein is encoded by a single copy gene which maps to a chromosome of about 900 kb . Northern blot analysis revealed that tcpgp2 gene is expressed as a polyadenylated transcript of approximately 5 kb in dividing amastigote and epimastigote forms; we did not detect the transcript in the non-dividing trypomastigote forms of the parasite . Gene transfection experiments in Leishmania tropica indicated that, under the conditions tested, tcpgp2 gene is not involved in drug resistance.

J Nat Prod, 1996 Jan, 59(1), 35 - 40
Bicinchoninic acid protein assay in the determination of adriamycin cytotoxicity modulated by the MDR glycoprotein; Hall AM et al.; The development of simultaneous resistance to structurally unrelated drugs in cancer cells is a major obstacle to effective cancer chemotherapy . This multidrug-resistance (MDR) phenomenon is largely attributed to overexpression of a 170 kD glycoprotein, which serves as a transmembrane efflux pump in extruding a variety of natural anticancer drugs such as vinblastine, doxorubicin, and taxol from cancer cells . It is desirable, therefore, to discover compounds that can block the efflux mechanism and thus reverse drug resistance . The bicinchoninic acid protein assay has been adapted for use in a microtiter plate, into an easy, indirect method for screening MDR efflux blockers in plant extracts . This spectrophotometric assay is used to determine the enhancement of adriamycin cytotoxicity against resistant cancer cells by plant extracts or pure compounds indirectly . We have shown that the optical density measured (amount of cellular protein present) correlates with the number of viable cells and that fluorescence of Adriamycin associated with the cell correlates with the concentrations of Adriamycin added to the media . In addition, the relative efficacy of MDR reversal by various alkaloids has been determined.

Chemotherapy, 1996, 42 Suppl 3, 24 - 9
Treatment of multidrug-resistant tuberculosis in Indonesia; Hadiarto M et al.; There is growing concern, even among developed countries, about the increasing incidence of multidrug-resistant tuberculosis (MDR-TB) . Results are reported from a study investigating ofloxacin used in the treatment of 57 patients with MDR-TB . Patients received ofloxacin 400 mg/day as well as three other sensitive anti-TB drugs based on susceptibility tests . Treatment duration was 9 months . Preliminary results of 35 evaluable patients show 55% of MDR-TB cases converted to smear and culture negative within 3 months of therapy . Ofloxacin in combination with other sensitive anti-TB medication shows promise in the treatment of MDR-TB and further studies are recommended.

Chemotherapy, 1996, 42 Suppl 3, 20 - 3; discussion 30-3
Treatment of multidrug-resistant tuberculosis in Taiwan; Suo J et al.; Eighty-seven patients with multidrug-resistant tuberculosis (MDR-TB) diagnosed between 1988 and 1990 were treated with isoniazid and at least three other effective second-line drugs based on in vitro susceptibility tests . Of these patients, 10% failed to adhere to the regimen and 43% remained sputum positive after 6 months of treatment . Only 47% showed sputum conversion within 6 months of treatment and 12% of them relapsed during the first year of follow-up . From September 1987 to July 1989, 36 patients with MDR-TB were treated with a regimen containing rifabutin, isoniazid and at least three other susceptible drugs . Only 47% achieved a sustained sputum conversion . Four died during treatment due to disease progression . From March 1992 to July 1993, 17 cases of MDR-TB were treated with an ofloxacin-containing anti-TB regimen for 12-24 months . Two failed to adhere to the regimen for more than 1 month during the first 6 months of therapy . Among the remaining 15, 26% failed to achieve sputum conversion, 73% achieved bacterial conversion, 9 within 1 month and the other 2 within 2 months . No significant adverse effect was associated with ofloxacin use . We concluded that ofloxacin is a better choice among the more toxic and less potent second-line drugs, and should be used along with other anti-TB drugs in treating patients with MDR-TB.

Chemotherapy, 1996, 42 Suppl 3, 16 - 9; discussion 30-3
Treatment of multidrug-resistant tuberculosis in China; Zhang LX; During the past decade the number and gravity of tuberculosis (TB) cases has continued to increase, both in developing and industrialized nations . Coupled with the recent emergence of multidrug-resistant tuberculosis (MDR-TB), the possibility that untreatable forms of the disease may become widespread has arisen . In China, the prevalence rate of smear-positive cases from three national surveys in 1979, 1984-1985 and 1990 was 187, 156 and 134/100,000, respectively, thus giving an annual average reduction rate of only 3.0% . This may be due to the accumulation of chronic cases, which is not surprising given that as many as 84.3% of new smear-positive cases received non-organized chemotherapy . To counteract this situation, a strategy was developed in Beijing to practice fully supervised chemotherapy for all new smear-positive cases . This is now 90% with a cure rate also of 90% . As a result, the prevalence rate of smear-positive cases has dropped, with an average annual reduction of 17% . Building upon this success, the World Bank Loan TB Control Project in China has been carried out in 12 provinces with 550 million people since 1992 . The main objective of this project is to provide fully supervised, 6-month short-course chemotherapy for all newly detected smear-positive cases . The cure rate based on cohort analysis was 88% in 1993 . Complete data are not available on resistance although the initial and acquired resistance rates were 28.1 and 41.1%, respectively . MDR-TB treated with ofloxacin has been increasing since 1992, with 317 cases reported during the period 1992-1995, of which 77% showed sputum conversion.

Chemotherapy, 1996, 42 Suppl 3, 10 - 5; discussion 30-3
Treatment of multidrug-resistant tuberculosis in Thailand; Maranetra KN; Tuberculosis (TB) has remained the 5th leading cause of death in Thailand for several years . There has been a slight change in the total number of TB cases notified since 1985 when the first case of HIV infection was reported . Although there is an increase in the incidence of TB in HIV-infected cases, the percentage of multidrug-resistant tuberculosis (MDR-TB) in this group is the same as in the HIV-negative group (2.7%) . The percentages of total initial drug resistance, four-drug resistance and MDR-TB have increased to 22.4, 1.4 and 4.8%, respectively . Comparable figures for acquired resistance are up to 2.5-, 10- and 6-fold, respectively . The rapid diagnosis and susceptibility pattern of MDR-TB are essential for improving therapeutic outcome . At present there is no defined standard regimen for MDR-TB and clinical practice has been to select a regimen of three to four sensitive or not previously exposed anti-TB drugs . Duration of treatment for 24-30 months depends on severity, previous therapy and the number of drug resistances . Surgery is suggested for persistent positive cases with localized lesions and a good cardiopulmonary reserve . The quinolone, ofloxacin, is a promising drug for MDR-TB, achieving a sputum conversion rate of 59-79% . A prospective study showed a success rate of 67% with no adverse effects . The current Bangkok multicenter trials on ofloxacin 600 mg daily combined with pyrazinamide, p-aminosalicylate, amikacin and ethambutol are ongoing . Good organization of ambulatory TB management combined with directly observed therapy will probably help to reduce the incidence of MDR-TBPIP: There has been a slight change in the total number of TB cases notified since 1985, when the first case of HIV was reported . Although there has been an increase in the incidence of TB in HIV-infected cases, the percentage of multidrug-resistant tuberculosis (MDRTB) in this group is the same as in the HIV-negative group (2.7%) . The multidrug-resistant (MDR) rate in 1988 was nearly 2% in Thailand, increasing to 5% in 1994 . The factors that promote MDRTB in Thailand include irregular drug taking, high initial drug resistance, the prescription of inappropriate regimens, and drug intolerance . The percentages of total initial drug resistance, four-drug resistance, and MDRTB have increased to 22.4%, 1.4%, and 4.8%, respectively . Comparable figures for acquired resistance are up to 2.5-, 10-, and 6-fold, respectively . Duration of treatment for 24-30 months depends on severity, previous therapy, and the number of drug resistances . Surgery is suggested for persistent positive case with localized lesions and good cardiopulmonary reserve . Quinolones are among the most promising drugs for second-line therapy against MDRTB . Quinolone and ofloxacin are promising drugs for MDRTB, achieving a sputum conversion rate of 59-79% . A prospective study showed a success rate of 67% with no adverse effects . However, when different regimens were examined, it was found that at least four anti-TB drugs are required . In current multicenter, controlled, prospective trials in Bangkok, 600 mg ofloxacin daily is combined with pyrazinamide, p-aminosalicylate, amikacin, and ethambutol, with a treatment duration of 18-24 months with a 2-year follow-up . No adverse effects were reported for the ofloxacin 300 mg/day regimen in several studies done . Optimal MDRTB treatment requires appropriate organization for planning and implementation and directly observed therapy . Guidelines developed in April 1996 call for at least 3-4 culture-sensitive drugs given for either 2-2.5 years or until negative sputum cultures have been present for at least 1 year .

Chemotherapy, 1996, 42 Suppl 3, 2 - 9; discussion 30-3
Multidrug resistance in the world: the present situation; Reichman LB; Tuberculosis (TB) is the leading cause of death attributable to a single infectious pathogen with one-third of the world population infected . In the USA, TB rates have fallen 3 years in succession after a sustained rise since 1985 . More important are the multidrug-resistant tuberculosis (MDR-TB) cases, which are thought to be largely due to the breakdown in the delivery of health care in the USA in conjunction with HIV infection . In countries facing the HIV epidemic, the overlap of these two populations leads to a rapid acceleration of active TB and the emergence of MDR-TB . In the USA the most recent published survey (1991) revealed that 3.5% of strains were resistant to isoniazid and rifampin . Worldwide, MDR-TB is also thought to be highly prevalent, not only because of a breakdown in health infrastructure but also because of inappropriate prescription, lack of drug availability and the use of combination capsules in which the drugs are not bioavailable . Key points in therapy are to order susceptibility tests, obtain a complete drug history, treat with an adequate number of effective drugs and never, ever add a single drug to a failing regimen.

Scand J Infect Dis, 1996, 28(5), 487 - 91
Drug resistance patterns among tuberculosis patients in Rome, 1990-1992; Girardi E et al.; Prevalence of, and risk factors for, drug-resistance of Mycobacterium tuberculosis were assessed among 407 hospitalized patients with tuberculosis in Rome, Italy, during the period 1990-1992 . Resistance to 1 or more drugs was detected in 106 isolates (26%) . Resistance to streptomycin was the most common (18.4%), followed by isoniazid (10.3%) and rifampin (7.9%) . 23 isolates (5.7%) were resistant to both isoniazid and rifampin . Resistance to at least 1 drug and resistance to both isoniazid and rifampin were significantly more common among recurrent cases (40.7% vs . 22.1%, p < 0.001; and 22.1% vs . 1.2%, p < 0.001) . Sex, country of origin and HIV infection were not significantly associated with prevalence of drug resistance . Among recurrent cases, prevalence of resistance to at least 1 drug and of resistance to both isoniazid and rifampin, was higher in subjects who had had a previous episode of tuberculosis later than 1969 . In the population studied the prevalence of drug-resistant tuberculosis was high, although the risk of initially becoming infected with a multidrug-resistant strain of M . tuberculosis in this area appears to be low . This study suggests the need for enhanced surveillance of drug-resistance of tuberculosis in our country and for implementation of intervention aimed to ensure adequate and complete therapy for patients with tuberculosis.

C R Seances Soc Biol Fil, 1996, 190(4), 455 - 66
{Resistance to antineoplastic treatments: mechanisms, clinical value}; Bonnal C et al.; Drug resistance is a major obstacle to successful chemotherapy for cancer . When it occurs, resistance to a wide range of agents is noted . Factors that rule this resistance can be defined as pharmacologic and cellular . Pharmacologic factors are those that prevent an adequate degree of tumor cell exposure and include considerations of dose and schedule of drugs . Cellular factors are those that imply the tumor cell itself and it is probable that multiple mechanisms co-exists: 1) the drug transport across the tumor cell membrane and the duration of the drug exposure, 2) the drug metabolism (activation, inactivation), 3) the cellular targets and the DNA repair processes . The pleiotropic multidrug resistance (mdr, mrp, lrp), alterations of a target enzyme (topoisomerase II, protein kinase C, glutathione S transferase, O6 alkylguanine-DNA alkyltransferase) and the protein modifications (heat shock protein, metallothioneins) are the principal mechanisms involved . Several methods have been established for the determination of the presence of these drug resistance mechanisms but variations in the results are observed with the different methods used . Therefore, the value and the relative importance of these mechanisms in human tumor resistance is not yet established . In the mean-time, strategies to prevent and to overcome this resistance are developed.

Breast Cancer Res Treat, 1996, 41(2), 111 - 22
The effects of cyclosporin A, tamoxifen, and medroxyprogesterone acetate on the enhancement of adriamycin cytotoxicity in primary cultures of human breast epithelial cells; Claudio JA et al.; Adriamycin (Adr), the single most active agent used in the treatment of breast cancer, may become ineffective as treatment progresses due to the development of multidrug resistant (MDR) tumors . A major mechanism associated with MDR is increased P-glycoprotein (Pgp) expression . This study examined the abilities of the anti-estrogen tamoxifen (TAM) and the progestin medroxyprogesterone acetate (MPA) as well as cyclosporin A (CsA), a known resistance modifier, to enhance the cytotoxic effects of Adr on human breast epithelial cells (HBEC) in primary culture . Pgp and estrogen receptor (ER) expression were determined in each of the cultures by immunocytochemical assays using the monoclonal antibodies C219 and H222 Sp gamma, respectively . The Adr-sensitive, Pgp-, ER+ MCF-7 cell line and the Adr-resistant, Pgp+, ER- MCF7-AdrR cell line were used as controls . Primary cultures were categorized as HBEC from tissues with or without previous chemotherapy . Pgp was detected in 1 of the 15 cell cultures from tissues without previous chemotherapy and in 5 of the 6 cell cultures from tissues previously exposed to chemotherapy . Incubation with either CsA or MPA plus Adr enhanced Adr toxicity in Pgp+ but not Pgp- cell cultures, whereas TAM had no effect on the sensitivity of any of the cultures . Of the 21 primary cultures of HBEC, 3 were ER+ . There was no correlation between the enhancement of Adr cytotoxicity and ER status . The data suggest that MPA as well as CsA may be useful as modifying agents in overcoming Pgp-associated multidrug resistance.

Oncol Res, 1996, 8(7-8), 287 - 93
Expression and characterization of the multidrug resistance-associated protein in insect cells infected with a recombinant baculovirus; Sun H et al.; The protein encoded by the multidrug resistance-associated protein (MRP) gene was examined after infection of SF21 insect cells with recombinant baculovirus containing a full-length MRP cDNA . The time course of appearance of the protein as determined by western blot analysis revealed that maximum levels occurred 2 days postinfection . The amount of MRP made in this system was somewhat variable, but levels that were about 4-fold greater than that found in HL60/ADR cells could be achieved . The protein appeared to be full-length but was present in a highly deglycosylated form . The P170 (MRP) was phosphorylated and located exclusively in membranes of infected cells . P170 (MRP) synthesized in this system was capable of carrying out the ATP-dependent transport of leukotriene C4 into isolated membrane vesicles . The results thus indicate that MRP synthesized in insect cells is functional and has properties similar to the authentic protein found overexpressed in certain multidrug-resistant isolates.

J Submicrosc Cytol Pathol, 1996 Jan, 28(1), 93 - 100
Ultrastructural features and P-glycoprotein immunolocalization in Saos-2/DX580 multidrug-resistant human osteosarcoma cells; Maraldi NM et al.; The multiple drug type of resistance to anticancer agents (MDR) is mediated by an over-expression of the MDR1 gene product, the P-glycoprotein . This is largely present at the cell surface of MDR cells, mediating the active efflux of cytotoxic molecules, but may be found also intracellularly . In this paper, using Saos-2 human osteosarcoma cells as a model, we provide further evidence of increased presence of P-glycoprotein at the plasma membrane and in the nucleus of MDR cells, where it is closely bound to the nuclear matrix . The structural changes observed in Saos-2 MDR cells, including an increase of the cell surface by the formation of blebs, and a peculiar clustering of chromatin, which are similar to those observed in other MDR cell lines, are likely to be associated with the observed overexpression of the P-glycoprotein at the cell membrane and nuclear level . These findings suggest the existence of more complex, still undetermined, mechanisms underlying the MDR phenomenon.

J Hepatol, 1996, 24 Suppl 1, 100 - 8
Role of multidrug resistance protein (MRP) in glutathione S-conjugate transport in mammalian cells; Muller M et al.; The human multidrug resistance protein (MRP), a 190-kDa member of the ABC-protein superfamily, is an ATP-dependent glutathione S-conjugate carrier (GS-X pump) and is present in membranes of many, if not all, cells . Overexpression of MRP in tumor cells contributes to resistance to natural product drugs and oxyanions . The function and specificity of the GS-X pump MRP is very similar to the canalicular multispecific organic union carrier (cMOAT) present in the canalicular membrane domain of hepatocytes and functioning in primary-active excretion of multivalent anionic conjugates into bile . In this review we discuss the function of MRP as GS-X pump in mammalian cells, its putative role in multidrug resistance, its relation to the hepatocyte cMOAT system and its dysfunction in the mutant TR- Wistar rat.

Invest New Drugs, 1996, 14(2), 169 - 80
In vitro cytotoxicity of S16020-2, a new olivacine derivative; Leonce S et al.; S16020-2 is a new olivacine derivative which has recently shown a marked antitumor activity in various experimental models . This study was undertaken in order to measure the inhibition of the proliferation of various sensitive and resistant tumor cell lines, by S16020-2, and to obtain information concerning its mechanism of action . For a continuous exposure, S16020-2 was as cytotoxic as adriamycin (ADR) (mean IC50 of about 28 nM) and on average, 46 fold more potent than elliptinium acetate (ELP), against a panel of 20 non-multidrug resistant cell lines . With a short exposure (1 hour) followed by a post-incubation of 95 hours in drug-free medium, S16020-2 was 5 and 6 fold more cytotoxic than ADR for human lung A549 and murine melanoma B16 cells, respectively . Furthermore, S16020-2 inhibited more actively the formation of colonies issued from proliferating cells, compared to colonies issued from quiescent A549 cells . Because quiescent cells demonstrated a 3 fold lower level of topoisomerase II alpha (topo II) than proliferating cells, these results suggest that this enzyme could be a potential target for S16020-2 . In addition, as demonstrated by flow cytometric studies, S16020-2 intercalated into DNA and induced a cell cycle arrest in G2 . Cell lines displaying the multidrug resistance (MDR) phenotype, P388/ADR-1, P388/ADR, P388/VCR-20, KB-A1, DC-3F/AD, S1/tMDR, and Colo320DM, were more sensitive to S16020-2 than to ADR or ELP, as shown by the mean resistance factors, 8, 201, and 23 respectively . In addition, the two cell lines displaying the pure classical MDR phenotype, linked exclusively to the P-glycoprotein (P-gp) overexpression (P388/VCR-20 and S1/tMDR), were as sensitive to S16020-2 as their sensitive parental counterparts, although they were resistant to ADR . S16020-2 is thus one of the most potent olivacine and ellipticine derivative yet characterized . The good cytotoxicity of S16020-2 against cells displaying a P-gp-mediated multidrug resistance, and its antitumor activity in vivo delineate an important chemotherapeutic potential for this drug.

Invest New Drugs, 1996, 14(2), 161 - 7
Efficacy of HMAF (MGI-114) in the MV522 metastatic lung carcinoma xenograft model nonresponsive to traditional anticancer agents; Kelner MJ et al.; Illudin analogs are cytotoxic to a variety of multidrug resistant cell lines, and display an unusual toxicity towards DNA helicase-deficient cell lines . Earlier illudin analogs demonstrated efficacy in several xenograft models, including a metastatic MV522 lung cancer model, resistant to conventional anticancer agents . These illudin analogs prolonged life span as compared to conventional agents, but did not induce complete remission of primary tumors . In vitro screening studies identified a semisynthetic derivative, hydroxymethylacylfulvene (HMAF, MGI-114), with increased selective cytotoxicity towards carcinoma cells . The HMAF analog was markedly effective in the experimental MV 522 metastasizing lung carcinoma xenograft system, a model refractory to treatment with existing anticancer agents . Treatment with paclitaxel, doxorubicin, or cisplatin failed to significantly inhibit primary tumor growth or prolong life span of MV522 tumor-bearing animals . Treatment with mitomycin C at the LD20 increased life span in surviving animals up to 61% (p = 0.04) . Treatment with HMAF induced primary tumor regression in all animals and increased life span greater than 150% (p < 0.001) . Thus, administration of HMAF inhibited development of lung metastasis in a model refractory to treatment with conventional anticancer agents . These results support further evaluation of HMAF as a therapeutic agent for treatment of solid tumors such as adenocarcinoma of the lung.

Invest New Drugs, 1996, 14(2), 139 - 46
Morpholinylanthracyclines: cytotoxicity and antitumor activity of differently modified derivatives; Ripamonti M et al.; The relationship between different chemical modifications on morpholinylanthracyclines and their ability to overcome multidrug resistance (MDR) has been evaluated testing all compounds in vitro on LoVo and LoVo/DX human colon adenocarcinoma cells and in vivo disseminated P388 and P388/DX murine leukemias . Results obtained led us to the following conclusions: 1) the insertion of the morpholinyl or the methoxymorpholinyl group on position 3' of the sugar moiety confers the ability to overcome MDR in vitro and in vivo; conversely, 4' morpholinyl compounds are effective on MDR cells only in vitro and result inactive in vivo on DX-resistant leukemia; 2) all chemical modifications performed on 3' morpholinyl or methoxymorpholinyl derivatives, that is substitutions on the aglycone or on position 2 of the morpholino ring, do not interfere with the activity of the compounds: all derivatives present in fact the same efficacy on sensitive and resistant models . It is concluded that position 3' in the sugar moiety plays a crucial role in the ability of morpholinyl-anthracyclines to overcome MDR.

Adv Exp Med Biol, 1996, 406, 83 - 97
The Epstein-Barr virus gene BHRF1, a homologue of the cellular oncogene Bcl-2, inhibits apoptosis induced by gamma radiation and chemotherapeutic drugs; McCarthy NJ et al.; Analysis of apoptosis, active and controllable cell death, has demonstrated that the size of a cell population can be regulated by changes in the cell death rate as well as in the rates of proliferation and differentiation . Factors which alter the rate of cell death, such as expression of the proto-oncogene bcl-2, can therefore directly affect the number of cells within a population . Bcl-2 has been shown to suppress apoptosis in response to a variety of stimuli and to act as a complementary survival signal for the random acquisition of other oncogenic mutations, such as deregulated c-myc . The Epstein Barr virus (EBV) gene BHRF1 was the first of a family of bcl-2 homologues now being identified . BHRF1 and bcl-2 share 25% primary amino acid sequence homology . Here we show that gamma radiation and several cytotoxic anticancer agents induce apoptosis in Burkitt's lymphoma (BL) cell lines, as has been found in several other systems . Using gene transfection studies we have also shown that expression of either BHRF1 or bcl-2 in BL cell lines significantly suppresses apoptosis in response to a variety of anticancer treatment . This has confirmed that BHRF1 is functionally homologous to bcl-2 in B-cells and suggests that BHRF1 may act to prevent apoptosis during EBV infection, maximising virus particle production, as has been suggested for other human and insect viral genes . Suppression of chemotherapeutic drug induced cell death by bcl-2 and BHRF1 as demonstrated in this cell system, results in resistance to a variety of different agents and may represent an alternative mechanism by which multidrug resistance arises during chemotherapy.

Electrophoresis, 1996 Jan, 17(1), 255 - 60
Use of internally controlled reverse transcriptase-polymerase chain reaction for absolute quantitation of individual multidrug resistant gene transcripts in tissue samples; Zhang F et al.; Research into the relative importance of P-glycoprotein (pgp) overexpression, in comparison to other mechanisms of multidrug resistance (mdr) phenotype development, has been hampered by difficulties of measurement in clinical samples . The small size, heterogeneity and often poor quality of clinical specimens renders the quantitation of mdr mRNA species by Northern analysis or RNAse protection difficult . The reverse transcriptase-polymerase chain reaction (RT-PCR) assay has both the sensitivity and specificity to make it suitable for the analysis of mdr mRNA levels in clinical samples . It also has a significant speed advantage over Northern and RNAse protection analysis . Unfortunately the variable nature of the reactions involved have made it difficult to obtain accurate and reproducible quantitative data . To overcome this difficulty we constructed internal RNA standards to each human and rat mdr mRNA species . When included in the RT-PCR reaction these internal standards allow both comparative and absolute quantitation of individual mdr family mRNA levels.

Free Radic Biol Med, 1996, 20(4), 601 - 6
Conferring drug resistance by MDR1 gene transfection increases susceptibility to irradiation and lipid peroxidation in 3T3 cell line; Mazzanti R et al.; This study was performed to test the hypothesis that conferring multiple drug resistance reduces cell susceptibility to irradiation and iron-stimulated lipid peroxidation . Multidrug resistant (PN1A) and parental drug sensitive (PSI-2) cell lines were exposed to ADP-Fe or Ascorbate-Fe complexes at 37 degrees C and to irradiation . Lipid peroxidation was estimated by the TBA test, whereas x-ray effect was estimated by clonogenic assay . Cell glutathione-S-transferase (GST), total and Se-dependent glutathione peroxidase (GSH-Px) activities, and glutathione and vitamin E were measured . PN1A produced more peroxides than PSI-2 after exposure to iron complexes and formed fewer colonies after irradiation . Higher activities of GST and total and Se-GSH-Px were observed in PN1A . Vitamin E and total glutathione did not differ in the two cell subclones . These data show that the induction of the mdr1 phenotype by transfection of mdr1 gene in 3T3 cells increases susceptibility to irradiation and iron stimulated lipid peroxidation.

J Cancer Res Clin Oncol, 1996, 122(11), 671 - 5
MDR1 expression correlates with mutant p53 expression in colorectal cancer metastases; de Kant E et al.; Overexpression of the multidrug resistance MDR1 gene is thought to contribute to drug resistance in non-responsive cancers like colorectal carcinoma . Little is known about the mechanisms by which expression of MDR1 is regulated in human tumours . However, there is growing evidence that regulation primarily takes place at the transcriptional level and that the process of tumour progression is related to activation of the MDR1 gene . Mutations in the p53 tumour-suppression gene occur in approximately 70% of colorectal cancers . As a transcriptional regulator, p53 might be involved in regulation of MDR1 expression in these tumours . We therefore determined MDR1 expression using the differential polymerase chain reaction technique in 30 colorectal tumours (4 primaries and 26 metastates) and correlated our results with previously reported data on p53 in the same group of patients . We found a significant positive correlation between p53 and MDR1 expression in p53-mutated tumours (P = 0.005; r = 0.596), but not in tumours without a p53 mutation . In addition, we observed a tendency towards higher MDR1 expression levels in tumours carrying p53 mutations (P = 0.14) compound to wild-type p53 tumours . These data indicate that mutant p53 may play a role in the regulation of MDR1 expression in human cholorectal cancer.

Oncol Res, 1996, 8(6), 249 - 57
Reduced daunomycin accumulation in drug-sensitive and multidrug-resistant human carcinoma KB cells following phorbol ester treatment: a potential role for protein kinase C in reducing drug influx; Drew L et al.; We have investigated the role of protein kinase C (PKC) in the multidrug resistance (MDR) phenotype of human KB carcinoma cell lines . The PKC activator 12-O-tetradecanoylphorbol-13-acetate (TPA) reduced daunomycin accumulation in both drug-sensitive KB-3-1 and MDR KB-C1 cells in a time-dependent manner . The inactive phorbol ester 4 alpha TPA did not reduce daunomycin accumulation, and the PKC inhibitor, Ro 31-8220, reversed the TPA effect . TPA had no effect on daunomycin efflux and did not induce Pgp expression in KB-3-1 cells or alter Pgp levels in KB-C1 cells . Linear, short-term daunomycin accumulation was reduced by pretreatment with TPA, an effect that could be reversed by Ro 31-8220 . The effects of TPA on PKC subspecies localisation and downregulation were also examined . TPA initially induced translocation of PKCs alpha and delta, and to a lesser extent, PKC epsilon to the membrane fraction; 8 h after TPA treatment, differential effects on downregulation of PKCs alpha and delta were observed between cell lines, although PKC epsilon was not reduced in either cell line . We therefore propose that the TPA-induced reduction in daunomycin accumulation in KB cells is due to a PKC-mediated process, which is maintained after depletion of certain PKC subspecies or is due to activation of downregulation insensitive PKC subspecies . These results suggest that PKC may regulate drug resistance by reducing drug influx in a Pgp-independent manner in KB cells . This may represent a mechanism of drug-resistance independent of, or in addition, to, Pgp-mediated drug efflux.

Recent Results Cancer Res, 1996, 142, 89 - 101
Molecular mechanisms and possibilities of overcoming drug resistance in gastrointestinal tumors; Dietel M; Primary and acquired resistance of tumor cells to antineoplastic drugs is a major cause of the limited efficiency of chemotherapy . Gastrointestinal (GI) tumors have proven to express cytostatic drug resistance at an unusually high rate . One major reason for this is the multidrug resistant (MDR) phenotype which is often found in carcinomas of the stomach, bile duct, pancreas, liver, and colon . MDR is due to the overexpression of a membrane-bound glycoprotein, the so called P-glycoprotein . However, this is not the only resistance mechanisms of GI tumor cells, but the intracellular compartmentalization of drugs with subsequent release to the microenvironment represents an additional potent mechanism of drug resistance . This is independent of P-glycoprotein and as yet cannot be reversed . Alterations of glutathione-S-transferase (GST) and topoisomerase I and II may be involved either . Analyses of cell lines for cross resistance against a battery of cytostatic drugs suggest even more mechanisms which may contribute to the marked resistance of gastrointestinal cancer . Only a detailed investigation of all different types of drug insensitivity, if ever possible, might offer a chance to fully understand this multifactorial orchestra of events and to develop complex strategies for overcoming drug resistance.

Cancer Treat Res, 1996, 87, 3 - 38
P-glycoprotein-mediated multidrug resistance: experimental and clinical strategies for its reversal; Ford JM et al.; The study of the cellular, biochemical, and molecular biology and pharmacology of MDR has provided one of the most active and exciting areas within cancer research and one that holds great promise for translation into clinical benefit . While convincing evidence for the functional role of P-gp in mediating clinical drug resistance in humans remains elusive, studies of the clinical expression of P-gp and trials of chemosensitizers with cancer chemotherapy suggest "resistance modification" strategies may be effective in some tumors with intrinsic or acquired drug resistance . However, even if P-gp-associated MDR proves to be a relevant and reversible cause of clinical drug resistance, numerous problems remain to be solved before effective clinical chemosensitization may be achieved . Such factors as absorption, distribution, and metabolism; the effect of chemosensitizers on chemotherapeutic drug clearance; toxicity to normal tissues expressing P-gp; and the most efficacious modulator regimens all remain to be defined in vivo . Clearly, the identification of more specific, potent, and less clinically toxic chemosensitizers for clinical use remains critical to the possible success of this approach . Nonetheless, the finding that a number of pharmacological agents can antagonize a well-characterized form of experimental drug resistance provides promise for potential clinical applications . Further study of chemosensitizers in humans and the rational design of novel chemosensitizers with improved activity should define the importance of MDR in clinically resistant cancer.

Oncol Res, 1996, 8(5), 207 - 18
alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro-6,7-dimethoxy-alpha- {(4-methylphenyl)thio}-2(1H)-isoquinolineheptanenitrile (CL 329,753): a novel chemosensitizing agent for P-glycoprotein-mediated resistance with improved biological properties compared with verapamil and cyclosporine A; Greenberger LM et al.; Agents that inhibit P-glycoprotein may restore sensitivity to some antitumor drugs in cancer patients . Optimization of the specificity and potency of one class of chemosensitizing agents related to verapamil has led to the identification of alpha-(3,4-dimethyoxyphenyl)-3,4-dihydro-6, 7-dimethoxy-alpha-{(4-methylphenyl) thio}-2(1H)-isoquinolineheptanenitrile, designated CL 329,753 . In vitro, 0.1 to 2.0 microM CL 329,753 restored sensitivity to drugs in the multidrug resistance (MDR) phenotype in cell lines that overexpress P-glycoprotein . CL 329,753 was greater than 10-fold more potent and efficacious than cyclosporine A or verapamil in vitro, particularly in cells that express high levels of P-glycoprotein . The enhanced activity of CL 329,753 may be related to its inability to be transported by P-glycoprotein, since low drug accumulation of cyclosporine or verapamil but not CL 329,753 was found in P-glycoprotein-containing cells, yet all three agents inhibited vinblastine binding to membranes containing P-glycoprotein and inhibited photoaffinity labeling of P-glycoprotein . In vivo, CL 329,753 resensitized drug-resistant tumors to vinblastine or doxorubicin in an ascitic or solid tumor model, respectively . No alteration in the plasma pharmacokinetic profile of doxorubicin by CL 329,753 has been found . Furthermore, the compound had 70-fold less calcium channel antagonistic activity compared with verapamil.

Invest New Drugs, 1996, 14(1), 55 - 67
Multidrug resistance in pediatric oncology; Kuttesch JF Jr; Cancer survival among children and adolescents has improved markedly due to evolution of multimodal treatment that incorporates combination chemotherapy, radiation therapy and/or surgery . However, 20-30% of children with malignancies will succumb to their disease or complications associated with their disease or treatment . A major limiting factor to improvement in survival among these patients is the occurrence of intrinsic and/or acquired resistance to our treatment interventions, chemotherapy and radiotherapy . Among these mechanisms, multidrug resistance, the focus of this review, is a well-documented phenomenon whose biochemistry, pharmacology and molecular biology has been extensively studied . A role for multidrug resistance in chemoresistance and therapeutic failure in childhood malignancies is suggested by the observation of clinical resistance to treatment regimes containing agents that are known substrates of multidrug resistance mechanisms . With the current results from studies in rhabdomyosarcoma, neuroblastoma, osteosarcoma, Ewing's sarcoma, leukemia and retinoblastoma, the role of multidrug resistance is still unclear . Earlier studies attempted to define a role for P-glycoprotein-mediated multidrug resistance; however, a limited number of reports suggest that the multidrug-associated resistance protein may play an active role in neuroblastoma . Further studies will be necessary using standardized and uniform approaches for the analyses of these mechanisms . Clinical trials directed toward reversal of multidrug resistance are premature since the exact role of P-glycoprotein is controversial in pediatric malignancies, the role of other mechanisms of multidrug resistance must be assessed and selective inhibitors of multidrug resistance have yet to be developed.

Adv Enzyme Regul, 1996, 36, 17 - 29
The function of the multidrug resistance proteins (MRP and cMRP) in drug conjugate transport and hepatobiliary excretion; Keppler D et al.; The MRP gene encodes a 190-kDa integral membrane glycoprotein which functions as a primary-active ATP-dependent export pump for amphiphilic anions . The MRP gene-encoded conjugate export pump and its canalicular isoform represent the transport activity which has been described earlier as multispecific organic anion transporter, non-bile acid organic anion transporter, glutathione S-conjugate export pump, or leukotriene export pump . Analyses of the substrate specificity of the human MRP pump were performed in plasma membrane vesicles from MRP-overexpressing drug-selected cells (7) and cells transfected with an MRP expression vector (8) . Substrates for MRP include thioether-linked conjugates of lipophilic compounds with glutathione, cysteinyl glycine, cysteine, and N-acetyl cysteine, but also glutathione disulfide, and glucuronate conjugates such as etoposide glucuronide . This broad-specificity ATP-dependent export pump is not only overexpressed in several multidrug resistant tumor cells and tissues, but is also present in most normal cells and tissues . The expression of cMRP and MRP in human liver and of cMrp and its homolog Mrp in rat liver was demonstrated by reverse transcription PCR, cDNA sequencing, and immunoblotting (13) . The important function of the cMRP gene-encoded broad-specificity conjugate export pump in hepatobiliary excretion is illustrated by the selective absence of this canalicular isoform from the hepatocyte canalicular membrane in transport-deficient mutant rats . This altered lack of cMrp is the basis for the hereditary detect of the hepatobiliary excretion of anionic conjugates in the mutant animals (13) . The absence of this canalicular Mrp in the mutants is analogous to the defect in the human Dubin-Johnson syndrome which is characterized by an impaired excretion of conjugated anions across the canalicular membrane.

Curr Opin Oncol, 1996 Jan, 8(1), 20 - 7
Biology and treatment of myeloma; Greipp PR et al.; New strategies for the treatment of multiple myeloma, particularly high-dose therapy with peripheral blood stem cell transplantation, can achieve complete response in up to 50% of cases . The patient's condition eventually relapses because transplantation does not completely eliminate myeloma . Posttreatment interferon may prolong responses achieved by transplantation or chemotherapy, but it does not necessarily prolong survival . New approaches are being developed to further eliminate myeloma cells after an incomplete response achieved by transplantation or other chemotherapy . These approaches aim at myeloma cell surface antigens, T-cell immunity, and biological mechanisms of myeloma growth and dissemination . Biological targets include interleukin-6 (the central myeloma growth factor), interleukin-1 beta (an amplifier of stromal and bone cell production of interleukin-6), and serum soluble interleukin-6 receptor (an enhancer of myeloma cell response to interleukin-6) . Efforts to eliminate circulating myeloma cells from peripheral blood stem cell harvests use positive selection or purging to provide a product that will engraft only normal stem cells, not myeloma cells . Multidrug resistance, a major factor in treatment failure, can be reversed by agents that inhibit the multidrug resistance protein on the myeloma cell surface . Finally, expanded knowledge of biologic mechanisms has led to the development of new prognostic factors . These aim to identify patients in clinical trials who can benefit from treatment regimens designed to overcome their impact on survival . Translating new biological advances into treatment programs is essential to improving therapy for patients with myeloma.

J Drug Target, 1996, 3(5), 399 - 409
The activity of doxorubicin niosomes against an ovarian cancer cell line and three in vivo mouse tumour models; Uchegbu IF et al.; Demonstration of the improved doxorubicin pharmacokinetics and tumoricidal activity, after a single intravenous dose of 10mg kg-1 doxorubicin sorbitan monostearate (Span 60) based niosomes in the mouse adenocarcinoma (MAC) tumour model (Uchegbu et al., 1995) preceded the present study in which the activity of doxorubicin C16G2 (a hexadecyl diglycerol ether) based niosomes was evaluated against naive and established MAC tumour models . C16G2 niosomes were equiactive with doxorubicin solution . It is concluded that while in some tumour models, niosomal formulations demonstrate some advantages over the free drug, caution is advocated in the extrapolation of these results . The activity of doxorubicin C16G2 and Span 60 niosomes was also studied against a human ovarian cancer cell line and its doxorubicin resistant subline . There was a slight reduction in the IC50 against the resistant cell line when the drug was encapsulated in Span 60 niosomes in comparison to the drug in solution . Taking into account the in-vitro release characteristics of the various niosomal formulations, it is concluded that the use of niosomal formulations against multidrug resistance shows sufficiently encouraging results to warrant further study.

Cytotechnology, 1996, 19(3), 253 - 6
Evaluation of P-glycoprotein expression in soft tissue sarcomas of the extremities; Serra M et al.; Soft tissue sarcomas comprise a heterogeneous group of mesenchymal tumors accounting for less than one-percent of adult neoplasms . In the last few years, the use of adjuvant chemotherapy has been proposed for the treatment of these lesions in order to obtain a better systemic control, but its usefulness is still controversial . In this study, we evaluated whether P-glycoprotein, a membrane protein strictly associated with multidrug resistance, is overexpressed in soft tissue sarcomas . By using human multidrug resistant sarcoma cell lines as controls, we analyzed P-glycoprotein expression in 34 primary and in 23 relapsed soft tissue sarcomas of the extremities . Overexpression of P-glycoprotein was found in 6 out of 34 primaries (18%) and in 8 out of 23 relapses (35%) . In particular, in malignant fibrous histiocytoma, the most frequent soft tissue sarcoma of adults, P-glycoprotein overexpression was found in 23% of primary untreated cases, in agreement with the reported relapse rate of this tumor after surgery and chemotherapy . These data suggest that, in soft tissue sarcomas, overexpression of P-glycoprotein may be of prognostic value and that the assessment of P-glycoprotein expression may be useful for the design of chemotherapy protocols.

Cytotechnology, 1996, 19(3), 247 - 51
Topology of MDR1-P-glycoprotein as indicated by epitope mapping of monoclonal antibodies to human MDR cells; Cianfriglia M et al.; The MDR1-P-glycoprotein binding sites of three different murine monoclonal antibodies (MM4.17, MM6.15 and MC57), directed towards living, intact human multidrug-resistant cells were investigated in order to study P-glycoprotein topology . By using synthetic peptide scanning, we demonstrated that well-defined regions localized on the predicted first, fourth and sixth extracellular loops are external . On the basis of the structure of MM6.15 epitope, which is distributed on the above three different extracellular loops (and thus is discontinuous), P-glycoprotein molecules result to be differently organized in the lipid bilayer . Moreover, the outcome of the MC57 and MM4.17 epitopes localization experiments, obtained through the use of phage-displayed peptide libraries, represent an additional challenge to the classical 12-transmembrane domain model of P-glycoprotein, since they agree with the novel topography of the molecule (10-transmembrane domain), which was recently proposed on the basis of biochemical and expression studies.

Cytotechnology, 1996, 19(3), 237 - 42
Absence of correlation between chemo- and radioresistance in a range of human tumour cell lines; Heenan M et al.; The correlation between cellular resistance to radiation and to chemotherapeutic drugs has been investigated in a number of solid tumour cell lines, and preliminary results indicate no direct relationship . The acquisition of a multidrug resistance (MDR) profile by adriamycin-selected variants of a human squamous lung carcinoma, an ovarian carcinoma, a cervical carcinoma and by a colchicine-selected variant of a Chinese hamster ovarian carcinoma resulted in alterations to their radiosensitivity . However, the degree of change in the radiosensitivity of the MDR cell lines could not be predicted from their level of resistance to adriamycin . Clonal populations derived from DLKP-A, an adriamycin-selected MDR variant of the human lung carcinoma cell line DLKP, exhibited individual radiosensitivity profiles, which did not correlate with their chemoresistance . Exposure of DLKP to consecutive increasing doses of radiation did not confer cross-resistance to chemotherapeutic drugs.

Cytotechnology, 1996, 19(3), 229 - 35
Anthracycline drugs and MDR expression in human leukemia; Pogliani EM et al.; We investigated the expression of P-glycoprotein (P-gp) in 50 adults with de novo acute myeloid leukemia (AML) at the initial diagnosis in order to further define the relationship between the presence of P-gp on leukemic cells and the efficacy of two different anthracycline drugs, Daunorubicin (DNR) and Idarubicin (IRR), in terms of remission, induction and survival . We found that 30 (60%) of the 50 patients were negative for P-gp expression (group 1) and 20 patients (40%) were positive (group 2) for P-gp expression by MRK16MoAb using a cut of 10% positive cells . Among the 50 patients, 35 (70%) obtained complete remission (CR); depending on P-gp expression the CR rate was 80% for group 1 and 45% for group 2 (p < 0.005) . The median duration of overall survival (OS) was 20 months for patients in group 1, compared to 10 months for patients in group 2 (p < 0.005) . Regarding the anthracycline used, no difference in CR has been observed in patients of group 1 (75% CTR with DNR versus 90% CR with IDR); on the contrary in group 2 we observed 40% CR with DNR versus 70% CR with IDR (p < 0.005) . No significant difference has been achieved in group 1 terms of median duration of overall survival between DNR and IDR regimen; on the contrary the median duration of OS in patients of group 2 treated with IDR regimen was significantly longer than DNR regimen (p < 0.005) . These results confirm the prognostic value of P-gp expression in AML at diagnosis and we suggest that Idarubicin could be a valid anthracycline drug for reversing multidrug resistance.

Cytotechnology, 1996, 19(3), 221 - 7
Induction of MRP/GS-X pump and cellular resistance to anticancer prostaglandins; Akimaru K et al.; We provide evidence that the expression of the human MRP/GS-X pump encoded by the MRP (multidrug resistance associated protein) gene is induced by cisplatin in human leukemia HL-60/R-CP (cisplatin-resistant) cells and modulates cell growth inhibition by delta(7)-prostaglandin A1 (PGA1) methyl ester . The MRP mRNA level in HL-60/R-CP cells increased remarkably after a 24-h incubation with 20 microM cisplatin; interestingly, however, no amplification of the MRP gene was detected . In cisplatin-sensitive HL-60 cells, which express the MRP/GS-X pump at low levels, c-myc expression was substantially suppressed by delta(7)-PGA1 methyl ester and the cell cycle was arrested in G1 phase . By contrast, in HL-60/R-CP cells overexpressing the MRP/GS-X pump, c-myc expression and cell proliferation were much less affected by delta(7)-PGA1 methyl ester . This suggests that induction of the MRP/GS-X pump may confer on cancer cells resistance to anticancer prostaglandins and that the resistance mechanism may involve the increased efflux of PG-glutathione conjugates, as active intermediates, from the cells via the MRP/GS-X pump.

Cytotechnology, 1996, 19(3), 207 - 14
Overexpression of P-glycoprotein in heat- and/or drug-resistant hepatoma variants; Pirity M et al.; We have earlier isolated a glucocorticoid-resistant, dedifferentiated rat hepatoma variant, the clone 2, which exhibited deficient stress activation of the major stress-inducible heat-shock protein hsp68 . Multidrug-resistant variants were isolated from clone 2 cells using increasing concentrations of colchicine . The induction deficiency of hsp68 was maintained in the colchicine-resistant clone 2 cells grown for several months in the presence of 1 microgram/ml colchicine (termed as highly multidrug-resistant variant) indicating that this heat-shock protein is not involved in the multidrug resistance . No alteration of the protein synthesis pattern was observed except the strong increase of the P-glycoprotein, which correlated with high level of corresponding mRNA . Stable heat-resistant variants of clone 2 were also isolated, which showed increased drug resistance to several drugs, i.e . they became moderately multidrug-resistant . This moderate multidrug resistance of the heat-resistant variants was further increased by stepwise selection with colchicine (highly multidrug-resistant heat-resistant variants) . The levels of P-glycoprotein mRNA and protein were elevated both in the heat-resistant, non drug selected, moderately drug-resistant and in heat-resistant, colchicine selected, highly drug-resistant variants . Decreased retention of antitumor drugs was observed in all multidrug-resistant variants indicating that P-glycoprotein was functional . Verapamil increased doxorubicin retention and cytotoxicity significantly . Our results showing that severely stressed hepatoma cells overexpressed the multidrug resistance gene(s) raise the possibility that the P-glycoprotein may participate in protection against environmental stress such as heat.

Cytotechnology, 1996, 19(3), 191 - 7
Relationship of LRP-human major vault protein to in vitro and clinical resistance to anticancer drugs; Izquierdo MA et al.; Multidrug resistance (MDR) has been related to two members of the ABC-superfamily of transporters, P-glycoprotein (Pgp) and Multidrug Resistance-associated Protein (MRP) . We have described a 110 kD protein termed the Lung Resistance-related Protein (LRP) that is overexpressed in several non-Pgp MDR cells lines of different histogenetic origin . Reversal of MDR parallels a decrease in LRP expression . In a panel of 61 cancer cell lines which have not been subjected to laboratory drug selection, LRP was a superior predictor for in vitro resistance to MDR-related drugs when compared to Pgp and MRP, and LRP's predictive value extended to MDR unrelated drugs, such as platinum compounds . LRP is widely distributed in clinical cancer specimens, but the frequency of LRP expression inversely correlates with the known chemosensitivity of different tumour types . Furthermore, LRP expression at diagnosis has been shown to be a strong and independent prognostic factor for response to chemotherapy and outcome in acute myeloid leukemia and ovarian carcinoma (platinum-based treatment) patients . Recently, LRP has been identified as the human major protein . Vaults are novel cellular organelles broadly distributed and highly conserved among diverse eukaryotic cells, suggesting that they play a role in fundamental cell processes . Vaults localise to nuclear pore complexes and may be the central plug of the nuclear pore complexes . Vaults structure and localisation support a transport function for this particle which could involve a variety of substrates . Vaults may therefore play a role in drug resistance by regulating the nucleocytoplasmic transport of drugs.

Cytotechnology, 1996, 19(3), 181 - 6
Multidrug resistance evaluation by confocal microscopy in primary urothelial cancer explant colonies; Cooper AJ et al.; Assessing functional multidrug resistance (MDR) status in clinical biopsy material using drug autofluorescence has potential applications to clinical management . The small size of many cystoscopy specimens has led us to develop, as an alternative to flow cytometry, a protocol for studying epirubicin accumulation in adherent colonies of primary bladder cancer cells viewed live and in situ by confocal microscopy . The limitations to quantitation inherent in this technique are compensated for by preservation of cellular organisation and the elimination of non-malignant cells . Biopsy material is disaggregated and explanted into culture-grade petri dishes . After incubation for three to seven days plaques of epithelial cells have developed . Classical patterns of sensitive and resistant drug distribution are observed . Cells of the rolled edges of the colony accumulate more drug than those of the inner epithelial monolayer . Some central areas of larger colonies give the appearance of drug arrested at the intercellular junctions to give a fenestrated pattern . These observations contribute to the understanding of mechanisms in MDR as well as forming the basis for a clinical urological MDR evaluation protocol.

Oncol Res, 1996, 8(2), 77 - 84
2-deoxy-D-glucose toxicity and transport in human multidrug-resistant KB carcinoma cell lines; Bentley J et al.; It is shown that a series of colchicine-selected multidrug-resistant (MDR) human KB carcinoma cell lines displayed increasing 2-deoxy-D-glucose collateral sensitivity, which correlated with increasing multidrug resistance . The relative resistance of MDR cell lines to 2-deoxy-D-glucose was reduced to 0.73 (KB-8-5), 0.3 (KB-8-5-11) and 0.2 (KB-C1) when compared with parental KB-3-1 (1.0) . 2-Deoxy-D-glucose accumulation was found to be reduced in the MDR cell lines in a manner that correlated with 2-deoxy-D-glucose collateral sensitivity . At 30 min 2-deoxy-D-glucose accumulation was reduced to 0.61 (KB-8-5), 0.41 (KB-8-5-11) and 0.22 (KB-C1) relative to KB-3-1 uptake (1.0) . The efflux of 2-deoxy-D-glucose was not significantly different between resistant and sensitive cell lines . Analysis of 2-deoxy-D-glucose uptake kinetics, by initial rate measurements, showed alterations in K(t) and J(max) for MDR when compared with KB-3-l cells . The levels of GLUT-1 facilitative transporter were found to be reduced significantly in the MDR cell lines in total cell homogenate and plasma membrane fractions by using Western blot analysis . Changes in the plasma membrane level of GLUT-1 correlated with 2-deoxy-D-glucose toxicity and uptake for MDR cell lines, where relative GLUT-i levels were reduced to 0.71 (KB-8-5), 0.43 (KB-8-5-1 1) and 0.27 (KB-Cl) relative to KB-31(1.0) . It is concluded that the response of human KB MDR cells to 2-deoxy-D-glucose involved alterations in the level and activity of the facilitative glucose transporter, GLUT-1, in a manner that is associated with the degree of multidrug resistance.

Arch Med Res, 1996 Autumn, 27(3), 421 - 5
Physiology and molecular biology of multidrug resistance in Entamoeba histolytica; Gomez MD et al.; In this paper, we present the most relevant facts on multidrug resistance (MDR) in the protozoan parasite Entamoeba histolytica . MDR in E . histolytica presents characteristics similar to transformed mammalian cells . E . histolytica drug resistant mutants show cross-resistance to several drugs, and as in mammalian cells the resistance is reverted by verapamil . Six P-glycoprotein-like genes (EhPgp) have been cloned and characterized . Apparently, four of these genes are transcribed in drug-resistant mutants (EhPgp1, EhPgp2, EhPgp5 and EhPgp6), although only EhPgp1, EhPgp5 and EhPgp6 transcripts were clearly detected . The open reading frame (ORF) of the four completely full length genes is about 1300 amino acids long . EhPgp1, EhPgp2 and EhPgp5 have between 64 and 67% of positional identity among them, while EhPgp6 shows 38 to 46% positional identity to the other ameba genes . Interestingly, the phylogenetic tree suggested that Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins . Differential gene expression in drug-resistant mutants was detected when specific probes for each Ehpgp gene were used . To understand the differential expression of EhPgp genes we initiated the characterization of the upstream flanking regions of EhPgp1 and EhPgp5 genes . Upstream sequences showed between 53 and 66% of positional identity to Dictyostelium discoideum promoters.

Oncol Res, 1996, 8(3), 111 - 20
Effect of the antitumor drug lonidamine on glucose metabolism of adriamycin-sensitive and -resistant human breast cancer cells; Fanciulli M et al.; The effect of lonidamine on glucose metabolism, hexokinase activity and adenylate pool of MCF-7 human breast cancer cells sensitive and resistant to adriamycin has been investigated . The following summarizes the results: 1 . In both cell types the greatest part of glucose was metabolized to lactate, whereas only a small proportion of glucose carbon atoms was incorporated into CO2, lipids, nucleic acids, and supporting structures . 2 . Glucose utilization, lactate production, and ATP content were higher in resistant cells due to a greater activity of mitochondrial hexokinase . 3 . Lonidamine decreased glucose utilization, aerobic glycolysis and ATP content in both cell types and the effect was significantly higher on resistant cells . 4 . The extent of inhibition in sensitive and resistant cells overlapped that found for mitochondrially bound hexokinase, thus indicating that the greater sensitivity of resistant cells to lonidamine was due to their higher amount of bound hexokinase . These findings confirmed a modified glucose metabolism in cells with resistant phenotype and suggested that lonidamine might be usefully used to reduce or overcome multidrug resistance of those cells with a reduced ability to accumulate and retain antitumor drugs.

Cancer Chemother Pharmacol, 1996, 38(6), 513 - 21
In vivo antitumor activity of S 16020-2, a new olivacine derivative; Guilbaud N et al.; The antitumor activity of S 16020-2, a new olivacine derivative, was investigated in vivo and compared with that of Adriamycin and elliptinium acetate in a panel of murine (P388 leukemia, M5076 sarcoma, Lewis lung carcinoma, and B16 melanoma) and human (NCI-H460 non-small-cell lung and MCF7 breast carcinomas) tumor models . S 16020-2 given i.v . was active against P388 leukemia implanted i.p., s.c., or intracerebrally . The therapeutic effect of an intermittent schedule (administration on days 1, 5, 9) was superior to that of single-dose treatment, allowing the i.v . administration of high total doses of S 16020-2 and resulting in the cure of 60% of mice in the i.p . P388 model . In this model, S 16020-2 was more active than elliptinium acetate and showed a better therapeutic index than Adriamycin: > or = 8 versus 2 . A good therapeutic effect of S 16020-2 was also observed in three P388 leukemia sublines displaying the classic multidrug-resistance phenotype, namely, P388/VCR, P388/VCR-20, and P388/MDRC.04, the latter being totally insensitive to vincristine and Adriamycin . However, S 16020-2 was not active against the P388/ADR leukemia, a model highly resistant to adriamycin in vivo . S 16020-2 was both more active than Adriamycin and curative in the M5076 sarcoma and Lewis lung carcinoma implanted s.c . In the B16 melanoma implanted i.p . or s.c., S 16020-2 was less active than Adriamycin . Against the NCI-H460 human tumor xenograft, S 16020-2 demonstrated activity superior to that of Adriamycin (T/C = 20% versus 43% on day 21) . Against the MCF7 breast cancer xenograft, S 16020-2 was active, but less so than Adriamycin (T/C = 23% versus 9% on day 21), whereas elliptinium acetate was marginally active (T/C = 49% on day 24) . The hematological toxicity of S 16020-2 given to B6D2F1 mice at pharmacological dose appeared to be less severe than that of Adriamycin, particularly in bone-marrow stem cells . These results demonstrate that S 16020-2 is a highly active antitumor drug in various experimental tumor models and is markedly more efficient than elliptinium acetate . Because of its pharmacological profile, which is globally different from that of Adriamycin, S 16020-2 is considered an interesting candidate for clinical trials.

Conn Med, 1996 Jan, 60(1), 9 - 14
Predictors of positive tuberculin skin test results in a jail population; Roberts CL et al.; The purpose of this analysis was to determine the prevalence and predictors of positive tuberculin skin tests (TSTs) in a jail population . TST results and demographic data were obtained for 996 male inmates of a Connecticut jail who were tested following identification of a case of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) . Inmates were predominantly young (median age, 26 years) and Black (51%) and were born in the United States (96%) . Overall 109 (11%) inmates had positive TST results . TST positivity was strongly associated with being born outside the United States (adjusted odds ratio {aOR} = 11.3, 95% confidence interval {95% CI} 4.9-25.8), being Puerto Rican born (aOR = 3.7, 95% CI 1.9-7.4), and increasing age (15-24 years aOR = l {referent}; 25-34 years aOR = 2.1 95% CI 1.2-3.5; 35-44 years aOR = 4.3 95% CI 2.4-7.7; > or = 45 years aOR = 6.4 95% CI 2.8-14.6) . The combination of being U.S.-born and Black was also associated with increased rates of positive TSTs . The prevalence of TST positivity was > 10% for all age groups of inmates born outside the United States or Puerto Rico and for Puerto Rican-born inmates aged > or = 25 years and U.S.-born inmates aged > or = 35 years . Analysis of routinely collected TST data allows predictors of TST positivity to be identified and may help determine population subgroups for whom anergy screening and preventive therapy should be considered.

Curr Genet, 1996 Jan, 29(2), 103 - 5
yAP-1- and yAP-2-mediated, heat shock-induced transcriptional activation of the multidrug resistance ABC transporter genes in Saccharomyces cerevisiae; Miyahara K et al.; We have examined whether the stress-induced transcriptional activation of YDR1/PDR5/STS1 is mediated by yAP-1 and yAP-2 . Of the stresses examined, heat shock-induced, rapid and transient PDR5 expression became very low in a yap1 yap2 double-gene disruptant, indicating that the yAP proteins mediate the response . Similar results were obtained with SNQ2, a close homologue of PDR5 . A set of 5'-truncation derivatives of the PDR5 gene identified the region from -484 to -434 as being sufficient for the response . A sequence similar to the yAP-1 recognition element recently identified in the stress-responsive yeast genes was found in this region and in the 5'-flanking sequences of SNQ2.

Stem Cells, 1996 Jan, 14(1), 56 - 63
Clinical reversal of multidrug resistance; Bates SE et al.; Reversal of drug resistance offers the hope of increasing the efficacy of conventional chemotherapy . We tested dexverapamil as a P-glycoprotein antagonist in combination with EPOCH chemotherapy in refractory non-Hodgkin's lymphoma . In a cross-over design, dexverapamil was added to EPOCH after disease stabilization or progression occurred . Objective responses were observed in 10 of 41 assessable patients . Biopsies for mdr-1 were obtained before EPOCH treatment and at the time of cross-over to dexverapamil . Levels of mdr-1 were low before EPOCH, but increased four-fold or more in 42% of patients in whom serial samples were obtained . Pharmacokinetic analysis revealed median peak concentrations of dexverapamil and its metabolite, nor-dexverapamil, of 1.66 mumol/l and 1.58 mumol/l, respectively . Since both are comparable antagonists, a median peak total reversing concentration of 3.24 mumol/l was achieved . Pharmacokinetic analysis of doxorubicin and etoposide levels confirmed a delay in the clearance of doxorubicin ranging from 5% to 24%; no change in the pharmacokinetics of etoposide was observed . This study provides sufficient rationale for testing dexverapamil in a randomized clinical trial.

Stem Cells, 1996 Jan, 14(1), 47 - 55
P-glycoprotein, multidrug resistance and protein kinase C; Fine RL et al.; The multidrug resistant (MDR) phenotype is a well-studied subject that has been recognized as a determinant underlying specific types of drug resistance in human cancer . Although it is clear that the P-glycoprotein plays a major role in MDR, it is not clear whether post-translational modifications such as phosphorylation have any major impact on its modulation . The laboratory of Dr . Bruce Chabner was one of the first to describe increased expression and activity of protein kinase C (PKC) associated with the MDR phenotype . Since that time, a similar correlation has been observed in many other MDR cell lines . Most of these studies have been performed with doxorubicin-selected cells that have acquired MDR and have shown increased PKC activity, mainly for PKC-alpha isoenzyme . Intrinsic MDR in human renal cell carcinoma lines has been shown to correlate directly with PKC activity, but further studies with intrinsic MDR cell lines are needed before any conclusions can be drawn . More recent evidence suggests that there is a complex biochemical process by which PKC isoenzymes differentially phosphorylate specific serine residues in the linker region of P-glycoprotein which may lead to alterations in P-glycoprotein ATPase and drug-binding functions . To further complicate matters, PKC plays an important role in anti-apoptotic pathways, which can confound the dissection and elucidation of drug-resistance mechanisms . However, these areas are still under active investigation and not fully answered . Further studies are needed to specifically answer the question of whether PKC directly modulates basal and/or drug-stimulated P-glycoprotein function . This manuscript reviews the majority of the literature on PKC and MDR, as well as offers caveats for interpretation of these studies to answer the above questions.

J Orthop Trauma, 1996, 10(5), 366 - 70
Hazards to the orthopaedic trauma surgeon: occupational exposure to tuberculosis . Risk reduction, testing, and treatment (a review article); Esterhai JL Jr et al.; Infection with tuberculosis (TB) in the United States has risen over the last decade . In the past 5 years, health care worker exposure to multidrug-resistant TB has lead to more than 100 skin-test conversions, 17 cases of active TB, and at least six deaths . As with human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, the orthopaedic traumatologist is at risk of exposure and infection because, in many cases, the medical histories of patients encountered in the trauma bay cannot be determined until well into the course of care . Risk depends principally on two factors: (a) likelihood of exposure (large urban settings, prisons, concentration of persons from countries with high TB prevalence), and (b) immune status of the surgeon . Prompt recognition, isolation, and appropriate treatment of patients with infectious TB; engineering controls; and the use of personal protective respiratory equipment can help prevent the transmission of TB to health care workers.

Kekkaku, 1996 Jan, 71(1), 65 - 9
{Manegement and countermeasures against tuberculous patients with chronic positive sputum}; Sato K; We studied measures for the prevention and treatment of chronic positive-sputum tuberculosis . Most physicians treating chronic intractable pulmonary tuberculosis are concerned about treatment and control measures . However, both the medical and social aspects of the disease must be dealt with . The study of the medical aspects of tuberculosis used data on patients at the Tokyo National Chest Hospital and other sanitoria in Japan . The socioeconomic study employed data from a health center in Tokyo . Recently, new cases of tuberculosis are concentrated in socioeconomically high risk groups, such as the homeless and illegal aliens, in a few large cities . Patients in these groups often have multidrug-resistant tuberculosis (MDRTB), including many patients with relapsing tuberculosis . However, it is dificult to keep such patients under treatment because of poor compliance and patient dropout . The results of our study are summarized as follows: 1 . Prevention and treatment of chronic intractable tuberculosis should involve both the medical and socioeconomic aspects of the disease . 2 . Surgical treatment offers benefits for patients with chronically positive sputum . Therefore, surgery should be recommended to patients with chronic intractable MDRTB . 3 . If resistance to both isoniazid and rifampin is demonstrated, it is better to replace all ineffective drugs with a new effective regimen than to add a single drug to a failing regimen.

Blood Cells Mol Dis, 1996, 22(1), 2 - 9; discussion 10
Anti-Fas/Apo-1 monoclonal antibody CH-11 depletes glutathione and kills multidrug-resistant human leukemic cells; Efferth T et al.; Apoptosis is a common pathway by which cells respond to noxious insults or growth regulatory factors . Since cellular glutathione (GSH) content has long been known to govern response to antineoplastic treatment we have compared induction of apoptosis in drug sensitive (HL-60 and K562/WT) and drug resistant (KG-1a and K562/ADM) human leukemic cell lines by the monoclonal antibody CH-11 (anti-Fas/Apo-1) . Fraction of apoptotic cells and cellular GSH were determined by flow cytometry . All cell lines were induced to undergo apoptosis by exposure to mAb CH-11 independent of resistance to conventional antineoplastic treatment . In conjunction with exposure to daunorubicin, vincristine, carboplatin, cytosine arabinoside, dexamethasone, or ionizing irradiation the effect of mAb CH-11 on induction of apoptosis was no more than additive . In contrast, preincubation with IFN-gamma markedly enhanced the induction of apoptosis by mAb CH-11 due to an increase of Fas-receptor expression . In each instance, GSH content decreased with increasing fraction of apoptotic cells indicating a crucial role of GSH in the apoptotic pathway.

J Cell Biochem Suppl, 1996, 24, 131 - 41
Biology of colorectal and gastric cancer cell lines; Park JG et al.; Cell lines established from the human colorectal and gastric cancers may provide very useful tools to the study of the disease and to develop and test new therapeutic approaches, and a large bank of well-characterized cell lines should reflect the diversity of tumor phenotypes and provide adequate models for the study of tumor heterogeneity . Colorectal lines are relatively easy to establish, while gastric cancer cell lines remain extremely difficult to propagate in long-term culture, and the number of cell lines is very limited . In this paper, we describe the up-to-date results of the characteristics of our nine colorectal cancer cell lines and four gastric cancer cell lines . Based on culture, xenograft, and ultrastructural morphologies, these cell lines could be subtyped into well-differentiated, moderately differentiated, poorly differentiated, and mucinous carcinomas . Basic properties concerning expression and secretion of antigens, neuroendocrine features, receptor binding of various gastrointestinal hormones and neurotransmitters, cytogenetic studies, gene amplification and expression, and chemosensitivity profiles are described . In particular, a greater number of receptors for hormones and neurotransmitters are expressed on human colorectal cancer cell lines compared to gastric cancer cell lines, raising the possibility that gastrointestinal hormones may have a greater autocrine effect on colon cancer cell growth . Despite major differences in the biology of colorectal cancer and gastric cancer as indicated by clinical studies, the multiple properties that we examined reveals marked similarities between the colorectal and gastric cancer cell lines . However, in vitro chemosensitivity patterns to cytotoxic drugs are very different in colorectal and gastric cell lines . Some of these observations may be due to the relatively low expression of the multidrug-resistance-associated (MDR1) gene in gastric cancer cell lines . In addition, colorectal cancer cell lines express receptors for peptide hormones more frequently.

Pneumologie, 1996 Jan, 50(1), 21 - 7
{Resistant lung tuberculosis in Berlin 1987-1993}; Schaberg T et al.; Resistance of Mycobacterium tuberculosis (M . tb) strains is an increasing problem worldwide . Since no public health data are available for urban populations in Germany, we investigated resistance in our hospitalized patients (n = 1011) during the last seven years . We evaluated clinical data and results of susceptibility tests (break-point technique/proportion method) for isoniazid, streptomycin, rifampin, pyrazinamide, protionamide, and ethambutol . Since 1987 there has been a relatively constant rate of 5.9% (3.9-7.8%) for single-drug resistance (SDR) but an increasing rate of multidrug-resistant (MDR) strains (> or = 2 first-line drugs) from 1.7% in 1987 to 5.8% in 1993 . 69% of patients with MDR strains showed resistance to two drugs and 31% to three or more drugs . Risk factors for SDR and MDR tuberculosis revealed previous therapy (odds ratio (OR) {CI95%} SDR: 2.2 {1.7-4.0}; MDR: 4.5 {2.3-8.8}) and foreign-born status (SDR: 2.2 {1.3-3.6}; MDR: 3.5 {1.8-6.8}) to be the most important factors associated with resistance (p < 0.006-0.0001) . Both primary and acquired resistance was higher in foreign-born than in German-born patients (p < 0.005) . There was a considerable increase in multidrug-resistant tuberculosis in our hospital during 1987 and 1993 . Since previously treated patients and patients born in countries with a high level of primary resistance had an increased risk of drug-resistant tuberculosis, we would advise a four-drug regimen as initial therapy in those patients.

Ann Surg Oncol, 1996 Jan, 3(1), 80 - 5
Modulation of multidrug resistance with antisense oligodeoxynucleotide to mdr1 mRNA; Sola JE et al.; BACKGROUND: P-glycoprotein (pgp), a 170-kDa adenosine triphosphate-dependent membrane drug efflux pump encoded by the mdr1 gene, mediates cross-resistance in tumor cells to structurally unrelated cancer drugs . We investigated the capacity for modulating multidrug resistance by selectively inhibiting synthesis of Pgp using an antisense oligodeoxynucleotide complementary to the initiation codon of mdr1 messenger RNA . METHODS: By continuous culture of K562 in 100 nM vincristine, a resistant cell line, K562/VCR100, was derived with high expression of Pgp (95.9% of cells) and an IC50 40-fold greater than that of the parental cell line . The K562/VCR100 cells were treated with 10 microM of 15-mer antisense and sense phosphorothioate oligodeoxynucleotides . Modulation of multidrug resistance was analyzed using a daunorubicin/tritiated thymidine incorporation assay and flow cytometric assessment of cellular rhodamine 123 accumulation . RESULTS: Treatment of K562/VCR100 with the antisense oligodeoxynucleotide led to a doubling in daunorubicin growth inhibition at 1 microgram/ml and a tripling of growth inhibition at 0.6 micrograms/ml (p < 0.0023); a 58% reduction in the daunorubicin IC50 (p < 0.02); and an increased rate of rhodamine-123 accumulation (p = 0.02) compared with treatment with sense oligodeoxynucleotide or media controls . CONCLUSIONS: These results suggest that antisense oligodeoxynucleotides may serve as a useful adjunct in the treatment and prevention of multidrug resistance during cancer chemotherapy.

Ann Surg Oncol, 1996 Jan, 3(1), 8 - 14
p-Glycoprotein expression as a predictor of breast cancer recurrence; Gregorcyk S et al.; BACKGROUND: Many new prognostic factors for breast cancer have been described, and yet the ability to predict patient outcomes remains poor . Overexpression of p-glycoprotein (p-gp), the multidrug resistance efflux pump, confers a worse prognosis to patients with certain leukemias and other tumors . The purpose of this study was to analyze the potential usefulness of p-gp expression as a prognostic factor in patients with breast cancer . METHODS: Paraffin blocks were obtained from 55 previously untreated patients who underwent surgery between 1987 and 1988 . To determine p-gp expression, tumor cell suspensions were incubated with the p-gp-specific C219 monoclonal antibody and analyzed using an indirect immunofluorescent flow cytometric assay . RESULTS: Twenty-four (44%) of the tumors were p-gp positive and 31 (56%) were p-gp negative . Among the p-gp positive patients, 65% had recurrence of their disease, whereas only 13% of the p-gp negative patients experienced recurrence (p = 0.0001) . The 5-year disease-free rate for p-gp positive patients was 39% compared with 83% for p-gp negative patients (p = 0.0001) . In univariate analysis examining 10 different variables, significant predictors of recurrence were p-gp, stage, and tumor size . Multivariate analysis using Cox Proportional Hazards regression showed that only p-gp and stage were significant independent predictors of recurrence (p = 0.0002) . CONCLUSIONS: p-gp is frequently expressed in patients with untreated breast cancer, with p-gp-positive patients being at significantly greater risk for disease recurrence . p-gp appears to be a useful prognostic factor in breast cancer and could potentially help guide management.

Cancer Detect Prev, 1996, 20(3), 180 - 4
Positive reaction of monoclonal antibody (TO73) with resistant leukemic cells; Kakihara T et al.; We previously reported the characteristics of a murine monoclonal antibody (TO73) against the multidrug-resistant (MDR) human leukemic cell line . In the present study, we have studied the clinical significance of TO73 by examining its reactivity with primary and relapsed leukemic cells . Of 20 patients with primary leukemia and 15 patients with relapsed leukemia, none and four cases, respectively, reacted with TO73 (p < 0.05) . Furthermore, three of the four positive cases were also positive for P-glycoprotein . These results indicate that TO73 preferentially reacts with leukemic cells resistant to chemotherapy and may be a new marker for refractory leukemia.

Acta Clin Belg, 1996, 51(3), 150 - 5
{Tuberculosis: multidrug-resistance in Belgium in 1992 and 1993}; Wanlin M et al.; The "Belgian TB Multidrug Resistance Working Group" determined in collaboration with 28 laboratories carrying out antibiograms for mycobacteria, the prevalence and incidence of multidrug resistance in Belgium in 1992-1993 . During this period, respectively 14 (1.1%) and 17 (1.3%) cases of multidrug resistance (i.e . resistance to at least isoniazid and rifampicin, according to the W.H.O . definition), were detected by these laboratories . Since 9 new cases of multidrug resistance were detected in 1992 and 10 in 1993, the incidence of multidrug resistance in Belgium can be estimated at 0.1 per 100.000 inhabitants . Among these 19 new cases, 2 are confirmed as primary resistance cases.

Cancer Chemother Pharmacol, 1996, 38(5), 481 - 6
Dose-dependent brain penetration of SDZ PSC 833, a novel multidrug resistance-reversing cyclosporin, in rats; Lemaire M et al.; This study quantitatively assessed the brain penetration of a potent P-glycoprotein inhibitor, SDZ PSC 833, and its effect on the blood-brain barrier (BBB) permeability (PS) of an anticancer agent, vincristine . At lower doses of SDZ PSC 833 the brain penetration, defined as the brain-to-blood partition coefficient (Kp), was very low in spite of the high lipophilicity of this compound . At higher doses, however, the brain penetration of SDZ PSC 833 was markedly increased . Since the blood pharmacokinetics of SDZ PSC 833 proved to be linear in the dose range studied, these results demonstrated a dose-dependent brain passage of SDZ PSC 833 . The brain passage of cyclosporin A was also found to be dose-dependent . However, the potency of SDZ PSC 833 in inhibiting the efflux mechanism at the BBB was higher than that of cyclosporin A since 10 times higher doses of cyclosporin A were required to obtain the same Kp values recorded for SDZ PSC 833 . Moreover, the coadministration of SDZ PSC 833 increased the brain penetration of cyclosporin A, whereas the latter did not modify that of SDZ PSC 833 . The increase in SDZ PSC 833 and vincristine PS values observed at high blood levels of SDZ PSC 833 are consistent with the hypothesis of a saturation of the P-glycoprotein pump present at the BBB . The involvement of P-glycoprotein in the brain passage of SDZ PSC 833 could be of great significance for clinical application of the drug in the treatment of brain cancers when it is given in combination with anticancer agents.

Cancer Chemother Pharmacol, 1996, 38(5), 476 - 80
Comparison of idarubicin and daunorubicin and their main metabolites regarding intracellular uptake and effect on sensitive and multidrug-resistant HL60 cells; Tidefelt U et al.; To study the effect of the main metabolites on the cytotoxic effect of daunorubicin and idarubicin in human HL-60 cells, drug-sensitive and multidrug-resistant HL60 cells were incubated with idarubicin and daunorubicin and their metabolites idarubicinol and daunorubicinol over a wide range of concentrations . The intracellular uptake of the drugs was determined by photofluorometry, and the cytotoxic effect in vitro was determined by a bioluminescence assay of intracellular adenosine triphosphate (ATP) after 4 days of culture in liquid medium . The effect of intracellular drugs was calculated from the incubation-concentration versus intracellular-uptake and cytotoxic-effect curves . The intracellular uptake of idarubicin was 6 times that of daunorubicin in drug-sensitive cells and 25 times higher in resistant cells . For idarubicinol as compared with daunorubicinol the corresponding factors were 25 and 7, respectively . As compared with the parent substances, the uptake of idarubicinol and daunorubicinol was 16% and 4%, respectively, in sensitive cells and 40% and > 100%, respectively, in resistant cells . An intracellular concentration of 0.5 nmol/mg protein of both parent substances caused a 50% growth inhibition in drug-sensitive cells as compared with 10 nmol/mg protein for drug-resistant cells . For the metabolites an intracellular concentration of 0.4 nmol/mg protein of idarubicinol and 2.0 nmol/mg protein of daunorubicinol was required to inhibit cells' growth by 50% in drug-sensitive HL60 cells . In the resistant HL60 cells the corresponding values were 30 nmol/mg protein for idarubicinol and 40 nmol/mg protein for daunorubicinol . These results confirm that idarubicinol may significantly contribute to the clinical effect of idarubicin . However, in combination with previous results that have shown low intracellular concentrations of the metabolites in vivo, it appears that the pharmacokinetic properties of the mother substances provide the major explanation for the clinical effect of idarubicin.

Cancer Chemother Pharmacol, 1996, 38(5), 417 - 24
Comparative pharmacokinetics of KRN8602, a new morpholino anthracycline, and adriamycin in tumor-bearing mice; Shinkai H et al.; It has been reported that KRN8602 shows antitumor effects similar or superior to those of Adriamycin (ADM) against several murine and human cell lines and has been found to be effective against multidrug resistant tumor cells . We investigated the pharmacokinetics of KRN8602, a new morpholino anthracycline, in comparison with ADM in mice bearing colon26 adenocarcinoma . After intravenous administration, both drugs disappeared triexponentially from the plasma and KRN8602 was eliminated faster than ADM . The rate of elimination of KRN8602 from tissues was also faster than than of ADM . The relative order of the area under the curve (AUC) of KRN8602 was spleen > tumor > small intestine > lung > kidney > heart > liver > brain > plasma, while that of ADM was spleen > kidney > lung > liver > heart > small intestine > tumor > plasma . ADM was not detectable in the brain . The AUC of KRN8602 was higher than that of ADM in the tumor and brain, but it was lower in other tissues . The tissue-to-plasma concentration ratio (KPapp) of KRN8602 was higher than that of ADM in the tumor, spleen, small intestine and brain . KRN8602 was metabolized to several metabolites . The concentrations of M1 and M2 (glycoside-type metabolites) was relatively high in the spleen . M3 (aglycone-type metabolite) showed a very high AUC ratio in the liver (34%) . In tumor, M1 and M2 concentrations were low and M3 was not detected . KRN8602 had a greater activity than ADM and M2 had a cytotoxic activity similar to KRN8602 against colon26 cells in an MTT assay . These results suggest that the strong antitumor effect of KRN8602 against colon26 is due not only to its strong cytotoxic activity but also to its marked transferability into tumors . KRN8602 shows better selective toxicity than ADM, because KRN8602 is more selective for tumors than ADM and less is transferred to normal tissues.

Yao Xue Xue Bao, 1996, 31(1), 1 - 5
{Induction of expression of MDR 1 gene by retinoic acid and DMSO and effects on rhodamine-123 efflux in HL-60 cell lines and resistant sublines}; Zhou WD et al.; Using dot blot hybridization and flowcytometry, the effects of differentiation inducers retinoic acid (RA) and dimethyl sulfoxide (DMSO) on the resistant level of HL-60 cells and its resistant subline cells were studied . When the cells were treated with RA 1 mumol.L-1 for 24 h, the expression of MDR 1 mRNA evidently increased in both HL-60 and its multidrug resistant subline cells . The efflux of Rho-123 in the multidrug resistant subline cells was slightly decreased . But, when the cells were treated with 2% DMSO for 24 h the efflux of Rho-123 increased obviously . The results suggest that RA can induce the expression of MDR1 gene but perhaps inhibit the function of pump glycoprotein 170 (Pgp-170) through phosphorylation/dephosphorylation pathway . However, DMSO could induce the expression of full function of Pgp.

Anticancer Drugs, 1996 Jan, 7(1), 60 - 9
Effect of modulators of the multidrug resistance pump on the distribution of vinblastine in tissues of the mouse; Lyubimov E et al.; Vinblastine at doses ranging from 0.2 to 6 mg/kg body weight was administered i.p . to mice in the absence or presence of the drugs PSC 833, cyclosporin A, mefloquine, quinidine and dipyridamole, all compounds that modulate the multidrug resistance pump and thus increase the accumulation of this cytotoxin in drug-resistant cells in cell culture . In the absence of modulators, vinblastine accumulated in tissues to different extents--lowest in brain, highest in pancreas and intestine . The extent of accumulation was directly proportional to the vinblastine dose in the range 0.2-6 mg/kg body weight . Both at high and low vinblastine doses, all the modulators except quinidine increased the ability of liver, kidney, intestine and lung to accumulate vinblastine by up to 5-fold, and with the further exception of mefloquine, also increased vinblastine levels in pancreas . Only dipyridamole had a marked effect also in brain . Cyclosporin A provided effective increases in the tissue distribution of vinblastine at plasma concentrations similar to those needed to block the multidrug pump in the case of cells in cell culture . For mefloquine, plasma concentrations three or four times higher were needed in vivo than were found to be effective in cell culture . The mouse system provides a quick and reliable in vivo method to assay modulators before they are tested in the clinic.

Medicina (B Aires), 1996, 56(1), 48 - 50
Hospital transmission of multidrug-resistant Mycobacterium tuberculosis in Rosario, Argentina; Aita J et al.; Multidrug-resistant tuberculosis has emerged over the last two years at Carrasco Hospital, located in Rosario city . Nosocomial transmission among 7 AIDS patients admitted into the same ward between June and December/94 was supported by temporal clustering of cases, matching drug susceptibility, and identical IS6110 fingerprints . Among 8 non-HIV chronic cases without evidence of reciprocal contact outside the hospital, two additional clusters of 2 and 4 cases, respectively, were identified . The latter was found to be generated by a strain genetically related to the one that infected AIDS patients . It is hypothesized that an ancestor strain, common to both, might have been brought into the hospital long before the outbreak was first suspected.

Medicina (B Aires), 1996, 56(1), 45 - 7
Multidrug resistant tuberculosis outbreak in Buenos Aires . DNA fingerprinting analysis of isolates; Morcillo N et al.; In order to determine the possible relationship among HIV patients coinfected with multidrug resistant tuberculosis strains who had been receiving clinical assistance in our Hospital, clinical and epidemiological information from 28 patients was collected . DNA fingerprinting by restriction fragment length polymorphism (RFLP) pattern was performed on the mycobacterial isolates from these patients, using the restriction enzyme Pvull and IS 6110 as genetic marker . A unique RFLP pattern was found in 10 isolates from 10 different patients who had a disease caused by a single strain . Our findings confirm RFLP as a reliable and useful tool to analyze TB transmission.

Ann Biol Clin (Paris), 1996, 54(1), 31 - 6
Detection of the multidrug resistance of P-glycoprotein in healthy tissues: the example of the blood-brain barrier; Lechardeur D et al.; The blood-brain barrier is formed by the cerebral capillary endothelial cells, joined together by tight junctions . These cells express the general endothelial cell markers as well as specific markers found on endothelial cells forming physiological barriers such as gamma-glutamyltranspeptidase, the glucose transporter Glut1 and the neutral amino-acid transporter . Using the monoclonal antibodies C219 and MRK16, we have revealed by Western blot and immuno-histochemistry the expression of the multidrug resistance P-glycoprotein on isolated rat cerebral cortex capillaries . On the other hand, P-glycoprotein was not detectable in brain cortex homogenates . P-glycoprotein thus appears to be a blood-brain barrier endothelium-specific marker which could regulate brain penetration of xenobiotics and thus participate in the neuroprotection of the brain.

Ann Biol Clin (Paris), 1996, 54(1), 25 - 9
{Modulation of chemoresistance: methodology of therapeutic trials}; Rossi JF; A certain percentage of cancers are primarily or subsequently resistant to chemetherapeutic agents . Several biological mechanisms are implicated in this phenomenon, including multidrug resistance/P-glycoprotein (mdr1/P-gp), resistance related proteins (P-95 and P-110), multidrug resistance associated protein (P-190), iso-enzymes of gluthatione S-transferase, topo-isomerases, glutathione peroxidase and others . mdr1/P-gp overexpression has been studied in many types of cancer . It represents an inducible, transferable and phylogenetically ancestral biological system . It is expressed at the surface of the cell, and in that way, it participates to several normal functions . The recent introduction of modulators/revertants of mdr1/P-gp may change some concepts in using chemotherapy for cancers . The first step is represented by a better knowledge of the cancers which overexpressed mdr1/P-gp, with determination of the best biological technique, including the gold standards . This allows the clinician to clarify the best impact of such a therapeutic way and to define the criteria of modulator selection . Such criteria includes in vitro selection using a panel of sensitive/resistant cell lines, in vivo tests including transgenic mice, nude or SCID mice, and toxicological studies . Choice of modulated drug is easier and depends on the biological target . For mdr1/P-gp, major drugs included doxorubicin and vinca-alkaloids . Due to the fact that some modulators have an influence on the pharmacokinetic parameters of chemotherapeutic drugs, it is important to verify such parameters . The last choice concerns the strategy of drug development with three levels of action: 1) modulation of clinical chemoresistance, intrinsic or acquired one; 2) modulation of biological resistance; 3) leading to the prevention of the amplification of low levels of chemoresistance . A new therapeutic way is born, which takes care of a dynamic aspect of the tumor, and necessitates a new use of chemotherapy.

Ann Biol Clin (Paris), 1996, 54(1), 17 - 9
{Predictive value of intracellular accumulation of daunorubicin and P-glycoprotein expression simultaneously determined by flow cytometry in adult acute myeloid leukemias}; Guerci A et al.; Multidrug resistance (MDR) phenotype expression was evaluated retrospectively in 87 patients with acute myeloid leukemia (AML), 69 with de novo AML, ten with relapsed AML and eight with AML secondary to myelodysplastic syndrome (MDS) . MDR phenotype, characterized by P-glycoprotein expression (MRK16 monoclonal antibody) and decrease in intracellular daunorubicin (DNR) accumulation was determined using flow cytometry . All patients received chemotherapy including cytosine-arabinoside and anthracycline (daunorubicin, zorubicin, idarubicin) or mitoxantrone, and quinine in ten cases . The predictive value of the MDR phenotype for clinical responsiveness was studied using uni- and multivariate analyses . Univariate analysis showed that DNR accumulation (p < 10(-4)), P-glycoprotein expression (p = 10(-4)) and disease status (de novo versus recurrent AML and acute MDS) (p = 10(-4)) were predictive of clinical responsiveness . The significance of these three parameters was maintained in multivariate analysis . When de novo AML was considered, only DNR accumulation was of predictive value (p < 10(-4)) for complete response to chemotherapy.

Ann Biol Clin (Paris), 1996, 54(1), 9 - 15
{Evaluation of multidrug resistance phenotype on medullary specimens from patients with acute leukemia by determination of nuclear efflux of tetrahydropyranyl-doxorubicin . Approach by confocal laser microspectrofluorometry}; Morjani H et al.; Confocal microspectrofluorometry allows the analysis of fluorescent molecules such as anthracylines in isolated living cells . An optical microscope fitted with a phase-contrast 100 X water-immersion objective enables simultaneous observation of the sample, focusing of the laser beam on the selected cell fraction (nucleus) and collection of the fluorescence emitted from the sample . The resulting intranuclear spectra are interpreted according to a quantitative model of the fluorescence spectra of both free and DNA-bound anthracycline . The intranuclear drug concentration can thus be determined . This technique has been applied to blast cells collected in patients with acute leukemia . Leukemic cells are aspirated from bone marrow, separated by Ficoll sedimentation and resuspended in RPMI-1640 containing 10% fetal calf serum and 200 nM tetrahydropyranyl-doxorubicin (THP-DOX) . After one hour, 20 cells are analyzed and the mean nuclear drug content is determined (C1) . Cells are then resuspended in the same medium but without anthracycline for 3 hours and the mean intranuclear drug concentration is then also determined (C3) . From C1 and C3 the retention rate (RR) is calculated . Firstly, the accuracy of the method was checked . In 4 AML patients, two different samples aspirated on the same day were divided into two portions . Thus, two measurements were made on each one (4 values per patient) . Coefficients of variation were satisfactory (4, 6, 12, and 12%) . Secondly, blast cells collected in patients with AML and ALL at diagnosis or in relapse were studied . P-glycoprotein (P-gp) and CD34 expression was also studied using respectively immunohistochemistry land flow cytometry . Results obtained from the first 21 patients showed that there was no correlation between RR and either P-gp or CD34 expression . This could result from the efflux of THP-DOX by other mechanisms and/or low sensitivity of the staining technique.

Ann Biol Clin (Paris), 1996, 54(1), 3 - 8
{Multidrug resistance and its reversal . General review of fundamental aspects}; Robert J; Among the mechanisms by which cancer cells evade chemotherapy, multidrug resistance (MDR) is certainly the best known . MDR is characterised by cross-resistance between numerous natural products used in cancer treatment, especially antibiotics and plant alkaloids . MDR results from a defect in cell accumulation of the drugs, which are actively effluxed from cells by a plasma membrane pump, which is a high molecular weight glycoprotein termed P-glycoprotein . This protein is encoded by a gene called mdr1, and can be inhibited by a variety of pharmacological compounds . The activation of the mdr1 gene can occur via numerous types of stimulation, especially anticancer drugs themselves, which can induce mdr1 gene transcription . P-glycoprotein is an ATPase transporter which is believed to extrude xenobiotics from the plasma membrane rather than from cytoplasm . Although potential sites of interaction of P-glycoprotein with its various ligands have been identified, especially at the level of putative transmembrane domains, the exact mechanism for drug pumping has never been elucidated . Reversal of MDR in vitro is easy to obtain and to characterise . An important development aims at identifying substances able to reverse MDR in the clinical setting, that are devoid of any pharmacological properties other than interaction with P-glycoprotein . Other targets can be postulated for these MDR modulators, whose combination could well lead to a synergistic reversal of drug resistance.

Hokkaido Igaku Zasshi, 1996 Jan, 71(1), 15 - 20
{Gene therapy for cancer}; Sakamaki S et al.; The strategies of gene therapy for cancer can be classified as: 1 . regulation of oncogenes and anti-oncogenes, 2 . immunogenetherapy, 3 . support of chemotherapy, 4 . suicide gene therapy, and 5 . gene marking . The first one is the strategy to inhibit the expression of oncogenes by their antisenses or rhybozymes, or to introduce anti-oncogenes into those tumor cells with the inactivate effector cells (lymphocytes) by transducing cytokine genes, etc., followed by retransfering the gene-modified effector cells to patients (adoptive immunotherapy) . The other one is to augment antigenicity of tumor cells . The immunogenetherapy method has been widely used for 70% of gene therapy of human cancer, because cells can be transduced ex vivo . The anti-tumor effects of human gene therapy using a GM-CSF gene by Muligan et al . or an IL-2 gene by Tahara and Lotze are expected . The third is the strategy to protect bone marrows from large dose of anti-cancer drug by transducing a multidrug resistance gene into those bone marrow cells or periphen blood stem cells, overcoming dose limiting of the drug . The fourth strategy is to transduce the herpes simplex virus thymidine kinase (KSV-TK) gene for activating the cytocydal prodrug (ganciclovir: GCV) into tumor cells in order to kill the tumor cells themselves following administration of GCV . At present, vectors most widely used for gene transduction are retroviruses and adenoviruses . However, the transduction using these vectors are primarily conducted ex vivo . The direct in vivo gene delivery method to target tumor cells are required.

Annu Rev Pharmacol Toxicol, 1996, 36, 161 - 83
P-glycoproteins and multidrug resistance; Bellamy WT; Multidrug resistance represents a major obstacle in the successful therapy of neoplastic diseases . Studies have demonstrated that this form of drug resistance occurs both in cultured tumor cell lines as well as in human cancers . P-glycoprotein appears to play an important role in such cells by acting as an energy-dependent efflux pump to remove various natural product drugs from the cell before they have a chance to exert their cytotoxic effects . Expression of the MDR1 gene product has been associated with a poor prognosis in clinical studies . It has been demonstrated in the laboratory that resistance mediated by the P-glycoprotein may be modulated by a wide variety of compounds . These compounds, which include verapamil and cyclosporin, generally have little or no effect by themselves on the tumor cells, but when used in conjunction with antineoplastic agents, they decrease, and in some instances eliminate, drug resistance . Clinical trials to modulate P-glycoprotein activity are underway at the present time to determine if such strategies will be feasible . Although the P-glycoprotein is expressed in many cell lines and occurs in patient tumors, its expression is not a universal feature of multidrug resistance, suggesting that other mechanisms are operating.

Oncol Res, 1996, 8(1), 27 - 35
Energy metabolism of adriamycin-sensitive and -resistant Ehrlich ascites tumor cells; Miccadei S et al.; Respiration, glycolysis, utilization of carbon from 14C-labeled glucose and the activities of some regulatory enzymes of the Krebs cycle and glycolysis in adriamycin-sensitive (EH-WT) and -resistant (EH-ADR) Ehrlich ascites tumor cells have been investigated . The following summarizes the results: 1 . Compared with wild-type cells, EH-ADR cells exhibited an enhanced rate of oxygen consumption as well as of ATP production (2-fold) . 2 . When the cells were supplied with glucose as the only added energy source, the aerobic lactate production was 30% higher in EH-ADR cells . However, in spite of the enhanced glycolysis, 50% of total cell ATP was still supplied by oxidative phosphorylation, whereas in EH-WT cells 65% of ATP was derived from glycolysis . 3 . The activities of the regulatory enzymes were remarkably more elevated in EH-ADR cells . 4 . The amount of glucose carbon atoms metabolized through the Krebs cycle and pentose phosphate pathway in EH-ADR cells was significantly higher than in EH-WT cells . 5 . These differences confirmed a modified energy metabolism in resistant cells and reflected metabolic adaptations associated with the development of multidrug resistance.

J Cancer Res Clin Oncol, 1996, 122(8), 465 - 75
Dexniguldipine hydrochloride, a protein-kinase-C-specific inhibitor, affects the cell cycle, differentiation, P-glycoprotein levels, and nuclear protein phosphorylation in Friend erythroleukemia cells; Patterson KK et al.; Dexniguldipine hydrochloride (DNIG) is a potent antineoplastic agent with well-documented anti-(protein kinase C) activity and an ability to reverse multidrug resistance . Given the importance of protein kinase C (PKC) activity in proliferation and differentiation, we examined the effect of DNIG on several parameters of Friend erythroleukemia cell (FELC) activity . Particular attention was paid to proliferation, hexamethylene-bisacetamide-(HMBA)-induced differentiation, nuclear localization of protein kinase C, and nuclear protein phosphorylation . P-glycoprotein expression was also followed as an indicator of changes in multidrug resistance . At 2.5 microM, DNIG caused a significant decrease in the rate of FELC proliferation, while maintaining a cellular viability of greater than 80%, whether exposure to the drug was continuous over 96 h or took the form of a 6-h pulse/chase . DNA synthesis was decreased in cells exposed to DNIG for 20 h . Flow cytometry showed a marked increase in the percentage of cells in S phase of the cell cycle . Phosphorylation studies revealed decreased phosphorylation of two nuclear proteins (80 kDa and 47 kDa) following a 4-h exposure to the drug . HMBA-induced differentiation was significantly inhibited with continuous exposure to DNIG, and this effect appears to be a pre-commitment one, as 6-h pulse/chase exposures also resulted in inhibition of differentiation . Cells induced to differentiate with HMBA also demonstrated a decrease in the quantity of the 80-kDa phosphoprotein . Western blotting revealed that, even in the face of decreased phosphorylation, exposure to this PKC inhibitor resulted in an increase in the amount of nuclear PKC alpha . Finally, levels of P-glycoprotein were decreased in the presence of this drug . Our work identifies several effects of the PKC inhibitor DNIG on FELC and suggests several roles for PKC in regulating FELC proliferation and differentiation . Additionally, these results suggest that this PKC inhibitor may increase the effect of other chemotherapeutic drugs, particularly S-phase-specific ones, by increasing the length of S phase and decreasing multidrug resistance . The possibility of combination therapy with DNIG and other antineoplastic agents should be investigated further in light of these findings.

Eur J Cancer, 1996 Jan, 32A(1), 86 - 92
MDR1 gene expression: evaluation of its use as a molecular marker for prognosis and chemotherapy of bone and soft tissue sarcomas; Stein U et al.; Successful chemotherapeutic treatment of malignant tumours is often limited by the intrinsic or acquired multidrug resistance (MDR) . The classical MDR phenotype is characterised by reduced drug accumulation within the cell, caused by overexpression of the MDR1 gene encoded P-glycoprotein . Some reports have been published evaluating MDR1 expression as a molecular marker for response to chemotherapy in human bone and soft tissue sarcomas . In this review, an attempt is made to summarise the accuracy of the measurement of MDR1 expression for use in prognosis, as well as in decisions on chemotherapeutic treatment of sarcomas . In addition, general problems for the performance of such studies is discussed.

Acta Oncol, 1996, 35(4), 473 - 8
Effects of interferons and tumour necrosis factor-alpha on human lung cancer cell lines and the development of an interferon-resistant lung cancer cell line; Suarez Pestana E et al.; Thirteen human lung cancer cell lines, 7 representing small cell lung cancer (SCLC) and 6 different types of non-SCLC, were tested for sensitivity to tumour necrosis factor alpha (TNF-alpha) and interferon alpha and gamma (IFN-alpha and gamma) using an automated fluorometric microculture cytotoxicity assay (FMCA) . One SCLC line (H-82) was found to be sensitive to IFN-alpha in short-term (72 h) culture, whereas after prolonged (5 days) culture two additional SCLC cell lines responded to IFN-gamma . TNF-alpha inhibited the growth of one large cell carcinoma cell line (H-157), whereas all SCLC lines were found to be insensitive . The combination of IFN-gamma and TNF-alpha produced no further response compared with the single agents used alone . By continuous cultivation of the IFN-alpha-sensitive cell line H-82 in the presence of increasing concentrations of IFN-alpha, an IFN-alpha-resistant subline (H-82) was established . This line displayed a high degree of resistance ( > 100 fold) to IFN-alpha and cross-resistance to IFN-gamma . There was no alteration in the number of IFN binding sites, in the growth rate, the expression of selected surface markers for SCLC or the expression of multidrug resistance markers in the H-82R subline compared with the parental H-82 cell line . The results demonstrate a heterogeneous response of SCLC cell lines to IFN-alpha and gamma and TNF-alpha with only a minority of the cell lines responding to these agents by growth inhibition . The IFN-alpha and gamma H-82R subline may serve as a valuable tool in future studies on the mechanisms of IFN antitumour activity.

J Cancer Res Clin Oncol, 1996, 122(7), 403 - 8
Modulation of multidrug resistance with dexniguldipine hydrochloride (B8509-035) in the CC531 rat colon carcinoma model; Van de Vrie W et al.; The chemosensitizing potency of dexniguldipine hydrochloride (B8509-035) on epidoxorubicin was assessed in a multidrug-resistant (MDR) tumour model, the intrinsic MDR rat colon carcinoma CC531 . In vitro in the sulphorhodamine B cell-viability assay the cytotoxicity of epidoxorubicin was increased approximately 15-fold by co-incubation with 50 ng/ml dexniguldipine . In vivo concentrations of dexniguldipine 5 h after a single oral dose of 30 mg/kg were 72 (+/- 19 SD) ng/ml in plasma and 925 (+/- 495 SD) ng/g in tumour tissue . Levels of the metabolite of dexniguldipine, M-1, which has the same chemosensitizing potential, were 26 (+/- 6 SD) ng/ml and 289 (+/- 127 SD) ng/g respectively . The efficacy of treatment with 6 mg/kg epidoxorubicin applied intravenously combined with 30 mg kg-1 day-1 dexniguldipine administered orally for 3 days prior to epidoxorubicin injection was evaluated on tumours grown under the renal capsule . Dexniguldipine alone did not show antitumour effects in vivo . Dexniguldipine modestly, but consistently, potentiated the tumour-growth-inhibiting effect of epidoxorubicin, reaching statistical significance in two out of four experiments . In conclusion, these experiments show that dexniguldipine has potency as an MDR reverter in vitro and in vivo in this solid MDR tumour model.

S Afr Med J, 1996 Jan, 86(1), 50 - 5
Rifampicin resistance in Mycobacterium tuberculosis--rapid detection and implications in chemotherapy; Pretorius GS et al.; OBJECTIVES: Tuberculosis treatment and susceptibility testing are cumbersome, especially in the case of multidrug-resistant (MDR) Mycobacterium tuberculosis . It is known that mutations in the rpoB gene of M . tuberculosis lead to resistance to rifampicin (RMP) . In this study, an attempt was made to apply molecular techniques for rapid detection of antibiotic resistance in clinical isolates of M . tuberculosis . DESIGN, SETTINGS AND SUBJECTS: RMP-resistant clinical isolates of M . tuberculosis from South Africa (N = 120) with unique resistant patterns were selected for calculation of resistance frequencies, and 74 MDR isolates of M . tuberculosis from different geographical origins were used for microbiological and molecular analysis . The polymerase chain reaction (PCR) technique was applied for amplification of a previously described region around a cluster of mutations in the rpoB gene, and single-stranded conformational polymorphism (SSCP) analysis was optimised to screen for mutations in the amplified region . RESULTS: The results showed that an optimised PCR-SSCP procedure could detect a cluster of mutations in the rpoB gene (for RMP resistance) in 95% of RMP-resistant isolates . This procedure could therefore be used in the prediction of RMP resistance . Evidence was obtained that these mutations can be screened for directly from BACTEC cultures or even directly from Ziehl-Neelsen-positive sputum samples . Statistical analysis also showed that this locus can be used to predict the presence of an MDR isolate, which may have important implications in decisions concerning chemotherapy . CONCLUSION: It is currently not feasible to test all tuberculosis cases, but application of the PCR-SSCP technology in the prediction of multidrug resistance in M . tuberculosis isolates may be important in patients, especially where frequencies are high for drug-resistant isolates This methodology could reduce the time required for sensitivity testing from approximately 6-12 weeks to a few days.

Pathologe, 1996 Jan, 17(1), 50 - 5
{P-glycoprotein expression in osteosarcoma}; Posl M et al.; One of the mechanisms by which multidrug resistance is mediated, is the mdr1 gene product, P-glycooprotein . Immunohistochemistry was performed for 63 osteosarcomas of 54 patients to investigate P-glycoprotein expression using the monoclonal antibody JSB-1 . Most of the patients were children or adolescents who had received treatment under the framework of the Cooperative Osteosarcoma Study Group . In addition P-glycoprotein expression was assayed in five growth plates . Of all cases 68.5% stained positive for P-glycoprotein . Cases that had received chemotherapy showed a higher incidence (80.9%) of positive P-glycoprotein immunostaining than cases that had not received chemotherapy (66.6%) . No relation could be established between P-glycoprotein expression and the response to chemotherapy, since the majority of P-glycoprotein positive biopsies showed a good response in the surgical specimen after chemotherapy . Furthermore, 42.9% of P-glycoprotein negative biopsies were classified as non-responders in the later surgical specimen . In addition to P-glycoprotein expression in osteosarcomas positive immunostaining was also detected in osteoblasts, osteocytes, osteoclasts as well as in some chondroblasts . The results indicate that P-glycoprotein expression in osteosarcomas also exists prior to chemotherapy and resembles the phenotype of normal bone tissue . However, the determination of P-glycoprotein by using immunohistochemistry in biopsies of osteosarcomas cannot predict the response to chemotherapy.

Bull Cancer, 1996 Jan, 83(1), 39 - 45
{Expression of the MDR1 gene in five human cell lines of medullary thyroid cancer and reversion of the resistance to doxorubicine by ciclosporin A and verapamil}; Massart C et al.; Medullary thyroid carcinoma (MTC) is frequently resistant to chemotherapy . Multidrug resistance (MDR) is one of the involved mechanisms . In this work we have studied the MDR1 gene expression in five MTC human cell lines that we have isolated and we have compared this expression to that of normal thyroid tissue . We have also tried to reverse the resistance to doxorubicin with verapamil (VRP) and ciclosporin A (CSA) . MDR1 ARNm expression was studied and quantified by polymerase chain reaction (PCR) in normal and pathological thyroid tissues . The doxorubicin-induced cytotoxicity was evaluated with the 3,-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) test, the neutral red (NR) uptake and with total glutathione (GSH) or intracellular lactate dehydrogenase (LDH) measurements . We found an increase of MDR1 ARNm in MTC as compared with normal tissues . Doxorubicin was cytotoxic after a 48-h coincubation with the cells . Three microM CSA and 10 microM VRP reversed the doxorubicin resistance only after a 48-h coincubation, generally followed with a 24 h-post-incubation . In these conditions, the GSH levels were decreased only by VRP in all the five cell lines . In conclusion, a chemoresistance related to the MDR1 gene overexpression was found in the five human MTC lines tested . VRP and CSA reversed the resistance to doxorubicin in all the MTC cell lines tested.

Arch Dis Child, 1996 Jan, 74(1), 44 - 6
Effects on growth of single short courses of fluoroquinolones; Bethell DB et al.; The aim of the study was to document the effects of short courses of fluoroquinolones given during an outbreak of multidrug resistant typhoid fever in southern Viet Nam on the growth of children over a period of two years . In a prospective cohort study, 326 Vietnamese children aged between 1 and 14 years were followed up for two years after receiving either ciprofloxacin (70 mg/kg given over 7 d) (n = 173) or ofloxacin (45-50 mg/kg given over 3-5 d) (n = 153) for suspected typhoid fever . Growth velocity and weight for height were compared with an age matched control group of children from the same locality (n = 223) who had not contracted typhoid or received any fluoroquinolones . In the ofloxacin and ciprofloxacin treated patients there was no evidence of acute joint toxicity, nor of any joint symptoms attributable to either of the fluoroquinolones . There was no difference in expected weight for height measurements between the three groups of children over the two year period . During the first year, height velocity in ciprofloxacin treated children was greater than in either ofloxacin treated children or untreated controls . Height velocity in the latter two groups was not significantly different . After two years height velocity was similar in the three groups . The results support the use of short course fluoroquinolone treatment in childhood typhoid, especially when caused by strains resistant to other antibiotics.

Am J Trop Med Hyg, 1996 Jan, 54(1), 62 - 6
Clinical studies of atovaquone, alone or in combination with other antimalarial drugs, for treatment of acute uncomplicated malaria in Thailand; Looareesuwan S et al.; The therapy of Plasmodium falciparum malaria continues to be a problem in many parts of Southeast Asia because of multidrug resistance to nearly all existing antimalarial drugs . Atovaquone is a novel hydroxynaphthoquinone with broad spectrum anti-protozoal activity . We recently evaluated the antimalarial activity of atovaquone in a series of dose-ranging studies in 317 patients with malaria at the Bangkok Hospital for Tropical Diseases . Originally, the drug was administered alone . Using atovaquone alone resulted in satisfactory, initial clinical responses in all patients; the mean parasite and fever clearance times were 62 and 53 hr, respectively . However, irrespective of the duration of therapy, overall cure rates were approximately 67% . In vitro sensitivity studies on parasites taken from patients prior to treatment and at the time of recrudescence showed a marked decrease in susceptibility to atovaquone in the recrudescent parasites . To improve cure rates, atovaquone was administered in combination with other drugs with antimalarial activity . Proguanil and tetracycline were chosen due to laboratory evidence of potentiation; doxycycline was selected because it has a longer half-life than tetracycline . Although pyrimethamine did not show laboratory evidence of potentiation with atovaquone, it was chosen as an alternative inhibitor of dihydrofolic acid reductase with a longer half-life than proguanil . The clinical studies with these drug combinations confirmed the laboratory results with marked improvement in cure rates for proguanil, tetracycline, and doxycycline; pyrimethamine showed only minimal improvement . Proguanil was subsequently selected as the preferred drug partner because of its long record of safety and the ability to use the drug in pregnant women and children . Of the 104 patients with falciparum malaria treated with atovaquone plus proguanil for 3-7 days, 101 were cured and had virtually no adverse side effects . The combination of atovaquone and proguanil also was effective in eliminating erythrocytic forms of P . vivax, but parasitemia recurred in most patients.

Cancer Chemother Pharmacol, 1996, 38(3), 210 - 6
Differential single- versus double-strand DNA breakage produced by doxorubicin and its morpholinyl analogues; Duran GE et al.; The morpholinyl analogues of doxorubicin (DOX) have previously been reported to be non-cross-resistant in multidrug resistant (MDR) cells due to a lower affinity for P-glycoprotein relative to the parent compound . In order to further investigate the mechanisms of action of these morpholinyl anthracyclines, we examined their ability to cause DNA single- and double-strand breaks (SSB, DSB) and their interactions with topoisomerases . Alkaline elution curves were determined after 2-h drug treatment at 0.5, 2 and 5 microM, while neutral elution was conducted at 5, 10 and 25 microM in a human ovarian cell line, ES-2 . A pulse-field gel electrophoresis assay was used to confirm the neutral elution data under the same conditions . Further, K-SDS precipitation and topoisomerase drug inhibition assays were used to determine the effects of DOX and the morpholinyl analogues on topoisomerase (Topo) I and II . Under deproteinated elution conditions (pH 12.1), DOX, morpholinyl DOX (MRA), methoxy-morpholinyl DOX (MMDX) and morpholinyl oxaunomycin (MX2) were equipotent at causing SSB in the human ovarian carcinoma cell line, ES-2 . However, neutral elution (pH 9.6) under deproteinated conditions revealed marked differences in the degree of DNA DSB . After 2-h drug exposures at 10 microM, DSBs were 3300 rad equivalents for MX2, 1500 for DOX and 400 for both MRA and MMDX in the ES-2 cell line . Pulse-field data substantiated these differences in DSBs, with breaks easily detected after MX2 and DOX treatment, but not with MRA and MMDX . DOX and MX2 thus cause DNA strand breaks selectively through interaction with Topo II, but not Topo I . In contrast, MRA and MMDX cause DNA breaks through interactions with both topoisomerases with a predominant inhibition of Topo I.

Tumori, 1996 Jan-Feb, 82(1), 12 - 21
Applications of 99mTc-sestamibi in oncology; Maffioli L et al.; Hexakis (2-methoxyisobutylisonitrile) technetium-99m (99mTc-SestaMIBI) is a radiopharmaceutical used in nuclear medicine for myocardial perfusion imaging . In the literature different non-cardiac applications of 99mTc-SestaMIBI have been reported . Clinical studies have been performed also in non-oncologic disease (such as thyroid adenoma, diabetic foot, osteomyelitis, pulmonary actinomycosis, aneurysmal bone cyst . Sudeck's atrophy) . Several models for the uptake mechanism of this radiopharmaceutical have been proposed such as binding to an 8-10 kDa cytosolic protein, simple lipid partitioning, or a membrane translocation mechanism involving diffusion and passive transmembrane distribution . Most evidence points in the direction of the third hypothesis . Many studies have indicated that uptake of hexakis (alkylisonitrile) technetium complexes is dependent on mitochondrial and plasma membrane potentials like other lipophilic cations . This explains the initial biodistribution of 99mTc-SestaMIBI to tissues with negative plasma membrane potentials and with relatively high mitochondrial content (like heart, liver, kidney and skeletal muscle tissue) . Malignant tumours also possess these properties in order to maintain their increased metabolism . This behaviour encouraged the study of 99mTc-SestaMIBI as an interesting tracer imaging various tumour types: osteosarcoma, brain, lung, breast, nasopharyngeal, parathyroid and thyroid cancer . Recent research on cell cellular physiology has further revealed an active transport of 99mTc-SestaMIBI out of the tumour cells, against the potential gradient . The same mechanism is also responsible for resistance to a structurally and functionally different group of cytotoxic agents such as vinca alkaloids, epipodophyllotoxins, anthracyclins and actinomycin D . This peculiar type of resistance is due to amplification of the mammalian MDR1 gene, located on chromosome 7 . For this reason the 99mTc-SestaMIBI uptake in vivo could permit the prediction of the response to the chemotherapy, when the decreased accumulation of 99mTc-SestaMIBI implies the presence of P-gp enriched tissues . In the next future a particular attention should be dedicated to this matter since one of the most important goals of the clinical trials is the demonstration of the usefulness of 99mTc-SestaMIBI for in vivo assessment of multidrug resistance.

Cancer Chemother Pharmacol, 1996, 38(2), 181 - 90
Kinetic parameters for reversal of the multidrug pump as measured for drug accumulation and cell killing; Lan LB et al.; We determined the kinetic parameters that describe the effect of 20 different modulators of the multidrug resistance pump on the reversal of cytotoxin accumulation in a resistant strain of P388 leukemia cells (P388/ADR), and on the reversal of cell killing for these cells . When measured by a direct comparison of the amplitude of the pertinent protocol (accumulation or cell killing), the Ki for reversal of accumulation was generally some four or five times larger than that for reduction of cytotoxicity . We showed that this was only an apparent discrepancy, since a full theoretical analysis of the two protocols allowed the intrinsic Ki to be obtained for the two procedures and these computed Ki values were then almost identical . We found that for six of the modulators studied (namely, cyclosporin A, quinidine, dipyridamole, propafenone, mefloquine, tamoxifen) the extent of pump reversal should be better than 90% at tolerated plasma levels culled from the literature.

Cancer Chemother Pharmacol, 1996, 38(2), 178 - 80
Lack of in vivo crossresistance with gemcitabine against drug-resistant murine P388 leukemias; Waud WR et al.; Gemcitabine, a novel pyrimidine nucleoside antimetabolite, has shown clinical antitumor activity against several tumors (breast, small-cell and non-small-cell lung, bladder, pancreatic, and ovarian) . We have developed a drug-resistance profile for gemcitabine using eight drug-resistant P388 leukemias in order to identify potentially useful guides for patient selection for further clinical trials of gemcitabine and possible noncrossresistant drug combinations with gemcitabine . Multidrug-resistant P388 leukemias (leukemias resistant to doxorubicin or etoposide) exhibited no crossresistance to gemcitabine . Leukemias resistant to vincristine (not multidrug resistant), cyclophosphamide, melphalan, cisplatin, and methotrexate were also not crossresistant to gemcitabine . Only the leukemia resistant to 1-beta-D-arabinofuranosylcytosine was crossresistant to gemcitabine . The results suggest that (1) it may be important to exclude or to monitor with extra care patients who have previously been treated with 1-beta-D-arabinofuranosylcytosine and (2) the lack of crossresistance seen with gemcitabine may contribute to therapeutic synergism when gemcitabine is combined with other agents.

Anticancer Res, 1996 Jan-Feb, 16(1), 407 - 12
Alterations of vinblastine influx in multidrug-resistant lymphoblastic leukaemic CEM cells; Colin M et al.; Typical multidrug-resistant (MDR) CEM/VLB100 cells exhibited reduced uptake of vinblastine (VLB) compared to their sensitive CEM counterparts mean results were respectively, 3.19 and 35.4 pmol per 10(6) cells . In CEM cells, the efflux of drug reached a steady state after 10-15 min while in CEM/VLB100 cells, the typical P170-mediated efflux was still efficient after 40 min . Nevertheless, the most striking difference observed in CEM/VLB100 cells was a dramatic decrease in early (30 sec) influx of drug which was 10 times lower than in sensitive cells, a characteristic still observed in the presence of Na azide and absence of glucose . MDR cells without little or no effect on sensitive cells . The Q10 entry of VLB into sensitive cells was 1.2 while it was 2.2 in CEM, suggesting that the mode of entry of VLB into MDR cells differed from that of CEM cells . Verapamil or nigericin, which rapidly increased the accumulation of VLB raised the Q10 to >2 . These results suggest that the primary defect in MDR cells would be an inhibition of influx, which might involve interactions with P170 through a reversible process.

Anticancer Res, 1996 Jan-Feb, 16(1), 365 - 8
Comparative evaluation of the intracellular accumulation and DNA binding of idarubicin and daunorubicin in sensitive and multidrug-resistant human leukaemia K562 cells; Bogush T et al.; BACKGROUND: Idarubicin is a new anthracycline, more potent in in vitro models than the common anthracyclines doxorubicin and daunorubicin . The intracellular accumulation and DNA binding of daunorubicin and idarubicin have been studied in human leukaemia K562 cells and their doxorubicin-resistant variant, which presents all features of multidrug resistance . METHODS: This was achieved by measuring the decrease of total drug fluorescence during short-term incubation of cell suspensions, directly in the cuvette of a spectrofluorometer; this quenching is due to intercalation of the drug into DNA and represents an evaluation of the active intracellular concentration of the drugs . RESULTS: Accumulation of both anthracyclines was reduced in resistant cells when compared to sensitive ones . Accumulation of idarubicin was significantly higher than that of daunorubicin in both cell types . The difference in anthracycline accumulation between sensitive and resistant cells was lower for idarubicin than for daunorubicin . Verapamil increased the accumulation of both anthracyclines in resistant cells, with a more pronounced effect for idarubicin . Preincubation of the cells with either drug did not influence the accumulation of the other one . CONCLUSION: It has been shown, with this very rapid technique, that the high potency of idarubicin could be explained, at least in part, by its high level of accumulation, both in sensitive and multidrug-resistant leukaemia cells . This is not due to a lack of activity of P-glycoprotein on idarubicin, but rather to an enhanced influx of this drug compared to daunorubicin.

Anticancer Res, 1996 Jan-Feb, 16(1), 289 - 96
Protein kinase C isoenzymes, p53, accumulation of rhodamine 123, glutathione-S-transferase, topoisomerase II and MRP in multidrug resistant cell lines; Utz I et al.; We investigated whether the expression of protein kinase C (PKC) isoenzymes, topoisomerase II alpha, II beta, multidrug resistance associated protein (MRP), p53 or the activity of glutathione-S- transferase (GST) are additional factors contributing to the resistance mediated by multidrug resistance gene 1 (mdr 1) . the cell lines employed for these studies were human lymphoblastoid CCRF cells selected for resistance with actinomycin D, vincristine and adriamycin, KB-3-1 and matched resistant KB-8-5 and KB-C1 cells (selected with colchicine), and a HeLa cell line, in which the resistance was obtained by transfection with the mdr1-gene . Analysis of PKC isozymes showed that there is no correlation of a specific isoenzyme with resistance, although minor differences in the expression were observed . In vincristine and adriamycin selected cells, topoisomerase II alpha- and II beta-MRNA levels were reduced, and in vincristine selected cells the MRP-mRNA was elevated compared with the sensitive line . In KB cells the levels of topoisomerase II alpha and II beta mRNA were increasing with the resistance . Expression of p53 did not correlate with Pgp levels . In summary, MRP and topoisomerase II may contribute to the mdr1 -mediated resistance in some cell lines, but PKC, p53 and GST seem to be of minor or no importance.

Anticancer Res, 1996 Jan-Feb, 16(1), 273 - 6
Growth regulation of multidrug resistant ovarian cancer cells by 1D7 monoclonal antibody; Yang X et al.; The small molecular weight protein (p7) was overexpressed in the human ovarian carcinoma cell lines, SKVLB600 (selected for vinblastine resistance from SKOV3 cells) . OVCAR 4/ADR100 (selected for doxorubicin resistance from NIH:OVCAR4) and (OVCAR4/VBL200 (selected for vinblastine resistance from NIH:OVCAR4) . Trace amounts of the protein were also found in the parent cell lines, SKOV3 and NIH: OVCAR4 . An anti-p7 monoclonal antibody (1D7) specifically inhibited the proliferation of the drug resistant cancer cells . In order to assess the function of p7, we established a new cell line, SKVLB600R, which was maintained in drug-free medium for 16 months . This cell line stopped expressing Pgp that is responsible for its resistance to cytotoxic drugs, but continued to overexpress p7 . The proliferation of SKVLB600R was also inhibited by 1D7 . Our results indicate that p7 may be specifically involved in the proliferation of multidrug-resistant cells.

Anticancer Res, 1996 Jan-Feb, 16(1), 209 - 11
Reversal of multidrug resistance by amitriptyline in vitro; Varga A et al.; Amitriptyline, a tricyclic antidepressant, was able to reverse the multidrug resistance efflux pump of human colon cancer subline SW 620 and multidrug resistant (mdr) mouse lymphoma cells by decreasing rhodamine 123 efflux . The inhibitory effect of amitriptyline on the efflux pump was dose dependent . An investigation was made of the effects of mouse tumour necrosis factor (TNF) alpha and interferon (IFN) gamma on the efflux pump activity of mdr cells together with amitriptyline compared to the par cells (mdr-) . After long-term cytokine pretreatment of mdr cells, the amitriptyline was more effective, due to some synergism between the amitriptyline and TNF-alpha.

Cancer Chemother Pharmacol, 1996, 37(6), 593 - 600
Reversal of doxorubicin, etoposide, vinblastine, and taxol resistance in multidrug resistant human sarcoma cells by a polymer of spermine; Gosland MP et al.; We have previously described the synthesis of a cytotoxic polymeric conjugate of spermine (Poly-SPM) which is able to inhibit the transport of polyamines (spermine, spermidine, and putrescine) into normal and malignant cells . Recent studies examining the toxicity of Poly-SPM in parental and multidrug resistant (MDR) cancer cells have revealed a cross-resistance in the MDR variant Dx5 to the toxic effects of the conjugate in the MDR-positive cells . There were also differences in spermine and putrescine uptake rates between parental and MDR-positive with the MDR-positive cells having a lower Vmax and a higher Km . The ability of this Poly-SPM to reverse MDR was examined in MDR variants (Dx5 cells) of the human sarcoma cell line MES-SA . The cells express high levels of the mdr1 gene product, P-glycoprotein, and are 25-to 60-fold resistant to doxorubicin (DOX), etoposide (VP-16), vinblastine (VBL), and taxol (TAX) . Cytotoxicity was measured by the MTT {3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide} assay . Poly-SPM (50 microM) lowered the drug concentration IC50 values in the Dx5 cells by 37-fold with VBL, 42-fold with DOX, 29-fold with VP-16, and 25-fold with TAX when compared to the control IC50 values without Poly-SPM . This reversal of resistance was concentration dependent, decreasing 17-fold with DOX, 6.1-fold with VBL, 19-fold with VP-16, and 5-fold with TAX when 25 microM Poly-SPM was used . No modulation was observed in the parental cell line MES-SA, which does not express the mdr1 gene . Poly-SPM had no influence on the IC50 of non-MDR chemotherapeutic agents such as cisplatin . The modulation studies correlated with the ability of Poly-SPM to reverse the cellular accumulation defect of {3H}-VBL and {3H}-TAX in the Dx5 but not MES-SA cells . Pretreatment of the Dx5 cell with alpha-difluoromethylornithine (DFMO at 2 and 5 microM) for 24 h increased the function of the MDR transporter to further decrease the cellular accumulation of VBL and TAX when compared to untreated cells . DFMO pretreatment is known to upregulate the polyamine transporter(s) . These findings show that, in addition to inhibiting polyamine transport, Poly-SPM reverses MDR in Dx5 cells, suggesting a potential relationship between the polyamine influx transporter and the MDR efflux pump . This potential functional link between the polyamine influx transporter(s) and the MDR efflux transporter (P-glycoprotein) offers a novel approach to inhibiting this form of drug resistance.

Cancer Chemother Pharmacol, 1996, 37(6), 556 - 60
On the mechanism of action of doxorubicin encapsulation in nanospheres for the reversal of multidrug resistance; Hu YP et al.; We had previously shown that doxorubicin encapsulation in polyisohexylcyanocrylate nanospheres could circumvent the P-glycoprotein-mediated multidrug resistance (MDR) exhibited by C6 rat glioblastoma in culture . We then investigated what could be the mechanism of such a circumvention . The cytotoxicity of free and encapsulated doxorubicin was evaluated in two MDR variants of the C6 cell line in a device allowing the separation of cells from drugs by a polycarbonate membrane of 0.2 micron pore size . We observed that the progressive disruption of the nanospheres allowed their doxorubicin content to reach the cell monolayer and exert its cytotoxicity in a fashion similar to that exhibited by free doxorubicin . However, no circumvention of MDR is obtained by doxorubicin encapsulation when drug-containing nanospheres are separated from the cells by the polycarbonate membrane . In addition, no effect on azidopine binding to P-glycoprotein-enriched membranes is exerted by unloaded nanospheres, even after their spontaneous degradation in cell-culture medium . Taken together, these results suggest that a physical contact between doxorubicin-containing nanospheres and the cells is required for the circumvention of MDR . The role of degradation products from the nanospheres as modulators of P-glycoprotein activity can be ruled out.

Gan To Kagaku Ryoho, 1996 Jan, 23(2), 191 - 201
{Novel targets for cancer chemotherapy}; Inoue S; Data resulting from new insights into targets for cancer chemotherapy have been stored after the educational symposium entitled "Novel Targets for Cancer Therapy" was held at the 27th Annual Meeting of the American Society of Clinical Oncology in 1991 . Novel targets for cancer chemotherapy, which have been found or assessed to be useful after the meeting, are summarized . The contents are as follows: 1) inhibitors of signal transduction pathway, 2) inducers of apoptosis and/or differentiation, 3) agents acting on DNA directly and indirectly, 4) inhibitors of telomerase, 5) agents acting on cytoplasmic microorgans, 6) inhibitors of cytoplasmic metabolism, 7) modulators of multidrug resistance, and 8) unknown or complex targets of drug action . Targets described in each passage may be applicable to the development of new anticancer agents . Agents acting on the targets have been optionally chosen and listed . The significance of important targets and agents in cancer chemotherapy is described in more detail.

Public Health Rep, 1996 Jan-Feb, 111(1), 26 - 31; discussion 32-3
The need for epidemic intelligence; Bloch AB et al.; The past decade has witnessed an unprecedented upturn in tuberculosis morbidity and outbreaks of difficult- to-treat and highly lethal multidrug-resistant tuberculosis . In the early 1990s, a national consensus developed among public health officials to define more comprehensively the problem, and in January 1993, expanded tuberculosis surveillance was implemented nationwide . Carefully selected epidemiologic and case management variables were added to the Report of Verified Case of Tuberculosis form . Information is collected on the health status and treatment of patients, including human immunodeficiency virus status, drug susceptibility test results, and the initial drug regimen . Completion of therapy and use of directly observed therapy are also monitored . The new surveillance system allows a comparison of the quality of care of patients in the public and private sectors . Additional epidemiologic variables include membership in high-risk groups (the homeless, residents of correctional or long-term care facilities, migrant workers, health care workers, and correctional employees) and substance abuse (injecting drug use, non-injecting drug use, and excess alcohol use) . The additional information derived from expanded tuberculosis surveillance is crucial to optimal patient management, policy development, resource allocation, as well as program planning, implementation, and evaluation at Federal, State, and local levels.

J Cancer Res Clin Oncol, 1996, 122(5), 307 - 12
Modulation of multidrug resistance by BIBW22BS in blasts of de novo or relapsed or persistent acute myeloid leukemia ex vivo; Schroder J et al.; The phenylpteridine derivative BIBW22BS (BIBW22) is a potent modulator of multidrug resistance (MDR) . We investigated BIBW22 in comparison to dexniguldipine and verapamil as modifier of MDR in blasts of de novo, relapsed or persistent acute myeloid leukemia (AML) in vitro . All patients with relapsed or persistent AML had been pretreated with idarubicin and cytosine arabinoside . The degree of MDR was determined by efflux kinetics of rhodamine 123 (R123), daunorubicin, and idarubicin measured by flow cytometry (FACS) . A total of 51 patients with AML, 25 de novo and 26 relapsed or persistent, were investigated . While only 6 out of 25 de novo AML blast populations showed moderate efflux of R123 and daunorubicin, 17 out of 26 blast populations of relapsed or persistent AML had an efflux between 20% and 44% within 15 min ex vivo . This efflux could be significantly inhibited by 1 microM BIBW22, 1 microM dexniguldipine, or 10 microM verapamil . For idarubicin we found an effusion of 40+/-9% within 15 min in all blast populations that could not be inhibited by the modulators . Clinically achievable drug concentrations causing only moderate side-effects are in the range of 0.5 microM dexniguldipine and 3 microM verapamil . Up to now, BIBW22 has not been investigated clinically . Thus the potential toxicity of concentrations of 0.5-1 microM BIBW22, sufficient for an optimal efflux inhibition ex vivo, is not known yet . We conclude from our ex vivo investigations in blast populations of de novo, relapsed or persistent AML that BIBW22 is a potent modulator of MDR.

J Cancer Res Clin Oncol, 1996, 122(5), 275 - 82
Vincristine induction of mutant and wild-type human multidrug-resistance promoters is cell-type-specific and dose-dependent; Stein U et al.; To investigate multidrug-resistance gene (MDR1) promoter efficacy and drug inducibility in cells with different multidrug-resistance phenotypes, multidrug-resistant HCT15 and drug-sensitive KM12 human colon carcinoma cell lines were transfected with constructs incorporating the chloramphenicol acetyltransferase (CAT) reporter gene, driven by wild-type and point-mutated MDR1 promoter regions . The basal CAT expression level in HCT15 cells was markedly elevated compared to KM12 cells . CAT induction by vincristine was dose-dependent over a broad concentration range (40-500 ng/ml) in both lines . The induction levels were related to the cells' MDR phenotype, with the multidrug-resistant HCT15 cells showing the greater effect . In both cell types, basal and drug-induced CAT expression were significantly enhanced by the point-mutated promoter regions . The findings support the possible exploitation of the MDR1 promoter for construction of drug-inducible and MDR-cell-targeted expression vectors for use in gene therapy.

Ann Hematol, 1996 Jan, 72(1), 17 - 21
Reversal effect of itraconazole on adriamycin and etoposide resistance in human leukemia cells; Kurosawa M et al.; Itraconazole is a triazole antifungal agent that inhibits cell membrane serol biosynthesis . Currently, itraconazole is a potent candidate for in vivo use to revert multidrug resistance in acute leukemias, with the added benefit of its antifungal effect . As previously reported, itraconazole, as well as verapamil, reversed adriamycin-resistant K562 cells (K562/ADR) and HL60 cells (HL60/ADR) in dosages compatible to the plasma levels achieved by the therapeutic dosages used for the treatment of fungal infections . By RT-PCR analysis of mdr1, mdr3, and mrp mRNA, these adriamycin-resistant cells showed a higher expression of the transcript of these genes than those of the parent cells . By FACS analysis, both the adriamycin-resistant cells showed a higher expression of P-glycoprotein on their cell surfaces . These results suggested the involvement of itraconazole in the mdr gene and/or mrp gene product-associated resistance . Furthermore, itraconazole partially reversed etoposide resistance in both the K562 and K562/ADR cells . The present study suggests that itraconazole may reverse multidrug resistance, at least in part, via a classical MDR-associated mechanism.

Cancer Chemother Pharmacol, 1996, 38(1), 81 - 7
Inhibition of etoposide elimination in the isolated perfused rat liver by Cremophor EL and Tween 80; Ellis AG et al.; Cremophor EL, a surfactant used in the clinical formulation of cyclosporine and paclitaxel, will reverse the multidrug resistance (MDR) phenotype in vitro . As other MDR modulators can alter the pharmacokinetics of cytotoxic drugs, the aim of this study was to examine the effect of Cremophor and another MDR-reversing surfactant, Tween 80, on the hepatic elimination and biliary excretion of etoposide . Using the isolated perfused rat-liver model with 80 ml recirculating perfusate containing 20% red blood cells and 4% bovine serum albumin, etoposide (1.6 mg) with and without Cremophor (800 or 80 mg) or Tween 80 (80 mg) was given into the perfusate reservoir, and perfusate and bile samples were collected for 3 h . Etoposide was measured by high-performance liquid chromatography (HPLC) and Cremophor was measured using a bioassay . Both surfactants changed the etoposide elimination profile from biphasic to monophasic . High-dose Cremophor increased the AUC (from 334 +/- 23 to 1540 +/- 490 microgram min ml(-1), P<0.05) and decreased the total clearance (from 4.8 +/- 0.3 to 1.1 +/- 0.3 ml/min, P<0.05) and biliary clearance (from 2.6 +/- 1.1 to 0.5 +/- 0.2 ml/min, p<0.05) but decreased the elimination half-life (from 62 +/- 17 to 40 +/- 5 min, P<0.05) and volume of distribution (from 424 +/- 85 to 65 +/- 19 ml, P<0.05) . Low-dose Cremophor and Tween 80 caused intermediate effects on these parameters that were statistically significant for total clearance, half-life, and volume of distribution . Cremophor had no adverse effect on liver function, whereas Tween 80 caused haemolysis and cholestasis . The initial high-dose Cremophor perfusate concentration was 0.8 mg/ml, which previous studies have shown to be clinically relevant and close to the optimal level for MDR reversal in vitro (1.0 mg/ml) . Cremophor may be a clinically useful MDR modulator, but it may alter the pharmacokinetics of the cytotoxic drug.

Cancer Chemother Pharmacol, 1996, 38(1), 65 - 70
Trifluoperazine as a modulator of multidrug resistance in refractory breast cancer; Murren JR et al.; Overexpression of P-glycoprotein (P-gp) has been implicated as the mechanism of multidrug resistance (MDR) in a number of human cancers, including carcinoma of the breast . We conducted a clinical trial to determine whether the P-gp inhibitor, trifluoperazine, could sensitize patients with refractory breast cancer to vinblastine chemotherapy . Adult patients with histologically confirmed, refractory, advanced breast cancer were treated with vinblastine at a dose of 1.7 mg/m2 per day by continuous infusion for five consecutive days . Patients who did not respond after two cycles were subsequently treated with vinblastine plus trifluoperazine at a dose of 8 mg twice daily during the five days of chemotherapy . In patients from whom tumor samples were available, the expression of P-gp was determined by immunocytochemistry . Of 35 patients enrolled, 30 were evaluable, 2 of whom (7%) achieved a partial response to vinblastine alone . Among the 16 patients treated with vinblastine plus trifluoperazine there was one response (6%) which lasted 16 weeks . Tumor samples were available from 16 patients, and 14 (87%) were immunoreactive for P-pg . P_pg expression was detected both in the patient who responded to vinblastine plus trifluoperazine and in one of the two patients who responded to vinblastine alone . Continuous-infusion vinblastine demonstrated limited activity in this study . Furthermore, trifluoperazine did not effectively reverse established resistance to vinblastine . This failure may be related the presence of multiple mechanisms of drug resistance in the heavily pretreated population, or because ineffective concentrations of the modulator were achieved in vivo . Future studies should evaluate more effective modulators, and attempt to reverse MDR earlier in the course of treatment, before other forms of resistance can develop.

Cancer Chemother Pharmacol, 1996, 38(1), 45 - 51
Glutathione S-transferase and glutathione peroxidase are essential in the early stage of adriamycin resistance before P-glycoprotein overexpression in HOB1 lymphoma cells; Lee WP et al.; We have previously established Adriamycin-resistant HOB1 cell lines showing the multidrug resistance (MDR) phenotype . For further study, we analyzed the free-radical scavengers glutathione S-transferase (GST) and glutathione peroxidase (GPX) by enzyme assays and Northern blots . Three cell lines, HOB1/ADR0.1, HOB1/ADR1.0, and HOB1/ADR5.0, represented HOB1 cells resistant to 0.1, 1.0, and 5.0 microM Adriamycin, respectively . The mdr1 transcript was overexpressed in HOB1/ADR0.1 cells, and the amount of its expression reached a maximum between HOB1/ADR1.0 and HOB1/ADR5.0 cell lines . The increases in GST activity and GST-pi expression were observed only in high-level-resistant cell line (HOB1/ADR1.0 and HOB1/ADR5.0), which also showed increased GPX activity and expression . For investigation of the cytotoxic effect of Adriamycin on HOB1 cells prior to the mdr1 overexpression, an appropriate number of parental HOB1 cells were treated with 0.1 microM Adriamycin for 7 days, and the viable cells (HOB1/ADR) were isolated and subjected to analyses for mdr1, GST-pi, and GPX expression and for GST and GPX activity . In comparison with HOB1/ADR0.1 cells, HOB1/ADR cells did not show mdr1 overexpression but had significant increases in the activity and expression of GST and GPX . The current study suggests that in the early phase of Adriamycin treatment, GST and GPX are more important than P-glycoprotein for the development in HOB1 cells of resistance against Adriamycin.

J Cancer Res Clin Oncol, 1996, 122(3), 161 - 65
Expression of multidrug-resistance-associated protein gene in human soft-tissue sarcomas; Oda Y et al.; We examined the mRNA expression of the multidrug- resistance-associated protein gene (MRP) in soft-tissue sarcomas and compared it with the expression of the multidrug resistance gene (MDR1), using the reverse transcriptase/polymerase chain reaction . We investigate 39 samples from 33 cases of soft-tissue sarcomas (11 liposarcomas, 9 malignant fibrous histiocytomas, 6 leiomyosarcomas, 4 malignant schwannomas, 3 fibrosarcomas, 3 synovial sarcomas, and 3 epithelioid sarcomas) and 7 benign soft-tissue tumors . All samples were obtained prior to chemotherapy . An expression of MRP mRNA was noted in 56% of soft-tissue sarcoma specimens . The co-expression of MRP and MDR1 was recognized in 15 samples (38%) (5/11 liposarcomas, 5/9 malignant fibrous histiocytomas, 3/6 leiomyosarcomas, 2/3 fibrosarcomas) and significantly correlated with histological grade (P=0.0165) . A positive and significant correlation was found between MRP and MDR1 expression in soft-tissue sarcomas(P=0.0013) . In benign soft-tissue tumors, 1 chemodectoma and 1 neurothekeoma showed low MRP expression; however, no case showed co-expression of MRP and MDR1.

Eur J Haematol, 1996 Jan-Feb, 56(1-2), 12 - 22
Prolongation of medium exchange is associated with a decrease in function but not expression of the P-glycoprotein pump in leukaemic cells; Hegewisch-Becker S et al.; Daunorubucin (DNR) accumulation studies as functional tests of the multidrug resistance (MDR1) gene product P-glycoprotein have produced diverging results when correlated to response to chemotherapy in acute leukaemia . To investigate possible reasons for this diversity a starvation experiment, based upon prolongation of medium exchange, was set up in the multidrug resistant cell line CEM/VBL100 . DNR accumulation (1 microgram/ml) was measured flow cytometrically in the presence or absence of Verapamil (10 micromol/l) . In cells permanently kept under ideal growth conditions, addition of Verapamil resulted in an average 90% increase in DNR enhancement in five successive experiments . In contrast, DNR accumulation increased by only 26% when the medium exchange was prolonged by 30 h to 42 h . This effect was not accompanied by changes in the MDR1 gene expression at the RNA or protein level . Consequently, 53 leukaemic blast samples of 30 newly diagnosed and 18 relapsed or refractory patients with acute leukaemia (ALL-18, AML-37) were processed without any delay and under the most stringent conditions possible . Evidence of the classical MDR phenotype was arbitrarily defined by a greater than 20% enhancement in DNR accumulation in response to Verapamil (10 micromol/l) or Cyclosporin A (3 micromol/l) . Using this cutoff point for analysis of newly diagnosed leukaemia we found DNR uptake better correlated to response to treatment (p = 0.002) than P-gp detection by means of immunocytochemistry, using a panel of monoclonal antibodies (p = 0.03) . We conclude that DNR accumulation studies are a sensitive method for predicting therapy outcome in acute leukaemia when performed with necessary precautions.

Br J Haematol, 1996 Jan, 92(1), 88 - 96
Studies of P-glycoprotein in chronic myelogenous leukaemia patients: expression, activity and correlations with CD34 antigen; Turkina AG et al.; Over-expression of the P-glycoprotein (Pgp), transmembrane drug efflux pump, has been shown to cause multidrug resistance of tumour cells (MDR) . To investigate the clinical significance of Pgp expression for chronic myeloid leukaemia (CML) diagnosis and monitoring we have studied 38 CML patients in various phases of the disease (chronic phase, CP; accelerated phase, AP; blast crisis, BC) . Anti-Pgp monoclonal antibody UIC2 and FACScan analysis were used . Pgp functional activity was investigated by evaluation of verapamil influence upon rhodamine 123 efflux from the cells . Correlations between Pgp and CD34 expression were investigated . In CP, Pgp-expressing cells were found in 2/14 patients; in one of them Pgp proved to be non-functional . There were few Pgp-expressing cells in AP cases . The group of BC patients consisted of cases resistant to chemotherapy . This gave us the opportunity to consider whether drug resistance of BC CML patients is preferentially connected with Pgp-mediated MDR . 11/22 BC patients had 20% or more of Pgp-expressing blasts in the peripheral blood . In all four Pgp+ BC cases studied for Pgp activity this protein was functional . Only 4/22 BC patients demonstrated large (40% or more) fractions of Pgp+ blasts . Moreover, sequential studies of 11 BC CML patients during treatment revealed an increase in the number of Pgp-expressing cells in only two cases . This suggests that Pgp+ cells did not often accumulate in BC CML patients due to chemotherapy and are the cause of drug resistance in only a few cases . A positive correlation between Pgp and CD34 expression was found (r = 0.69; P = 0.0004) . 3/22 BC CML patients had large fractions of both Pgp+ and CD34+ blasts in their peripheral blood . The BC CML patients with this immunophenotype of blast cells may represent a subtype of BC CML resistant to treatment due to Pgp overexpression.

Leukemia, 1996 Jan, 10(1), 48 - 55
Expression of multidrug resistance-associated protein (MRP) mRNA in blast cells from acute myeloid leukemia (AML) patients; Ross DD et al.; A specific and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay was developed for measuring the mRNA of the multidrug resistance-associated protein (MRP) . A region corresponding to bp 3897-4471 of MRP cDNA is amplified, which encompasses approximately half of the second nucleotide-binding domain (NBD2) . In two multidrug resistant (MDR) sublines of the HL-60 human acute myeloid leukemia (AML) cell line which overexpress MRP but not P-glycoprotein, the assay detects elevated levels of MRP mRNA (4- to 8-fold) relative to the drug-sensitive parental cells (designated HL-60/W) . Blast cells from 24 patients with AML were also studied for MRP expression using this RT-PCR method . Expression of MRP was normalized for that of beta-actin in the blast cells, which was also determined by RT-PCR . All of these blast cell samples had MRP expression that was detectable after 35 PCR cycles . Eighteen of these patients samples had levels of expression of MRP mRNA equal to or less than that expressed by HL-60/W cells . In six patient blast cell specimens, the expression of MRP mRNA was up to 1.7-fold higher than that of HL-60/W cells . In 21 specimens, the steady-state intracellular accumulation of daunorubicin (1 microgram/ml, 3h) was also determined . The blast cells with MRP mRNA expression higher than HL-60/W had a lower median accumulation of daunorubicin compared to those whose MRP expression was less than HL-60/W, suggesting a functional defect in drug transport in the cells with higher MRP expression; a similar trend toward lower daunorubicin accumulation was also noted in the one-third of samples that displayed the highest expression of MDR1 mRNA (also determined by RT-PCR) . These studies illustrate the range of expression of MRP in AML blast cell specimens . The identification of MRP overexpression in MDR AML cell lines and in some AML patient blast cells with low intracellular daunorubicin accumulation warrants further study of MRP as a component of clinical drug resistance in AML.

J Cell Physiol, 1996 Jan, 166(1), 43 - 8
Isolation of a polyamine transport deficient cell line from the human non-small cell lung carcinoma line NCI H157; Shao D et al.; In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogue, N1,N12-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NCI H157 . The mutant line exhibits identical growth rates in the presence or absence of the analogue . However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells . In contrast to the parental cells, where a superinduction of spermidine/spermine N1-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity . BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected . This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin . Treatment of these cells with methylglyoxal bis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth . Our data suggest that these mutant cells are deficient in polyamine transport . Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylornithine (DFMO) induced growth inhibition in the mutant cells.

Cancer Chemother Pharmacol, 1996, 37(4), 305 - 16
Development of an orthotopic SCID mouse-human tumor xenograft model displaying the multidrug-resistant phenotype; Bellamy WT et al.; Multiple myeloma is a plasma cell malignancy which is generally incurable in spite of a high initial response to chemotherapy . While animal models of myeloma are known, the recent developments of human xenografts in nude and SCID mice suggests a promising experimental model . The SCID model, in particular, holds promise because these animals readily accept hematopoietic and lymphoid transplantation and do not generally develop graft versus host reaction . We have developed two drug-resistant variants of the human multiple myeloma cell line ARH-77 by in vitro exposure to gradually increasing concentrations of doxorubicin (ARH-D60) or mitoxantrone (ARM-80) . When injected into irradiated SCID mice, the ARH-D60 cell line grew in an orthotopic pattern with the development of osteolytic lesions . This is in contrast to the 8226/C1N human myeloma cell line which grows in a disseminated but nonorthotopic manner in the SCID mouse . Both the ARH-D60 and ARM-80 cell lines are resistant to doxorubicin and cross-resistant to mitoxantrone, vinca alkaloids, taxol and m-AMSA while maintaining sensitivity to antimetabolites and alkylating agents . Growth characteristics and cell cycle kinetics, including S-phase, were not altered in the resistant sublines . The ARH-D60 and ARM-80 cell lines both displayed a classic multidrug-resistance (MDR) phenotype which was partially reversed by the addition of verapamil . These two cell lines represent the first MDR human myeloma cell lines which have demonstrated an orthotopic growth pattern in the SCID mouse and thus may be of value in studying the pathophysiology of this disease.

Cancer Chemother Pharmacol, 1996, 37(4), 297 - 304
Transport mechanism of anthracycline derivatives in human leukemia cell lines: uptake and efflux of pirarubicin in HL60 and pirarubicin-resistant HL60 cells; Nagasawa K et al.; We studied the transport mechanism of pirarubicin (THP) in HL60 and its THP-resistant (HL60/THP) cells, which showed no expression of mdr1 mRNA on Northern blot analysis . Under physiological conditions, the uptake of THP by both types of cell was time- and temperature-dependent . The amount of drug transport in the resistant cells was significantly less than that in the parent cells within 3 min of incubation . THP uptake was significantly higher in the presence than in the absence of 4 mM 2,4-dinitrophenol (DNP) in glucose-free Hanks' balanced salt solution in both HL60 and HL60/THP cells and the increases were approximately equal . In the presence of DNP, the uptake of THP by both types of cell was concentration-dependent, and there were no significant differences in the apparent kinetic constants (Michaelis constant (Km), maximum velocity (Vmax) and Vmax/Km) for THP uptake between HL60 and HL60/THP cells . Additionally, THP transport was competitively inhibited by its analogue doxorubicin . The efflux of THP from HL60/THP cells was significantly greater than that from HL60 cells, and the release from both types of cell was completely inhibited by decreasing the incubation temperature to 0 degrees C and by treatment with DNP in glucose-free medium . In contrast, the P-glycoprotein inhibitors verapamil and cyclosporin A did not inhibit THP efflux . However, genistein, which is a specific inhibitor of multidrug resistance-associated protein (MRP), increased the THP remaining in the resistant cells, and the value was approximately equal to that of the control group in the sensitive cells . These results suggest that THP is taken up into HL60 and HL60/THP cells via a common carrier by facilitated diffusion, and then pumped out in an energy-dependent manner . Furthermore, the accelerated efflux of THP by a specific mechanism, probably involving MRP, other than the expression of P-glycoprotein, resulted in decreased drug accumulation in the resistant cells, and was responsible, at least in part, for the development of resistance in HL60/THP cells.

Blood, 1996 Jan 1, 87(1), 42 - 50
A bicistronic retrovirus vector containing a picornavirus internal ribosome entry site allows for correction of X-linked CGD by selection for MDR1 expression; Sokolic RA et al.; Chronic granulomatous disease (CGD) is an inherited hematologic disorder involving failure of phagocytic cell oxidase to produce superoxide (O2-.), resulting in recurrent infections . The success of retrovirus gene therapy for hematopoietic diseases will be limited both by the efficiency of ex vivo transduction of target cells and by the ability of corrected cells to replace uncorrected cells in vivo . Using MFG-based retrovirus vectors containing oxidase genes, we have previously demonstrated in vitro correction of CGD, but transduction rates were low . In the present study we explore a strategy for providing a selective growth advantage to transduced cells, while retaining the single promoter feature of MFG responsible for high virus titer and enhanced protein production . We constructed a bicistronic retrovirus producing a single mRNA encoding both the therapeutic gene for the X-linked form of CGD (X-CGD), gp91phox, and the selectable human multidrug resistance gene, MDR1 linked together by the encephalomyocarditis virus internal ribosome entry site (IRES) . As a control we constructed a bicistronic vector with the polio virus IRES element and using the bacterial neomycin resistance gene (neor) as the selective element . In Epstein-Barr virus transformed B (EBV-B) cells from an X-CGD patient, a tissue culture model of CGD, we show correction of the CGD defect and complete normalization of the cell population using either of these vectors and appropriate selection (vincristine for MDR1 and G418 for neor) . Using a chemiluminescence assay of O2- . production, populations of cells transduced with either vector demonstrated initial correction levels of from less than 0.1% up to 2.7% of normal EBV-B cell oxidase activity . With either construct, cell growth under appropriate selection enriched the population of transduced cells, resulting in correction of X-CGD EBV-B cells to a level of O2- . production equalling or exceeding that of normal EBV-B cells . These studies show that a therapeutic gene can be linked to a resistance gene by an IRES element, allowing for selective enrichment of cells expressing the therapeutic gene . Furthermore, the use of MDR1 as a selective element in our studies validates an important approach to gene therapy that could allow in vivo selection and is generalizable to a number of therapeutic settings.

Br J Cancer, 1996 Jan, 73(2), 183 - 8
Modulation of vinblastine cytotoxicity by dilantin (phenytoin) or the protein phosphatase inhibitor okadaic acid involves the potentiation of anti-mitotic effects and induction of apoptosis in human tumour cells; Kawamura KI et al.; Cellular insensitivity to vinca alkaloids is suggested to be primarily due to drug efflux by P-glycoprotein (P-gp) . The anti-epileptic phenytoin (DPH), which does not bind to P-gp, can selectively enhance vincristine (VCR) cytotoxicity in wild-type (WT) or multidrug-resistant (MDR) cells . We now demonstrate that the protein phosphatase inhibitor okadaic acid (OKA) can mimic the effect of DPH by selectively enhancing cytotoxicity of vinblastine (VBL), but not taxol and doxorubicin, in human leukaemia HL-60 cells . Both DPH and OKA potentiate the anti-mitotic effects of VBL by enhanced damage to the mitotic spindle, resulting in prolonged growth arrest . Also, unlike VBL alone, in human leukaemia or non-small-cell lung carcinoma cells treated with VBL plus DPH, recovery from damage to the mitotic spindle is compromised in drug-free medium and cell death by apoptosis in interphase ensues . Since protein phosphatases are involved with the regulation of metaphase to anaphase transit of cells during the mitotic cycle, enhanced VBL cytotoxicity in the presence of DPH or OKA may involve effects during metaphase on the mitotic spindle tubulin leading to growth arrest and apoptosis in interphase . These novel results suggest that DPH or OKA could be powerful tools to study cellular effects of vinca alkaloids and possibly for the development of novel therapeutic strategies.

Br J Cancer, 1996 Jan, 73(2), 169 - 74
Inheritance of chromosome 7 is associated with a drug-resistant phenotype in somatic cell hybrids; de Silva M et al.; A major form of drug resistance in tumour cells known as classical multidrug resistance (MDR) is associated with the overexpression of the mdr1 gene product, the membrane protein P-glycoprotein (P-gp), which acts as an energy-dependent drug efflux pump . In this study the inheritance of P-gp expression was examined using hybrids formed after somatic cell fusion between a drug-sensitive human T-cell leukaemia cell line, CEM/CCRF, and a drug-resistant derivative, CEM/A7, which is characterized by a clonal chromosomal duplication dup(7)(q11.23q31.2) . Fourteen hybrids, chosen at random, were analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) and by binding studies involving the monoclonal antibody MRK16, which recognises an external P-gp epitope . Only two hybrids were positive for both MRK16 antibody labelling and mdr1 mRNA . Partial karyotypic analysis of all hybrids revealed that only the MRK16-positive hybrids contained the duplication in chromosome 7 seen in the CEM/A7 parental MDR line . Therefore, P-gp overexpression in the MRK16-positive hybrids may be linked to the inheritance of chromosome 7 from CEM/A7 and possibly associated with the chromosome 7 abnormality.

J Cancer Res Clin Oncol, 1996, 122(1), 27 - 40
Membrane interactions of some catamphiphilic drugs and relation to their multidrug-resistance-reversing ability; Pajeva IK et al.; The multidrug-resistance (MDR)-reversing ability of the catamphiphilic drugs could be mediated through their interaction with the membrane phospholipids . This could lead directly (through changes in membrane permeability and fluidity) and/or indirectly (through inhibition of P-glycoprotein phosphorylation via inhibition of the phosphatidylserine-dependent protein kinase C or changes in the conformation and functioning of the membrane-integrated proteins via changes in the structure organization of the surrounding membrane bilayer) to the reversal of MDR . Using differential scanning calorimetry and NMR techniques and artificial membranes composed of phosphatidylcholine or phosphatidylserines we found a significant correlation between the MDR-reversing activity of the drugs in doxorubicin-resistant human breast carcinoma MCF-7/DOX and murine leukaemia P388/DOX tumour cells (data taken from the literature) and their ability to interact with phosphatidylserines . Trans- and cis-flupentixol were found to interact most strongly with both the phospholipids, followed by trifluoperazine, chlorpromazine, triflupromazine, flunarizine, imipramine, quinacrine and lidocaine . Differences in the interaction of trans- and cis-flupentixol with the phospholipids studied are suggested to be responsible for their different MDR-reversing ability . Verapamil showed moderate membrane activity, assuming that the membrane interactions are not the only reason for its high MDR-reversing ability . Amiodarone showed very strong interactions with phosphatidylserines and is recommended for further MDR-reversal studies.

Am J Respir Crit Care Med, 1996 Jan, 153(1), 317 - 24
Outcome of MDR-TB patients, 1983-1993 . Prolonged survival with appropriate therapy; Park MM et al.; We analyzed the clinical and laboratory findings and outcome of 173 patients hospitalized at our institution from 1983 to 1994 with multidrug-resistant tuberculosis (MDR-TB) and evaluated outcome . The 173 patients (mean age 40 +/- 1 yr) were predominantly male (92%), African American or Hispanic (80%), and mostly undomiciled . Over half (52%) were known to be HIV-infected . HIV-positive MDR-TB patients had significantly more pulmonary and constitutional symptoms, more extrapulmonary disease, and fewer cavitary lesions on chest radiographs . Fifty-five percent of the patients in the cohort have died; mortality was significantly greater for HIV-positive than HIV-negative (72% versus 20%, p < 0.01) . The median duration of survival of MDR-TB patients was 22 +/- 1 mo . Overall, extrapulmonary involvement was a risk factor for shorter survival, while a cavitary lesion on initial chest film and institution of appropriate treatment were positive predictors of survival . In HIV+ patients, only appropriate therapy was associated with prolonged survival (median of 14.1 mo) . Interestingly, there was a trend toward better outcome in the first half of the decade reviewed . We conclude that although mortality from MDR-TB is high in both HIV-positive and HIV-negative patients, institution of appropriate therapy is the factor most strongly associated with a favorable outcome . Development of new diagnostic and therapeutic strategies for MDR-TB are urgently needed.

J Acquir Immune Defic Syndr Hum Retrovirol, 1996 Jan 1, 11(1), 53 - 8
Mycobacterial infection in Mexican AIDS patients; Molina-Gamboa JD et al.; To describe the characteristics of mycobacterial infection in Mexico, we reviewed records from patients who were seen at the AIDS Clinic of the National Institute of Nutrition in Mexico City from 1983 to 1992 . Of 460 AIDS patients, 118 (25.6%) were found to have mycobacterial infections by positive Ziehl-Neelsen stain, culture, or both . Cultures were completed for 66 of the 118 stain-positive specimens . Mycobacterium tuberculosis was the most common species found (n = 13), followed by M . avium complex (n = 12); 21 infections were identified a nonspecific mycobacteria other than tuberculosis (MOTT) and 20 infections were from species other than tuberculosis . Susceptibility testing was performed in only two tuberculosis cases, with one strain showing multidrug resistance . We conclude that mycobacterial infection is common among our AIDS population, and MOTT may be at least as common as M . tuberculosis . Previous reports of the rarity of MOTT could be related to the lack of adequate diagnostic methods in developing countries.

Am J Med, 1995 Dec 29, 99(6A), 40S - 44S
Clinical drug resistance: the role of factors other than P-glycoprotein; Kaye SB; Clinical drug resistance is a major obstacle to successful chemotherapy for cancer . When it occurs, resistance to a wide range of agents is noted . This clinical observation should not be confused with so-called "multidrug resistance," which is a laboratory-based phenomenon, whereby cross-resistance in experimental models to structurally unrelated compounds is seen and is due to increased expression of P-glycoprotein (PGP) . In the majority of cases of clinical drug resistance in solid tumors it is likely that other factors will play a major role . These other factors can be defined as pharmacologic or cellular . Pharmacologic factors are those that prevent an adequate degree of tumor cell exposure and include considerations of dose and schedule of drug, and also of drug metabolism, which may relate to concomitant medication and to genetic variations . Clinical maneuvers to circumvent drug resistance by increasing dose are so far of unproven value . Cellular factors are those that apply at the tumor cell itself, and it is probable that multiple mechanisms exist . These include considerations of drug uptake, activation/inactivation, and changes in target enzymes and in DNA repair processes . After DNA damage has occurred, a key determinant of the sensitivity of the tumor cell is its ability to undergo apoptosis . It is conceivable that failure to engage this process is a key factor in resistance to a number of drug classes, although there is little clinical evidence to support this at present . However, the genetic controls for the process of apoptosis are now being unraveled, and if this notion proves correct, the possibility will exist for the design of more rational means of circumventing drug resistance to a wide range of agents . In the meantime, strategies that should be pursued further in order to overcome this key clinical problem include further exploration of alternating or sequential drug schedules and using new non-cross-resistant agents, such as taxoids.

Gene, 1995 Dec 29, 167(1-2), 221 - 5
Similar cleavage efficiencies of an oligoribonucleotide substrate and an mdr1 mRNA segment by a hammerhead ribozyme; Holm PS et al.; Hammerhead ribozymes (Rz) can specifically recognize and cleave target RNAs in trans . This makes them attractive in antisense RNA approaches for specific gene inactivation in vivo . A severe limitation is the poor cleavage efficiency of large RNA substrates, in contrast to the high activities observed with small oligoribonucleotides (oligos) as model substrates . It was suggested that the low efficiency is caused by poor accessibility of the target sequence in the structure of the long RNA substrates . This means it should be possible to overcome this limitation by judicious choice of the target sequence, although experimental proof was lacking . We observed similar cleavage efficiencies of small and large RNA substrates with a hammerhead Rz directed against multidrug resistance-encoding mdr1 mRNA . Accordingly, large RNAs can also be good substrates, if an optimal target sequence is selected.

FEBS Lett, 1995 Dec 27, 377(3), 285 - 9
The catalytic cycle of P-glycoprotein; Senior AE et al.; P-glycoprotein is a plasma-membrane glycoprotein which confers multidrug-resistance on cells and displays ATP-driven drug-pumping in vitro . It contains two nucleotide-binding domains, and its structure places it in the 'ABC transporter' family . We review recent evidence that both nucleotide-sites bind and hydrolyse Mg-ATP . The two catalytic sites interact strongly . A minimal scheme for the MgATP hydrolysis reaction is presented . An alternating catalytic sites scheme is proposed, in which drug transport is coupled to relaxation of a high-energy catalytic site conformation generated by the hydrolysis step . Other ABC transporters may show similar catalytic features.

Biochim Biophys Acta, 1995 Dec 27, 1264(3), 369 - 76
Differences between nuclear run-off and mRNA levels for multidrug resistance gene expression in the cephalocaudal axis of the mouse intestine; Chianale J et al.; P-glycoprotein is a multidrug transporter encoded by the mdr3 gene in the mouse intestinal epithelium . The aims of this study were to characterize the mdr3 gene expression in the cephalocaudal axis of the intestine in adult animals and during perinatal development, and to define the molecular mechanism responsible for the heterogeneous expression of the gene along the cephalocaudal axis . RNA extracted from stomach, duodenum, jejunum, ileum, cecum and colon was hybridized by slot blot and Northern blot using a mdr3 cDNA probe . The regulation of gene expression was investigated examining the rate of transcription by nuclear run-off analysis . Transport studies of rhodamine 123, a substrate of P-glycoprotein, were performed in everted jejunum and ileum . The level of mdr3 mRNA and P-glycoprotein found in ileum was 6-fold higher than the level found in duodenum . The regional pattern of mdr3 gene expression is established in the intestine of 10-day-old animals . Similar mdr3 hybridization signal in nuclear run-off assay was found in nuclei of enterocytes isolated from jejunum and ileum, suggesting that the heterogeneous expression of the mdr3 gene in the cephalocaudal axis of the small bowel may be predominantly regulated at the post-transcriptional level . Transport rate of rhodamine 123 from the serosal to mucosal side in everted ileum was higher than the rate of transport found in jejunum . These results indicate that enterocytes of the ileum may be more actively involved in the P-glycoprotein-mediated transport of xenobiotics into the intestinal lumen.

Biochem Biophys Res Commun, 1995 Dec 26, 217(3), 825 - 31
The MDR1 downstream promoter contains sequence-specific binding sites for wild-type p53; Strauss BE et al.; We have examined the interaction of the wild-type p53 protein with the downstream promoter of the human multidrug resistance gene-1 (MDR1) . Our findings indicate that wild-type p53 inhibits reporter activity driven by the MDR1 downstream promoter (base pairs -189 to +133 relative to the major transcriptional initiation site) in a dose-dependent manner in cotransfection assays in the BHK and the Saos-2 cell lines . A 123 base-pair segment of DNA (-119 to +4 relative to the major transcriptional initiation site), a 193 base-pair segment (-189 to +4), and a 135 base-pair segment (-2 to +133) have been isolated from the MDR1 downstream promoter which, like the full promoter, are negatively controlled by wild-type p53 . In addition, we show sequence-specific binding of wild-type p53 protein to the MDR1 downstream promoter . These in vitro results suggest that the presence of wild-type p53 negatively affects expression of the MDR1 gene product, p-glycoprotein, at the transcriptional level.

J Med Chem, 1995 Dec 22, 38(26), 5066 - 70
Novel inhibitors for multidrug resistance: 1,3,5-triazacycloheptanes; Sawanishi H et al.; 1,3,5-Triazacycloheptanes were synthesized and examined for reversal of the multidrug resistance dependent on P-glycoprotein . Most of these compounds increased the intracellular uptake of vinblastine in multidrug-resistant mouse leukemia P388/ADR cells without influence upon the vinblastine accumulation in P388/S cells . The efficacy of 1,5-dibenzyl-1,3,5-triazacycloheptanes in increasing the vinblastine accumulation was in the order of 2,4-dithioxo (5) > 2-oxo-4-thioxo (4) approximately 4-(methylthio)-2-oxo (6) > 2,4-dioxo (2) . The efficacy was further increased when the benzyl group was converted to a chlorobenzyl group . Among these compounds, 6c {1,5-bis(4-chlorobenzyl)-1,5,6,7-terahydro-4-(methylthio)-2H-1,3,5 - triazepin-2-one} potentiated the in vitro cell growth-inhibitory effect of vinblastine, adriamycin, and mitomycin C on P388/ADR cells and prolonged the life span of P388/ADR-bearing mice in combined therapy with vinblastine more than vinblastine alone.

FEBS Lett, 1995 Dec 18, 377(2), 232 - 6
Phylogenetic classification of the major superfamily of membrane transport facilitators, as deduced from yeast genome sequencing; Nelissen B et al.; From the approximately 5000 open reading frames presently identified by systematic sequencing of the yeast genome, 100 Saccharomyces cerevisiae transport proteins belonging to the major facilitator superfamily (MFS), were assigned to 17 families on the basis of extensive database searches and binary comparisons . These families include multidrug resistance proteins and transport proteins for sugars, amino acids, uracil/allantoin, allantoate, phosphate, purine/cytosine, proteins, peptides, potassium, sulfate, and urea . Four new families of unknown function have been identified . For the sugar and amino acid transport proteins, alignments were made and phylogenetic trees were constructed allowing the identification of several clusters of proteins presumably exhibiting similar transport functions.

FEBS Lett, 1995 Dec 18, 377(2), 201 - 7
The pharmacological profile of the vesicular monoamine transporter resembles that of multidrug transporters; Yelin R et al.; Vesicular neurotransmitter transporters function in synaptic vesicles and other subcellular organelles and they were thought to be involved only in neurotransmitter storage . Several findings have led us to test novel aspects of their function . Cells expressing a c-DNA coding for one of the rat monoamine transporters (VMAT1) become resistant to the neurotoxin N-methyl-4-phenylpyridinium (MPP+) {Liu et al . (1992) Cell, 70, 539-551} . The basis of the resistance is the VMAT1-mediated transport and sequestration of the toxin into subcellular compartments . In addition, the deduced sequence of VMAT1 predicts a protein that shows a distinct homology to a class of bacterial drug resistance transporters (TEXANs) that share some substrates with mammalian multidrug resistance transporters (MDR) such as the P-glycoprotein . These findings induced us to test whether compounds that are typically transported by MDR interact also with vesicular transporters . The use of {3H}reserpine binding to determine drug interactions with VMAT allowed assessment of the ability of various drugs to bind to the substrate site of the transporter . Cytotoxic compounds such as ethidium, isometamidium, tetraphenylphosphonium, rhodamine, tacrine and doxorubicin, interact specifically with vesicular monoamine transporters . Verapamil, a calcium channel blocker, is also a competitive inhibitor of transport . In the case of rhodamine, fluorescence measurements in digitonin-permeabilized cells demonstrated ATP-dependent VMAT-mediated transport . The results imply that even though the bacterial and vesicular transporters are structurally different from the P-glycoprotein, they share a similar substrate range . These findings suggest a novel possible way of protection from the effects of toxic compounds by removal to subcellular compartments.

Int J Cancer, 1995 Dec 11, 63(6), 798 - 803
High rate of MDR-1 and heterogeneous pattern of MRP expression without gene amplification in endometrial cancer; Esteller M et al.; The present study was undertaken to determine the pattern of expression of 2 molecular markers of multidrug resistance, the MDR-1 and MRP genes, in endometrial carcinomas from 50 untreated patients using a PCR semi-quantitative assay . In addition, PCR analysis of MDR-1 and MRP gene amplification was obtained in each case . High rate of MDR-1 expression without gene amplification was detected in all endometrial-carcinoma samples, as well as in 10 adenomatous hyperplasias and 50 samples of normal endometrium . On the other hand, MRP appeared to be expressed at moderate levels in normal endometrial tissue, while relatively high levels of MRP expression without gene amplification were occasionally observed in endometrial-carcinoma samples.

Biochem Biophys Res Commun, 1995 Dec 5, 217(1), 333 - 40
The region 3' to the major transcriptional start site of the MDR1 downstream promoter mediates activation by a subset of mutant P53 proteins; Strauss BE et al.; We have examined the response of the human multidrug resistance gene-1 (MDR1) downstream promoter to various mutants of human p53 in a reporter assay system . Our findings indicate that mutant 175H inhibits reporter activity driven by the MDR1 downstream promoter (base pairs -189 to +133 relative to the major transcriptional initiation site) in a dose-dependent manner in cotransfection assays in the BHK and the Saos-2 cell lines . A 123 base-pair segment of DNA (-119 to +4 relative to the major transcriptional initiation site) and a 193 base-pair segment (-189 to +4) have been isolated from the MDR1 downstream promoter which, like the full promoter, are negatively controlled by mutant 175H . However, a 135 base-pair segment (-2 to +133) of the promoter is activated by mutant 175H as well as mutant 248Q, but not by mutants 213Q and 234H . Thus some mutants of p53 are able to activate transcription from the 3' region of the MDR1 downstream promoter, an activity that characterizes these p53 mutants as "gain of function" mutants.

Ugeskr Laeger, 1995 Dec 4, 157(49), 6859 - 63
{Tuberculosis in low prevalence countries . Back to the future?}; Pedersen JT et al.; The incidence of tuberculosis has declined in all European countries in this century but in several countries the decline has come to a stop . Microscopy a.m . Ziehl-Neelsen and culture are still the mainstays in the diagnosis of tuberculosis but new DNA-technics such as PCR and RFLP are increasingly used . The tuberculin test is hitherto the only practically feasible test for identification of subjects infected with M . tuberculosis . BCG is the most used vaccine in the world but the protective mechanisms are not fully understood and the efficacy controversial . Immunotherapy with M . vaccae may be used as an adjuvant to chemotherapy . Few cases of tuberculosis are observed in AIDS-patients in Denmark . Modern standard treatment of tuberculosis comprises a six-month regimen with isoniazid, rifampicin, pyrazinamide and possibly ethambutol . Multidrug resistance in tuberculosis is rare in Western Europe . Chemoprophylaxis with isoniazid is debatable . Patients with smear-positive sputum may be infectious whereas patients with extrapulmonary tuberculosis normally pose no risk.

Cell Growth Differ, 1995 Dec, 6(12), 1505 - 12
Coordination of transcription factors, NF-Y and C/EBP beta, in the regulation of the mdr1b promoter; Yu L et al.; The expression of mdr genes that encode P-glycoprotein, an integral membrane drug transporter, has been associated with the emergence of the multidrug resistance phenotype during treatment with cancer chemotherapeutic drugs . To understand the regulation of the mdr genes, the murine mdr1b promoter has been isolated and characterized in our laboratory . Three nuclear protein binding sites that interact with nuclear proteins present in both drug-sensitive and -resistant murine macrophage-like 1774.2 cells have been localized to the promoter . In this report, transcription factor NF-Y has been identified as binding to the Y-box sequence in site 1 and as a major factor in the regulation of the murine mdr1b promoter in the mouse adrenal cell line, Y-1, that endogenously expresses the mdr1b gene . The expression of CCAAT/enhancer binding protein beta (C/EBP beta) in Y-1 cells augmented mdr1b promoter activity and resulted in an increased level of mdr1b mRNA . The effect of C/EBP beta expression on mdr1b promoter activity was sensitive to mutations in the Y-box, suggesting that coordination of NF-Y with C/EBP beta is required for further activation of the mdr1b promoter . Our studies have indicated that NF-Y is a critical factor for the mdr1b promoter, and its coordination with other factors, such as C/EBP beta, could be an important mechanism involved in mdr1b gene expression.

Int J Clin Pharmacol Ther, 1995 Dec, 33(12), 664 - 9
Bioavailability and pharmacokinetic characteristics of dexniguldipine-HCl, a new anticancer drug; Goedhals L et al.; Dexniguldipine-HCl is a new dihydropyridine derivative with antineoplastic activity and potency for overcoming multidrug resistance . In this pharmacokinetic study the bioavailability of 3 doses of an oral formulation of dexniguldipine was to be determined . Fourteen patients with malignant disease not eligible for higher priority treatment and sufficient general condition were included . In 12 patients all pharmacokinetic investigations were available for evaluation . A single 4-h infusion of 2 mg per kg body weight of dexniguldipine was given as reference . Thereafter 3 increasing oral dosages (750, 1,500, 2,250 mg/d) were given on a 3-time daily basis for 3 consecutive weeks . On day 7 (under steady state conditions) of each period, a pharmacokinetic profile was done . Absolute bioavailability at the 3-dose levels was 3, 4, and 5%, respectively, thus slightly increasing with dose, but generally low . After intravenous administration terminal half life was 22.4 h, clearance 36.9 l/h and volume of distribution 1,193 1 . Toxicity was tolerable with main adverse events being loss of appetite, nausea, and vomiting . Cardiovascular effects and a decrease in serum calcium were reported in several patients . Patients were allowed to continue treatment if a benefit was expected, and 2 patients showed tumor regression during treatment . One patient with renal cell carcinoma achieved a partial remission . Bioavailability of this oral formulation seems too low for routine clinical use, despite the fact that clinical effects have been observed.

Baillieres Clin Haematol, 1995 Dec, 8(4), 831 - 44
Modulation of multidrug resistance in multiple myeloma; Sonneveld P; Primary or acquired drug resistance is the major cause of failure of chemotherapy in MM . MDR is associated with expression of a membrane P-gp, which acts as an efflux pump for natural product drugs, such as doxorubicin and vincristine . P-gp expression is observed in extensively treated patients and is a cause of refractoriness to VAD chemotherapy . Among several other non-cytotoxic drugs, cyclosporins modulate the function of P-gp in refractory myeloma cells . Early clinical trials with verapamil and cyclosporin have shown that these can be combined with VAD . In patients treated with drug-resistance modifiers, there is, besides an effect on the tumour cell, also an increased plasma exposure to several cytostatic drugs, which is mediated through inhibition of biliary efflux . Thus, the clinical effect of drug modulation may result from inhibition of tumour P-gp and from altered drug pharmacokinetics . Several trials are now in progress in order to evaluate the clinical benefit of resistance modulators in myeloma.

Anticancer Drugs, 1995 Dec, 6(6), 727 - 35
Saturation reversal of the multidrug pump using many reversers in low-dose combinations; Lyubimov E et al.; Multidrug resistance in cancer cells, in cell culture and in the clinic, is often associated with a membrane protein (the multidrug resistance pump or P-glycoprotein) that pumps out anti-cancer drugs as fast as they enter the cell . This pump is blocked by a range of well-known pharmaceuticals that reverse drug resistance . We have investigated whether effective reversal of drug resistance could be achieved by using many reversers together, each at a low dose relative to its maximal tolerated plasma level . We measured in cell culture, using resistant P388 cells in suspension, the extent of reversal of the accumulation of two labeled cytotoxins (vinblastine and daunomycin) . We fitted the data to a modified Michaelis-Menten equation and extracted the half-inhibition constants for 18 reversers acting on the pump . We measured also the reversal of resistance in a cell growth assay using incorporation of labeled thymidine . We showed that these drugs in groups of up to 18 together, each drug being at a low dose, in many cases well-tolerated in humans, had additive effects so that the combination was as effective as any of the drugs present singly . This was the case both for reversal of cell accumulation and for the effects of cytotoxins on cell growth . Our data show that a low-dose multidrug approach to saturation reversal of the multidrug pump is feasible in cell culture and provide the initial experimental basis for the development of an effective regime of such combination reversal therapy.

Diagn Cytopathol, 1995 Dec, 13(5), 411 - 22
Tumor genes and their proteins in cytologic and surgical specimens: relevance and detection systems; Leong AS et al.; Oncogenesis is the consequence of a series of genetic alterations that allow unrestrained cellular growth, tissue invasion, and eventual metastases . Tumor-related genes can be classified into functional categories . Proto-oncogenes/oncogenes have a stimulatory role in cell growth, and the inactivation of cancer-suppressor genes/antioncogenes results in the loss of cell cycle regulation . More recently, three other groups of tumor-related genes have been recognized . They include the antiapoptosis genes which protect from programmed cell death, the antimetastasis genes, and multidrug resistance genes . Besides aiding in tumor diagnosis, the detection of such tumor-associated genes and their products allows the identification of individuals with an inherited predisposition to neoplastic growths, and the overexpression of many of these oncogene products has been shown to be a potential marker of tumor behavior and a predictor of treatment outcome and response . The ability to utilize DNA and RNA probes for nucleic acid hybridization and polymerase chain reaction procedures in cell and tissue preparations of solid tumors and lymphoid proliferations expands and complements the information provided by immunohistochemical techniques . These probes allow direct visualization and correlation of specific genes and their protein products with cytomorphologic features, and form a powerful addition to the armamentarium of the cytopathologist and surgical pathologist.

Zentralbl Bakteriol, 1995 Dec, 283(2), 225 - 38
On the development of mycobacterial infections II . Changing mycobacterial infection spectrum in the Cologne area 1983-1993; Schutt-Gerowitt H; The overall detection rate of mycobacteria in the Cologne area did not change between 1983 and 1993 . But a significant shift of the rate of Mycobacterium tuberculosis to nontuberculous mycobacteria, especially Mycobacterium avium-intracellulare complex, could be observed . Whereas the portion of Mycobacterium tuberculosis fell during these years from about 90% to 50-70% the portion of Mycobacterium avium-intracellulare increased steadily from 0 to 23% of all mycobacteria positive patients . The portion of tuberculosis patients positive for HIV remained constantly at 5-7% until 1992 . Whether the 10% in 1993 signalizes a definitive increase remains to be proven . HIV positive patients with the diagnosis mycobacteria show another development . Whereas Mycobacterium tuberculosis is isolated constantly from about 2-3% of all HIV positive patients, the Mycobacterium avium-intracellulare complex increased from 0 to 10% . As methodical factors play a special role in the diagnostic procedure of mycobacteria, we evaluated some of the newer techniques for our routine laboratory: The well-known high sensitivity and specificity of the DNA probes (Gen-probe system) was confirmed, and by the radiometric culture system, in fact, the ubiquitous mycobacteria were detected much better . Otherwise from one third of special materials Mycobacterium tuberculosis and Mycobacterium bovis grew only on the conventional solid media . The susceptibility testing of 925 Mycobacterium tuberculosis strains revealed a rate of 10% (95 strains) with resistance to one antituberculotic drug only and 3% (29 strains) with resistance to two or more drugs . Only 0.9% (8 strains) were resistant to isoniacid and rifampicin (multidrug-resistant strains) indicating that drug resistance of Mycobacterium tuberculosis is still no serious problem in our region.

J Antimicrob Chemother, 1995 Dec, 36(6), 1079 - 83
A comparative clinical trial of artemether and the sequential regimen of artemether-mefloquine in multidrug resistant falciparum malaria; Karbwang J et al.; A randomized, comparative clinical trial for assessment of the efficacy of two antimalarial regimens, artemether alone and the sequential regimen artemethermefloquine, was carried out in 109 Thai male patients with acute uncomplicated, multidrug resistant falciparum malaria who were admitted to the Bangkok Hospital for Tropical Diseases . Fifty-three patients received oral artemether at a total dose of 700 mg (300 mg initially, followed by 100 mg daily for 4 days), and 56 patients received the sequential regimen of artemether-mefloquine (300 mg oral artemether initially, followed by 750 mg oral mefloquine after 24 h) . Patients in both groups had a rapid initial response to treatment, with a median parasite clearance time of 40 h compared with 43.5 h for the sequential regimen . Median fever clearance times were 42.5 h and 32.5 h for artemether and the sequential regimen respectively . Parasitaemia reoccurred in patients of both groups during the follow up period, six in the artemether and three in the sequential regimen (cure rates were 88 and 94%) . No serious adverse effects were observed in either group of patients.

Leuk Lymphoma, 1995 Dec, 20(1-2), 143 - 52
Is P-glycoprotein a sufficient marker for multidrug resistance in vivo? Immunohistochemical staining for P-glycoprotein in children and adult leukemia: correlation with clinical outcome; Kaczorowski S et al.; Seventy-eight patients: 45 children, 33 adults and 27 normal healthy donors were enrolled in the study . Expression of P-glycoprotein (P-gp) was evaluated with three monoclonal antibodies (MAb's) directed to intra-(C219, JSB-1) and extra-cellular (MRK-16) epitopes of P-gp and immunocytochemical (IC) APAAP staining method . Twenty-seven healthy donors peripheral blood mononuclear cells (PBMC) were investigated by means of IC and FACScan analysis . Positive staining for P-gp was detected in 31% children's and 33% adults' leukemia samples . No reactivity of three MAb's was observed with peripheral blood mononuclear cells (PBMC) by means of IC . Flow cytometry analysis with C219 MAb revealed staining for P-gp present on sub-population of lymphocytes and monocytes . P-gp (+) as well as P-gp (-) cases were compared in respect to clinical outcome, FAB classification and blood group . Complete remission (CR) was achieved in 12/14 (85%) children's and 9/11 (81%) adults' P-gp (+) leukemia cases . Within the P-gp (-) leukemia cases CR was observed in 24/29 (82%) and 18/22 (81%), respectively . Partial remission, relapse, resistance and death were noticed in 14% children's and 18% adults' P-gp (+) samples . In P-gp (-) cases these parameters were observed in 17% and 18%, respectively . These results raise the question whether the expression of P-gp can be used as single prognostic marker to detect multidrug resistance (MDR phenomenon) in vivo?

Stem Cells, 1995 Dec, 13 Suppl 3, 106 - 13
Blood stem cell transplantation and gene therapy of cancer; Korbling M; Based on the concept of circulating hematopoietic stem cells with indefinite self-renewal capacity that gives rise to all three cell lineages, peripheral blood progenitor cells (PBPCs) have widely replaced the use of bone marrow (BM) progenitors for autologous transplantation purposes in patients with malignant hematological disorders and selected solid tumors . Ex vivo purification of normal CD34+ cell subsets contained in the patient's apheresis product possibly eliminates clonogenic tumor cells, but also serves as a target cell population for gene transduction . Genetic tagging of PBPC autografts has proven that: 1) NEOR gene expression is sustained for more than 18 months and 2) clonogenic tumor cells contaminating the autograft contribute to relapse . A second generation of gene transduction studies includes new treatment strategies such as the induction of chemoprotection (multidrug resistance gene-1), chemotherapy sensitization (p53), cancer vaccination and genetic chemosensitization . Most recently allogeneic PBPC transplantation has successfully been introduced with the intention of improving the graft-versus-leukemia effect without inducing a higher incidence or more severe graft-versus-host disease (GVHD) than what is expected after BM transplantation . Introducing the herpes virus thymidine kinase cDNA into activated donor T cells makes them susceptible to gangciclovir, thus allowing the in vivo inactivation of GVHD-inducing T cells . With the close interaction of molecular genetics and clinical oncology/hematology, genetic engineering of stem cell grafts will lead into a new stage of stem cell transplantation technology.

Stem Cells, 1995 Dec, 13 Suppl 3, 93 - 9
Retroviral transfer of the multidrug resistance-1 gene into lineage-committed and primitive hemopoietic cells; Fruehauf S et al.; Transfer of the multidrug resistance-1 (MDR1) gene to hemopoietic cells for myeloprotection against cytostatic agents is a new and rapidly developing field in "cancer gene therapy." Before clinical application, safety and efficacy criteria need to be met . The retroviral producer cell lines and the retroviral supernatant need to be tested for replication-competent retrovirus and contamination with adventitious agents . The cell source needs to contain sufficient hemopoietic cells with repopulating ability . We used CD34(+)-selected mobilized peripheral blood progenitor cells (PBPC) for MDR1 transductions in order to obtain a favorable vector to target cell ratio . An analysis of 249 patients who had undergone PBPC harvesting revealed that primarily solid tumor and non-Hodgkin's lymphoma patients are eligible for CD34+ selection . They can be expected to retain sufficient CD34+ cells for rapid and sustained engraftment after myeloablative therapy if the CD34+ cell loss (approximately 50%) during the procedure is taken into account . Clinical MDR1 gene therapy protocols focus on these two patient groups . Next we characterized MDR1 gene transfer into lineage-committed and primitive hemopoietic cells . Provirus-specific polymerase chain reactions showed a high efficiency gene transfer into colony-forming-units granulocyte-macrophage and long-term culture cells . The level of the conferred P-glycoprotein expression was estimated by fluorescence-activated cell sorting analysis to be up to 3 log above mock-transduced controls . The cobblestone area forming cell assay, which is a stroma-dependent long-term culture assay measuring frequencies of stem cell subsets in a limiting-dilution set-up, allowed demonstration of sustained expression of the MDR1 gene in the progeny of primitive hemopoietic cells . This is a favorable basis for a clinical MDR1 gene therapy trial.

Bull Cancer, 1995 Dec, 82(12), 987 - 95
Homoharringtonine: an effective new natural product in cancer chemotherapy; Zhou DC et al.; Homoharringtonine (HHT) is a cytotoxic alkaloid isolated from the evergreen tree cephalotaxus harringtonia native to the southern provinces of China . The principal mechanism of action of HHT is the inhibition of protein synthesis in a dose- and time-dependent manner by acting on the ribosomes of cancer cells . It blocks the progression of cells from G1 phase into S phase and from G2 phase into M phase . It is synergestic or additive in vitro with AraC, amsacrine, actinomycin D and dexamethasone . Clinical studies have indicated that HHT is effective in treating acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS), but not acute lymphoblastic leukemia (ALL) and solid tumors . The dose limiting toxicities are hypotention and myelosuppression . Homoharringtonine has relatively mild extramedullary toxicities and no anthracycline-like cardiac toxicity, which make it a suitable candidate for the treatment of aged patients . Pharmacological studies indicate that HHT belongs to the category of multidrug resistance (MDR)-related drugs . The cells resistant to HHT are cross-resistant to anthracycline, vinca alkaloids, mitoxantrone, but not cis-platine and AraC . Multiple mechanisms, including the sequential emergence of overexpression of multidrug resistance-associated protein (MRP) and MDR1 genes, are involved in the cross-resistance of tumor cells to HHT.

Gen Pharmacol, 1995 Dec, 26(8), 1673 - 84
Mechanisms of resistance to topoisomerases poisons; Prost S; 1 . Drug resistance remains a major obstacle to cancer treatment . Resistance to chemotherapy can be intrinsic, characterised by the nonresponsiveness of the tumour to the initial treatment . Alternatively, cancers that initially respond to chemotherapy can relapse after various times because of acquired resistance . 2 . Resistance to drugs used as single agents is generally accompanied by the development of resistance to other drugs that can be structurally and functionally different . 3 . Among the drugs commonly used in cancer treatment there are compounds that have been shown to inhibit DNA topoisomerases (Topos) . These critical enzymes regulate the topological conformation of the DNA and participate in essential cellular processes . 4 . This paper reviews the Topos' cellular functions, their catalytic activities and the mechanisms of resistance to inhibitors of Topos, with particular attention to the atypical multidrug resistance phenotype.

Pharmacol Res, 1995 Dec, 32(6), 355 - 62
Effects of different sampling strategies on predictions of blood cyclosporine concentrations in haematological patients with multidrug resistance by Bayesian and non-linear least squares methods; Wu G et al.; The Bayesian method (BM) can use previous information for the optimization of dosage regimen . However, Bayes' law remains true when the parameters are obtained from the infinite population . Therefore a bias might exist in the previous information and affect BM predictive performance . To overcome this shortcoming, the blood drug concentration of a patient can be used to individualize his pharmacokinetic parameters . Until now, at least two sampling strategies, i.e . steady-state and non-steady-state sampling strategies, have been developed to individualize and predict blood drug concentration . In the present study we used five sampling strategies: (1) all samples; (2) post-infusion samples; (3) during-infusion samples; (4) samples within 95% confidence interval/interquartile range of a steady-state concentration; (5) the sample of the mean/median at the mid-time-point of a steady-state to individualize and predict blood cyclosporine concentrations in haematological patients with multidrug resistance . We investigated the effects of different sampling strategies on BM and the nonlinear least squared method (NLLSM) predictive performances . The results showed that BM predictive performance was better than NLLSM . But the results did not prove that the steady-state sampling strategies were superior to the non-steady-state ones.

Hum Cell, 1995 Dec, 8(4), 157 - 61
{Biological features determining the chemosensitivity of gastric cancer}; Ichiyoshi Y et al.; We analysed the relationship between several biological properties of gastric cancers and their chemosensitivity determined by MTT assay . Higher chemosensitivity was associated with poor differentiation, aneuploidy, and higher proliferative activity . Lymph node metastasis was more chemosensitive than primary lesion, while liver metastasis was less . Gastric cancer expressing multidrug-resistance associated protein (MRP) showed lower sensitivity to several anticancer drugs, including adriamycin and etoposide . p53 status and susceptibility to apoptosis were also associated with chemosensitivity . Thus, chemosensitivity of clinical gastric cancer might be increased if these characters can be modified by some new biologic therapy.

Eur J Cancer, 1995 Dec, 31A(13-14), 2354 - 61
The dithiane Ro 44-5912 enhances vinblastine sensitivity of drug resistant and parental KB lines in vivo; Eliason JF et al.; The multidrug resistance modifying activity of a dithiane analogue of tiapamil, Ro 44-5912, was examined in vivo . Results of acute toxicity studies in mice indicated that lethal toxicity occurred with doses greater than 1 mmol/kg of body weight . In a preliminary pharmacokinetic investigation, Ro 44-5912 appeared to have a longer half-life in mice than did its (R) enantiomer Ro 44-5911 (3.15 +/- 0.02 h versus 2.15 +/- 0.14 h) as measured by total radiolabel in plasma . In non-tumour bearing mice, Ro 44-5912 enhanced the toxicity of vinblastine in a manner that was dependent on the dose of both drugs . Vinblastine did not have a significant effect on tumour growth when given to nude mice bearing the parental cell line KB-3-1 at a dose of 1.5 mg/kg once per week for 3 weeks . Combination treatment with Ro 44-5912 markedly enhanced the antitumour activity of vinblastine . Similar results were seen when KB-3-1 tumours were treated with the combination of vinblastine plus cyclosporin A . Another tiapamil analogue, Ro 11-2933, had no enhancing activity with this tumour when used at an equitoxic combination dose . Ro 44-5912 also significantly enhanced vinblastine activity with P-glycoprotein-expressing KB-8-5 tumours . In three independent experiments, Ro 44-5912 enhanced the growth inhibiting activity of vinblastine by a mean of approximately 40% . Neither Ro-11-2933 nor cyclosporin A, at the maximal tolerated doses in combination with vinblastine, led to significant inhibition of KB-8-5 tumour growth compared to treatment with the two vehicles alone . These results show that Ro 44-5912 is an active modulator of drug resistance in vivo.

Eur J Cancer, 1995 Dec, 31A(13-14), 2313 - 9
The influence of BIBW22BS, a dipyridamole derivative, on the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in vitro and in human tumour xenografts; Jansen WJ et al.; Dipyridamole is known as a potent inhibitor of facilitated diffusion-mediated nucleoside transport as well as a modulator of 'classical' multidrug resistance . BIBW22BS, a derivative of dipyridamole, has been found to be 20- to 100-fold more potent in the reversal of multidrug resistance when compared to the parent compound . In parallel, we studied the efficacy of BIBW22BS in the modulation of the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in human cancer cell lines . BIBW22BS, at non-toxic concentrations up to 1.0 microM, increased the antiproliferative effects of 5-fluorouracil 2- to 6-fold in seven of the eight colon cancer cell lines tested in a dose-dependent manner . The addition of 1.0 microM BIBW22BS to methotrexate resulted in a slight increase in the antiproliferative effects, but inhibited the activity of gemcitabine 30- to 100-fold in various cancer cell lines . In vitro, no notable difference was found between BIBW22BS and dipyridamole in their capacity to modulate the activity of the antimetabolites studied . BIBW22BS did not affect the growth inhibition induced by 5-fluorouracil or gemcitabine in human tumour xenografts grown subcutaneously in nude mice . We confirmed the higher potency of BIBW22BS when compared to dipyridamole in the reversal of drug resistance in the Pgp-positive COLO 320 cell line.

Cancer, 1995 Dec 1, 76(11), 2351 - 6
Frequent expression of P-glycoprotein/MDR1 by nasal T-cell lymphoma cells; Yamaguchi M et al.; BACKGROUND . Lethal midline granuloma is now considered to be a malignant lymphoma derived from peripheral T cells or from natural killer cells . The therapeutic outcome of nasal T-cell lymphoma (NL) treated by conventional chemotherapy for non-Hodgkin's lymphoma is poor, although some patients have a good response to radiotherapy . To clarify the mechanisms of drug resistance, the expression of P-glycoprotein (P-gp)/MDR1, which is the product of the multidrug resistance (MDR) 1 gene, and MDR3 mRNA in NL cells, were examined . METHODS . Ten Japanese patients with NL were studied . Nine of these patients were examined before therapy . P-glycoprotein expression and phenotypes of lymphoma cells were examined by immunohistochemical staining using UIC2 as an anti-P-gp monoclonal antibody . In one case, the Rhodamine-123 efflux test was performed . MDR1 and MDR3 mRNA were detected by reverse transcription polymerase chain reaction . RESULTS . Nine of the 10 patients were P-gp positive . In one of nine, functional P-gp expression was observed . MDR1 mRNA was detected in all seven examined patients with P-gp positive NLs, whereas MDR3 mRNA was negative . Retrospectively, patients who received chemotherapy alone had poorer outcome than those treated by combination chemotherapy after irradiation . CONCLUSION . The poor prognosis for patients with NL treated with chemotherapy may be explained by P-gp expression of the NL cells.

Leuk Res, 1995 Dec, 19(12), 927 - 31
Prognostic value of rhodamine-efflux and MDR-1/P-170 expression in childhood acute leukemia; Tafuri A et al.; The evidence that mechanisms other than P-170 expression may influence its "pump" and the retention/efflux of chemotherapeutic agents, prompted us to investigate the value of a functional multidrug resistance (MDR) assay in a series of childhood acute leukemia samples . Forty acute leukemia cases, mainly of lymphoid origin (ALL), were evaluated for MDR expression using a functional test based on rhodamine-123 efflux (Rhd-E) . This was correlated with the quantification of P-170 external epitopes based on the positivity with the 4E3.16 and MRK16 monoclonal antibodies (MAbs) . When compared with the status of the disease and response to treatment, the mean (m) Rhd-E value was significantly lower in patients at diagnosis (m = 7.1% versus m = 22.4% at relapse) and in patients who achieved a complete remission (m = 8.81% versus 31.5% in resistant cases) . In the 22 samples analyzed, an overall correlation was found between the functional assay and the P-170 expression (r = 0.6), despite the much lower level of MDR positivity recognized by the immunocytometric method (m = 0.78% and 0.9% in cases at diagnosis versus m = 3.7% and 4.1% at relapse, with the 4E3.16 and MRK16 MoAbs) . These data suggest that the assessment of the clinical impact of MDR expression in pediatric ALL should be based on methodological approaches capable of providing information extended to the P-170 pump function, rather then only on its gene and protein expression.

Leukemia, 1995 Dec, 9(12), 2042 - 8
Cell surface expression of the multidrug resistance P-glycoprotein (P-170) as detected by monoclonal antibody MRK-16 in pediatric acute myeloid leukemia fails to define a poor prognostic group: a report from the Childrens Cancer Group; Sievers EL et al.; Expression of the multidrug resistance (MDR-1) gene product, P-glycoprotein (P-170), and the stem cell antigen, CD34, at diagnosis were determined using monoclonal antibodies (MoAbs) MRK-16 and 12.8 respectively, in 130 pediatric acute myeloid leukemia (AML) patients entered onto Childrens Cancer Group (CCG) study CCG-2891 . Fluorescein isothiocyanate (FITC) as a second step reagent was employed for the measurement of P-170 expression since it is commonly used in clinical laboratories . Nine of 30 (30%) infant ( < 1 year of age) de novo specimens expressed P-170 at levels > or = 20% of control cells . In contrast, eight of 100 (8%) AML samples from older children ( > or = 1 year of age) expressed the multidrug resistance surface protein at diagnosis . With the exception of one infant, all de novo samples that expressed P-170 also expressed CD34 . Pediatric patients of any age with positive P-170 expression using MoAb MRK-16 with a FITC-conjugated second step reagent fared no worse than remaining patients treated on the same treatment with regard to induction failure, incidence of relapse, event-free survival, or overall survival . Further investigation is necessary to determine whether P-170 assay systems with greater sensitivity will distinguish pediatric AML patients with poor prognosis.

Semin Oncol, 1995 Dec, 22(6 Suppl 13), 29 - 34
Taxoids: effective agents in anthracycline-resistant breast cancer; Ravdin PM; The results of recent clinical trials have shown that docetaxel (Taxotere; Rhone-Poulenc Rorer, Antony, France), like paclitaxel (Taxol; Bristol-Myers Squibb Oncology, Princeton, NJ), has high levels of activity in patients with anthracycline-resistant breast cancer . Agents that are at least partially non-cross-resistant with anthracyclines are especially promising for the treatment of breast cancer; the taxoids (docetaxel and paclitaxel) are such agents . Although preclinical evaluations shows clear instances of strong cross-resistance (particularly in cells lines expressing the P-glycoprotein, multidrug resistance), high response rates have been reported in patients with prior anthracycline exposure and/or anthracycline resistance . Phase I studies of anthracycline and taxoid combinations have been conducted . Excellent response rates have been noted in some of these studies . In some studies using regimens combining doxorubicin and paclitaxel, unanticipated toxicities have occurred, such as typhlitis, as well as congestive heart failure at lower than expected cumulative doses of doxorubicin . Phase II and III studies of regimens including both anthracyclines and taxoids have been initiated . Docetaxel and paclitaxel appear to be valuable agents for use in anthracycline-resistant breast cancer patients, and may find a place in anthracycline-containing combination regimens.

Tuber Lung Dis, 1995 Dec, 76(6), 575 - 7
Exogenous reinfection with multidrug-resistant Mycobacterium tuberculosis in an immunocompetent patient; Shafer RW et al.; We used restriction-fragment-length-polymorphism (RFLP) DNA fingerprinting to document exogenous reinfection with a multidrug-resistant strain of Mycobacterium tuberculosis in an immunocompetent patient . Molecular epidemiologic studies using RFLP analysis may elucidate the epidemiology of exogenous reinfection with M . tuberculosis.

J Mol Evol, 1995 Dec, 41(6), 974 - 8
Repetitive DNA sequences located in the central region of the human mdr1 (multidrug resistance) gene may account for a gene fusion event during its evolution; Pauly M et al.; The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy . Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves . According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure . The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA).poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17 . These repeated sequences most likely represent "molecular fossils" of ancient DNA elements which were involved in such a recombination event.

Arch Pharm (Weinheim), 1995 Dec, 328(11-12), 755 - 9
Sensitizing properties of new N-(2-arylalkyl) propionamides in drug-sensitive and multidrug-resistant tumor cells; Ros F et al.; The propionamides 5 and 6 have been synthesized and tested for stimulation of antitumor drug activity . 5 and 6b increase vincristine cytotoxicity in drug-sensitive murine tumor cells; 5 also increases the toxicity in multidrug resistant cells . Dissimilar trends in sensitive and resistant cells have been observed for the stimulating activity of several propionamides of this family and structurally related verapamil with their molar refractivity, suggesting different size requirements for the sensitizers in sensitive and resistant cells.

Pneumologie, 1995 Dec, 49 Suppl 3, 653 - 6
Tuberculosis and HIV infection worldwide; Murray JF; The incidence of HIV-associated tuberculosis is increasing worldwide and will continue to increase during the foreseeable future, especially in developing countries . HIV infection appears to increase the opportunity for M . tuberculosis to succeed in causing infection after inhalation into the lungs . Moreover, there is persuasive evidence that in the presence of HIV infection, new-onset tuberculous infection will progress rapidly to clinically significant disease and the likelihood that latent tuberculous infection will reactivate is enormously increased . The accelerating and amplifying influence of HIV infection is contributing to the increasing incidence of disease caused by multidrug-resistant strains of M . tuberculosis . Neither clinical or radiographic features reliably distinguish the majority of patients with HIV-associated tuberculosis from those who are non-HIV-infected . The remainder, however, may have atypical manifestations and be difficult to diagnose . Six months of chemotherapy with conventional antituberculosis drugs cures most patients, but many die during or after treatment of other AIDS-related complications.

Am J Physiol, 1995 Dec, 269(6 Pt 1), G961 - 73
Posttranscriptional regulation of mRNA levels in rat liver associated with deoxycholic acid feeding; Kren BT et al.; We investigated the effects of bile acid feeding on the mRNA levels and transcriptional activity of genes involved in various facets of hepatic cell function . Rats were maintained for 10 days on standard diet supplemented with combinations of 1 and 0.4% deoxycholic acid and ursodeoxycholic acid . Significant reductions in mRNA levels for liver fatty acid binding protein, albumin, the asialoglycoprotein receptor, connexins 32 and 26, and cytochromes P-450IIB1 and P-450IIE1 were associated with 1% deoxycholic acid feeding . Conversely, the 1% deoxycholic acid-fed animals exhibited increased mRNA levels for cholesterol 7 alpha-hydroxylase, 3-hydroxy-3-methylglutaryl-CoA reductase, multidrug resistance, procollagens, extracellular matrix, protooncogenes, tumor suppressors, and cyclins . The 0.4% deoxycholic acid-fed animals exhibited increased mRNA levels for c-jun, H-ras, p53, cyclins D1 and D3, fibronectin, and procollagens alpha 1(I) and alpha 1(III) . Transcriptional rate changes could not account for the observed changes in steady-state mRNA levels . Ursodeoxycholic acid feeding had no significant effect on gene expression and almost completely inhibited the changes associated with 1% deoxycholic acid when coadministered . The results indicate that dietary ingestion of deoxycholic acid profoundly affects hepatic gene expression in the rat, and regulation occurs primarily at the posttranscriptional level.

Versicherungsmedizin, 1995 Dec 1, 47(6), 212 - 6
{Therapy and prognosis of tuberculosis}; Fischer B et al.; Treatment and prognosis of tuberculosis . Worldwide the so-called short-course chemotherapy has become the standard treatment for tuberculosis . The 6-month regimen consists of isoniazid, rifampin, and pyrazinamid given for 2 month followed by isoniazid and rifampin for 4 month . Ethambutol or streptomycin is added in the first 2 month in patients with advanced disease . This recommendation applies to both HIV-infected and uninfected persons . The major determinant of the outcome of treatment is patient adherence to the drug regimen . In susceptible strains the success rate with the 6-month regimen in sputum conversion is far beyond 90% within the first two month of therapy . The relapse rate after 3 to 5 years is about 0-3% . Multiple-drug-resistant tuberculosis (i.e., resistance to at least two drugs) presents difficult treatment problems . Treatment must be individualized and based on susceptibility studies . For patients with tuberculosis that is resistant to rifampin and isoniazid, even the best available treatment is often unsuccessful . The role of new agents such as the quinolone derivatives and amikacin in the treatment of multidrug-resistant disease is not known, although these drugs are commonly being used in such cases.

Exp Parasitol, 1995 Dec, 81(4), 480 - 90
Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene; Gueiros-Filho FJ et al.; A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine . This phenotype was partially reverted by the calcium channel antagonist verapamil . Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux . Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells . An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

J Histochem Cytochem, 1995 Dec, 43(12), 1187 - 92
Multidrug resistance P-glycoprotein monoclonal antibody JSB-1 crossreacts with pyruvate carboxylase; Rao VV et al.; Multidrug resistance (MDR) is associated with overexpression of a 170 KD plasma membrane P-glycoprotein (P-gp), a putative energy-dependent efflux transporter that reduces intracellular accumulation of chemotherapeutic agents . For detection of P-gp expression in normal and malignant tissues, an MDR1-specific monoclonal antibody (MAb) JSB-1 has been used extensively . In this report we show that MAb JSB-1 crossreacts with a protein of M(r) approximately 130,000 present in rat liver mitochondrial inner membrane/matrix fractions . Peptide mapping and microsequencing identify this protein as pyruvate carboxylase (PC), an abundant mitochondrial enzyme . MAb JSB-1 also crossreacts with purified PC from bovine liver . Under immunoblotting conditions, this crossreactivity is partially abolished by pre-incubation of MAb JSB-1 with a 1000-fold molar excess of MAb C494 epitope-specific peptide (PNTLEGN), indicating that the epitope of MAb JSB-1 may either overlap with or be in close proximity to that of MAb C494 . Immunohistochemical cross-reactivity was also demonstrated in cryosections of human skeletal muscle, a tissue known not to express P-gp . MAb JSB-1 strongly immunostained Type 1 fibers, the subtype known to contain abundant mitochondria . Use of MAb JSB-1 for detection of MDR1 P-gp expression should be approached with caution.

Ann Hematol, 1995 Dec, 71(6), 311 - 2
Aggravation of vincristine-induced neurotoxicity by itraconazole in the treatment of adult ALL; Bohme A et al.; In four of 14 patients with acute lymphoblastic leukemia (ALL) who received induction chemotherapy containing weekly injections of vincristine and simultaneous antifungal prophylaxis with itraconazole, unusually severe and early vincristine-induced neurotoxicity was observed . In these patients (three female, one male) paresthesia and muscle weakness of the upper/lower extremities and paralytic ileus occurred after the first or second vincristine injection . In one patient a laryngeal nerve paresis required mechanical ventilation . The neurotoxic complications were more serious than those seen in a previous series of 460 ALL patients under the identical cytostatic regimen but without itraconazole prophylaxis . The underlying mechanism is unclear . Interaction with the cytochrome P-450 system, reversal of multidrug resistance, and influence of estrogens are to be considered.

Appl Environ Microbiol, 1995 Dec, 61(12), 4223 - 9
Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2; Clennel AM et al.; A 3.5-kb region of plasmid pTF-FC2, which contains a transposon-like element designated Tn5467, has been sequenced, and its biological activity has been investigated . The transposon is bordered by two 38-bp inverted repeat sequences which have sequence identity in 37 of 38 and in 38 of 39 bp to the tnpA distal and tnpA proximal inverted repeats of Tn21, respectively . Within these borders, open reading frames with amino acid similarity to a glutaredoxin-like protein, a MerR regulatory protein, and a multidrug-resistant-membrane transport-like protein were found . The gene for the glutaredoxin-like protein was expressed in Escherichia coli and enabled growth of a glutathione-requiring E . coli trxA gshA mutant on minimal medium and the reduction of methionine sulfoxide to methionine . In addition, there were two regions which, when translated, had homology to 85% of the N-terminal region of the Tn21 resolvase (tnpR) and to 15% of the C terminus of the Tn21 transposase (tnpA) . A region containing res-like sites was located immediately upstream of the partial tnpR gene . Neither the partial transposase nor the resolvase genes of Tn5467 were biologically active, but Tn5467 was transposed and resolved when the Tn21 transposase and resolvase were provided in trans . Tn5467 appears to be a defective transposon which belongs to the Tn21 subgroup of the Tn3 family.

Mol Cell Biol, 1995 Dec, 15(12), 6875 - 83
Expression of an ATP-binding cassette transporter-encoding gene (YOR1) is required for oligomycin resistance in Saccharomyces cerevisiae; Katzmann DJ et al.; Semidominant mutations in the PDR1 or PDR3 gene lead to elevated resistance to cycloheximide and oligomycin . PDR1 and PDR3 have been demonstrated to encode zinc cluster transcription factors . Cycloheximide resistance mediated by PDR1 and PDR3 requires the presence of the PDR5 membrane transporter-encoding gene . However, PDR5 is not required for oligomycin resistance . Here, we isolated a gene that is necessary for PDR1- and PDR3-mediated oligomycin resistance . This locus, designated YOR1, causes a dramatic elevation in oligomycin resistance when present in multiple copies . A yor1 strain exhibits oligomycin hypersensitivity relative to an isogenic wild-type strain . In addition, loss of the YOR1 gene blocks the elevation in oligomycin resistance normally conferred by mutant forms of PDR1 or PDR3 . The YOR1 gene product is predicted to be a member of the ATP-binding cassette transporter family of membrane proteins . Computer alignment indicates that Yor1p shows striking sequence similarity with multidrug resistance-associated protein, Saccharomyces cerevisiae Ycf1p, and the cystic fibrosis transmembrane conductance regulator . Use of a YOR1-lacZ fusion gene indicates that YOR1 expression is responsive to PDR1 and PDR3 . While PDR5 expression is strictly dependent on the presence of PDR1 or PDR3, control of YOR1 expression has a significant PDR1/PDR3-independent component . Taken together, these data indicate that YOR1 provides the link between transcriptional regulation by PDR1 and PDR3 and oligomycin resistance of yeast cells.

Laryngoscope, 1995 Dec, 105(12 Pt 1), 1294 - 9
P-glycoprotein expression in the squamous cell carcinoma of the tongue base; Rabkin D et al.; P-glycoprotein (PGP), which is a product of the multidrug resistance gene (MDR1), is an active transmembrane efflux pump responsible for detoxifying normal cells as well as rendering tumor cells resistant to chemotherapy . It has also been implicated to be expressed by more aggressive cancers . It has not been well described in squamous cell carcinoma of the head and neck . In this investigation, an attempt was made to characterize advanced squamous cell carcinoma of the base of tongue with respect to expression of PGP . Using immunohistochemical techniques two anti-PGP monoclonal antibodies (JSB1 and C494) were used to detect PGP in these lesions, and an attempt was made to correlate levels of PGP staining and various tumor parameters . Usefulness of PGP in predicting survival and time to recurrence was also examined for these advanced lesions . All 33 base of tongue lesions showed staining for PGP with these monoclonal antibodies . This was the first study examining utility of C494 in detecting PGP in squamous cell carcinoma at this site . Increased level of PGP expression was seen in better-differentiated tumors as well as in tumors with diploid DNA . A trend of higher PGP expression and decreased survival emerged . This may represent a true relationship, but inherent heterogeneity of PGP expression within cells cannot be excluded . Both antibodies examined appear to be useful in the investigations of PGP distribution in squamous cell carcinomas of the head and neck sites by immunohistochemical techniques . Prognostic value of the level of PGP expression remains to be seen.

Int Arch Allergy Immunol, 1995 Dec, 108(4), 309 - 12
Vaccination against tuberculosis; Lowrie DB et al.; Recent findings in mice have changed our perception of how protective immunity works in tuberculosis and hold promise for the rapid development of new vaccines . For example, we now know: (1) that a single mycobacterial protein antigen can be sufficient to generate powerful protective immunity, provided that it is presented to the immune system in the right way; (2) that the expression of protection depends on cytotoxic antigen-specific T cells; (3) that the identity of the antigen may be less important than the mode of presentation, and (4) that injection of DNA encoding the antigen (DNA vaccination) is a superior way of raising protective immunity compared to injection of the antigen itself . These advances are timely because there is an urgent need for a new vaccine against tuberculosis . There continue to be about 3 million deaths from tuberculosis every year worldwide and increasingly the causative bacteria are multidrug resistant.

J Biol Chem, 1995 Dec 1, 270(48), 28790 - 6
Effect of ionizing radiation on AP-1 binding activity and basic fibroblast growth factor gene expression in drug-sensitive human breast carcinoma MCF-7 and multidrug-resistant MCF-7/ADR cells; Lee YJ et al.; We studied the effect of ionizing radiation on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in drug-sensitive human breast carcinoma (MCF-7) cells and its drug-resistant variant (MCF-7/ADR) cells . Northern blot and gel mobility shift assays showed that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells . In MCF-7 cells, however, we observed little/no induction of bFGF gene expression and AP-1 binding activity after the stress . Nevertheless, MCF-7 cells transfected with plasmids containing c-jun gene contain high levels of bFGF protein . H-7 (60 micrograms/ml), a potent protein kinase C (PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells . Corroborating this observation, overexpression of PKC alpha induced bFGF gene expression in MCF-7 cells . Taken together, these results suggest that stress-induced bFGF gene expression is mediated through the activation of PKC and AP-1 transcription factors . Differences in the levels of PKC activity and AP-1 binding factors may be responsible for differential expression of bFGF among breast cancer cell lines . Although there are large differences in response to ionizing radiation between MCF-7 and MCF-7/ADR cell lines, we observed no significant differences in radiocytotoxicity between them.

Am J Pathol, 1995 Dec, 147(6), 1633 - 48
Transcription factor and liver-specific mRNA expression in facultative epithelial progenitor cells of liver and pancreas; Dabeva MD et al.; The pattern of mRNA expression for liver-specific proteins and liver-enriched transcription factors was studied in two models of facultative gut epithelial progenitor cells activation: D-galactosamine (GalN)-induced liver injury and dietary copper depletion leading to pancreatic acinar atrophy . After 5 weeks of copper deficiency (CuD), pancreatic acini of Fischer 344 rats underwent atrophy, associated with intense proliferation of small duct-like cells with oval-shaped nuclei . These cells resemble morphologically epithelial progenitor cells of the liver that proliferate after GalN administration . Activated pancreatic epithelial cells express mRNAs for liver-specific genes normally expressed in fetal liver, including alpha-fetoprotein, albumin, alpha-1 antitrypsin, glucose-6-phosphatase, and others, but not genes that are turned on after birth such as serine dehydratase, tyrosine aminotransferase, and multidrug resistance gene-1b . They express mRNAs for liver-enriched transcription factors including HNF-1 alpha, HNF-3 beta and gamma, HNF-4, and members of the CCAAT-enhancer binding protein (C/EBP) family . The only mRNA for a liver-enriched transcription factor not detected in the pancreas of CuD animals was HNF-3 alpha . Expression of HNF-3 alpha, beta, and gamma, and C/EBP-beta mRNA was highly activated in proliferating liver epithelial cells on days 2 and 3 after GalN injury . Increased expression of C/EBP-delta was observed first in the liver on day 1 after GalN administration and in the pancreas at 4 weeks after initiating CuD . We suggest that C/EBP-delta could be involved in the initial activation of epithelial progenitor cells and that HNF-3 alpha, beta, and gamma, and C/EBP-beta might participate in their maturation . We conclude further that pancreatic epithelial progenitor cells undertake differentiation through the hepatocyte lineage but cannot complete the differentiation program within the pancreatic milieu.

Am J Pathol, 1995 Dec, 147(6), 1545 - 52
Membrane transport proteins associated with drug resistance expressed in human melanoma; Schadendorf D et al.; Melanoma cells often display a multidrug-resistant phenotype, but the mechanisms involved are largely unknown . We have studied here the recently identified transport-associated proteins, MRP and LRP, and the well-known drug resistance marker P-glycoprotein using a panel of 16 human melanoma cell lines and 71 benign and malignant melanocytic tissue samples . By flow cytometry and immunohistochemistry, expression of P-glycoprotein was not detectable on the protein level in the 10 cell lines analyzed, although by reverse transcriptase polymerase chain reaction, MDR-1 gene expression was demonstrated in 2 of 10 cell lines . In addition, immunohistology revealed P-glycoprotein expression in only 1 of 71 melanocytic lesions . In contrast, MRP was detected in a subset of melanoma cell lines by reverse transcriptase polymerase chain reaction and immunohistology (4 of 10) . LRP expression was observed in 8 of 10 melanoma cell lines by immunochemistry and in 10 of 10 by reverse transcriptase polymerase chain reaction . Furthermore, MRP was detected immunohistologically in almost 50% of primary and metastatic melanoma specimens, although no significant differences were found between metastases taken before or after chemotherapy . Expression of LRP was detected in a subset of nevi with nevus cells exhibiting up to 25% positive LRP reactivity . In 13 of 21 primary melanomas and 23 of 37 metastases, more than 25% of tumor cells were stained by the LRP-56 monoclonal antibody . Particularly in the group of metastases with more than 50% of LRP-positive cells, 7 of 11 of the metastases had been previously exposed to chemotherapeutic drugs . Although the expression of membrane transport proteins may explain only the chemoresistance toward lipophilic, natural compounds and not resistance against alkylating agents, the lack of P-glycoprotein expression after chemotherapeutic treatment and the significant expression of MRP and LRP in melanoma cells provide first insights into the drug-resistant phenotype in melanoma . Additional studies analyzing the role of MRP and LRP in chemoresistance of melanoma are warranted.

J Virol, 1995 Dec, 69(12), 7541 - 7
Novel retroviral vectors for efficient expression of the multidrug resistance (mdr-1) gene in early hematopoietic cells; Baum C et al.; We present data that retroviral gene expression in early hematopoietic cells is subjected to transcriptional controls similar to those previously described for embryonic stem cells . Transient transfection experiments revealed that both the viral enhancer region in the U3 region of the long terminal repeat as well as a repressor element coincident with the primer binding site of Moloney leukemia viruses are limiting for expression in hematopoietic cells in a differentiation-dependent manner . Within the group of Moloney leukemia virus-related viruses, only the myeloproliferative sarcoma virus showed high enhancer activity in myeloid (including erythroid) cells . In contrast, enhancer regions related to the Friend mink cell focus-forming viruses mediate much higher gene expression levels in both multipotent and lineage-committed myeloid cells . In addition, transcriptional repression related to sequences in the primer binding site of Moloney leukemia virus-derived vectors is also found in early hematopoietic cells and can be overcome by using the corresponding sequences of the murine embryonic stem cell virus . On the basis of these results, two types of novel retroviral hybrid vectors were developed; they combine the U3 regions of either the Friend mink cell focus-forming virus family or the myeloproliferative sarcoma virus with the primer binding site of the murine embryonic stem cell virus . When used to express the human multiple drug resistance gene, these vectors substantially improve protection to cytostatic drugs in transduced hematopoietic cell lines FDC-Pmix, TF-1, and K-562 in comparison with Moloney leukemia virus-derived vectors presently used for the stem cell protection approach in somatic gene therapy.

Blood, 1995 Dec 1, 86(11), 4286 - 94
Anti-B4-blocked ricin synergizes with doxorubicin and etoposide on multidrug-resistant and drug-sensitive tumors; O'Connor R et al.; Anti-B4-blocked ricin (anti-B4-bR) is an immunotoxin directed against CD19-positive cells that is currently being tested in several B-cell leukemia/lymphoma clinical trials . To explore the possibility of using anti-B4-bR in combination with chemotherapy protocols, we investigated the in vitro and in vivo cytotoxic effects of combining it with doxorubicin or etoposide using the lymphoma cell line Namalwa and a P-glycoprotein-expressing cell line, Namalwa/mdr-1, obtained by retroviral infection of Namalwa cells with the mdr-1 gene . Namalwa/mdr-1 cells were slightly more sensitive to anti-B4-bR than Namalwa cells; IC37 values were approximately 4 pmol/L and 8 pmol/L, respectively . When anti-B4-bR was combined simultaneously with doxorubicin or etoposide, additive to supra-additive killing of Namalwa and Namalwa/mdr-1 cells was observed . In xenografts of Na-malwa/mdr-1 cells in severe combined immunodeficiency (SCID) mice, doxorubicin and etoposide at their maximum tolerated doses (3 mg/kg x 3 or 15 mg/kg x 3) showed no therapeutic effect . However, treatment with 5 daily bolus injections of anti-B4-bR (50 micrograms/kg) followed by treatment with doxorubicin or etoposide significantly increased the life span of the mice by 129% and 115%, respectively . After treatment with anti-B4-bR, the Namalwa/mdr-1 population expressed lower levels of P-glycoprotein, and this decrease may account for the synergistic action of the drug combinations . These results suggest that anti-B4-bR could be used to good effect in combination with current treatment regimens and further hint at a promising role for this immunotoxin in treatment of disease at the minimal residual disease stage, where cells may be resistant to chemotherapy.

Biochim Biophys Acta, 1995 Nov 30, 1269(3), 260 - 6
Activation of silent MDR1 genes in revertant cells by fusion with multidrug-resistant cells; Yusa K et al.; We isolated revertant and resistant clones from multidrug-resistant K562/ADM cells and evaluated the expression of P-glycoprotein and the DNA copy number of MDR1 . The 9 revertant clones contained 2- to 26-fold DNA copies of MDR1; however, they expressed an extensively decreased P-glycoprotein compared with K562/ADM, while the 10 multidrug-resistant clones contained 4- to 48-fold DNA copies, and the expression level of P-glycoprotein was dependent on the copy number of MDR1 DNA . The decreased expression of P-glycoprotein in the revertants was not due only to the loss of the copy number of MDR1 DNA . To elucidate the mechanism of P-glycoprotein expression decrease in the revertants, a revertant clone (R1-5) was fused with a multidrug-resistant clone (A2-1) or with a drug-sensitive clone isolated from K562 . Compared with K562 clone, the A2-1 contained 32-fold MDR1 DNA copies and showed 131-fold resistance to Adriamycin . The revertant clone R1-5 contained 26-fold MDR1 DNA copies but expressed only 5% the P-glycoprotein of A2-1 cells and showed only 2-fold resistance to Adriamycin . For selection of intraspecific hybrids, a neomycin-resistant or a blasticidin S-resistant gene was introduced into clones by electroporation of pSV2neo or pSV2bsr . The introduction of these resistant genes did not alter the copy number or expression of MDR1 in the clones . Hybrid cells between R1-5bsr and A2-1neo were found to express 136 +/- 15% of the P-glycoprotein of A2-1 cells evaluated by quantitive flow cytometry . These hybrid cells contained 41- to 48-fold MDR1 copies and showed the multidrug-resistant phenotype, such as decrease of rhodamine123 accumulation and 120- to 210-fold resistance to Adriamycin (compared with K562), indicating that the 'silent' MDR1 genes in the revertant clone R1-5 were activated by cell fusion with an MDR clone . R1-5bsr x K562neo hybrids were found to contain 8- to 11-fold MDR1 copies and there was no increase in P-glycoprotein expression as compared with R1-5.

Cancer Lett, 1995 Nov 27, 98(1), 115 - 20
Gain and loss of hypersensitivity to resistance modifiers in multidrug resistant Chinese hamster ovary cells; Warr JR et al.; Some, but not all, multidrug resistant cell lines exhibit collateral hypersensitivity to resistance modifiers . We have examined the relationship between levels of P-glycoprotein expression and resistance modifier hypersensitivity in Chinese hamster ovary cell lines . We have defined a model system for the gain and loss of such sensitivity which indicates that its presence is not simply proportional to P-glycoprotein levels, but is acquired only above a certain level of P-glycoprotein expression . We also show that previously reported differences in such sensitivity between lines is not attributable to differences in genetic background of the cell lines used for selection of drug resistance.

FEBS Lett, 1995 Nov 27, 376(1-2), 95 - 8
Circumvention of multidrug resistance in neoplastic cells through scavenger receptor mediated drug delivery; Mukhopadhyay A et al.; A conjugate of the antineoplastic drug daunomycin (DNM) with maleylated bovine serum albumin (MBSA-DNM) was taken up with high efficiency by a multidrug resistant variant, JD100, of the murine-macrophage tumour cell line, J774A.1, through the scavenger receptors resulting in cessation of DNA synthesis . In contrast, free DNM at similar concentrations did not affect the incorporation of {3H}thymidine by these cells . These results suggest that receptor-mediated intracellular delivery of antineoplastic drugs could be a viable and new approach for overcoming the problem of multidrug resistance in chemotherapy of neoplastic diseases.

J Immunol Methods, 1995 Nov 16, 187(1), 127 - 37
Establishment and evaluation of MRK16-magnetic cell sorting assays for detecting low expression of multidrug resistance P-glycoprotein using human leukemia cell lines and peripheral blood cells from healthy donors; Okochi E et al.; Two types of magnetic cell sorting assays, termed MRK16-MACS and MRK16-MACS-FACS, have been established to detect low expression level of P-glycoprotein (P-gp) using a monoclonal antibody MRK16, which recognizes a cell surface epitope of P-gp . With K-562 and U-937 cell lines, which are known to express low levels of P-gp and hence routinely used as negative control cell lines in conventional flow cytometry, both assays gave significantly positive reactivities indicating improved specificity and sensitivity of these assays . The findings in the dilution test, where P-gp-positive cells were added to P-gp-negative cells at various ratios, demonstrated that the MRK16-MACS assay is quantitative and capable of detecting small numbers of P-gp-positive cells as few as 2.5% of the total cells tested . Furthermore, specific enrichment of P-gp-expressing cells in magnetic cell sorting assays was verified by reverse transcription-polymerase chain reaction (RT-PCR) analysis and functional assay for P-gp with Rhodamine 123 . The availability of such magnetic cell sorting assays may offer an approach to quantitate low level of P-gp expression.

Cancer Res, 1995 Nov 15, 55(22), 5342 - 7
Expression of multidrug resistance-associated protein in NIH/3T3 cells confers multidrug resistance associated with increased drug efflux and altered intracellular drug distribution; Breuninger LM et al.; Multidrug resistance is a major obstacle to cancer treatment . Using an expression cDNA library transfer approach to elucidating the molecular basis of non-P-glycoprotein-mediated multidrug resistance, we previously established that expression of multidrug resistance protein (MRP), an ATP-binding cassette superfamily transporter, confers multidrug resistance (G . D . Kruh et al., Cancer Res., 54: 1649-1652, 1994) . In the present study, we generated NIH/3T3 MRP transfectants without using chemotherapeutic drugs to facilitate the pharmacological analysis of the MRP phenotype . MRP transfectants displayed increased resistance to several lipophilic drugs, including doxorubicin, daunorubicin, etoposide, actinomycin D, vincristine, and vinblastine . However, increased resistance was not observed for Taxol, a drug for which transfection of MDR1 confers high levels of resistance . Verapamil increased the sensitivity of MRP transfectants relative to control transfectants, but reversal was incomplete for doxorubicin and etoposide, the drugs for which MRP conferred the highest resistance levels . For the latter two drugs, MRP transfectants, which were approximately 8- and approximately 10-fold more sensitive than control cells in the absence of verapamil, exhibited 3.8- and 3.3-fold relative sensitization with 10 microM verapamil, respectively, but remained approximately 2 and approximately 3-fold more resistant than control cells . Analysis of drug kinetics using radiolabeled daunorubicin revealed decreased accumulation and increased efflux in MRP transfectants . Confocal microscopic analysis of intracellular daunorubicin in MRP transfectants was consistent with reduced intracellular drug concentrations, and also revealed an altered pattern of intracellular drug distribution characterized by the initial accumulation of drug in a perinuclear location, followed by the development of a punctate pattern of drug scattered throughout the cytoplasm . This pattern was suggestive of a process of drug sequestration, possibly followed by vesicle transport . Both increased drug efflux and perinuclear drug accumulation are consistent with the reported localization of MRP in plasma and cytosolic membranes (N . Krishnamachary and M . S . Center, Cancer Res., 53: 3658-3663, 1993; M . J . Flens et al., Cancer Res., 54: 4557-4563, 1994) . These results thus indicate that the drug specificity of MRP is quite similar to that of MDR1, but also suggest potential differences in Taxol specificity and the level of verapamil sensitivity . In addition, these results indicate that MRP functions to extrude drug from the cell, but additionally suggest the intriguing possibility that drug sequestration contributes to drug resistance by protecting cellular targets and/or contributing to drug efflux.

Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 602 - 9
Drug-stimulated ATPase activity of a deletion mutant of the human multidrug-resistance protein (MDR1); Welker E et al.; The baculovirus-insect cell system has been used for the functional expression of the human multidrug resistance protein (MDR1) and a mutant MDR1 variant lacking a twenty amino acid segment from the first extracellular loop (delta aa78-97 MDR1) . Both MDR1 proteins were found to be correctly inserted into the insect cell membrane as indicated by their interaction with MRK 16 antibody . The removal of the 78-97 segment from the first extracellular loop dramatically altered drug-stimulated ATPase activity . Rhodamine 123 or vinblastine were not able to stimulate the mutant protein and Calcein AM had also little effect . In contrast, verapamil increased the ATPase activity of the mutant almost to the same maximal level as that of the wild type . However, the verapamil concentration needed for the half maximal stimulation of the ATPase activity was found to be about hundred times higher than that for the wild type MDR1 . These results indicate that a partial deletion of an extracellular loop modulates the affinity of MDR1 for its transportable substrates in a variable fashion.

Br J Hosp Med, 1995 Nov 15-Dec 12, 54(10), 494 - 500
Drug-resistant tuberculosis: mechanisms and management; Hayward CM et al.; Infection with Mycobacterium tuberculosis remains a major global health problem and the recent outbreaks of multidrug resistant (MDR) tuberculosis have been a major cause for concern . An accurate picture of the extent of this problem is not possible because only a limited number of countries have reliable surveillance programmes . However, the experience in the USA reinforces the need for strict adherence to standard public health measures and good clinical practices to minimise the impact of MDR tuberculosis in the human immunodeficiency virus era.

J Biol Chem, 1995 Nov 10, 270(45), 26956 - 61
Both P-glycoprotein nucleotide-binding sites are catalytically active; Urbatsch IL et al.; The technique of vanadate trapping of nucleotide was used to study catalytic sites of P-glycoprotein (Pgp) in plasma membranes from multidrug-resistant Chinese hamster ovary cells . Vanadate trapping of Mg- or Co-8-azido-nucleotide (1 mol/mol of Pgp) caused complete inhibition of Pgp ATPase activity, with reactivation rates at 37 degrees C of 1.4 x 10(-3) s-1 (t1/2 = 8 min) or 3.3 x 10(-4) s-1 (t1/2 = 35 min), respectively . UV irradiation of the inhibited Pgp yielded permanent inactivation of ATPase activity and specific photolabeling of Pgp . Mild trypsin digestion showed that the two nucleotide sites were labeled in equal proportion . The results show that both nucleotide sites in Pgp are capable of nucleotide hydrolysis, that vanadate trapping of nucleotide at either site completely prevents hydrolysis at both sites, and that vanadate trapping of nucleotide in the N- or C-terminal nucleotide sites occurs non-selectively . A minimal scheme is presented to explain inhibition by vanadate trapping of nucleotide and to describe the normal catalytic pathway . The inhibited Pgp-Mg-nucleotide.vanadate complex is probably an analog of the catalytic transition state, implying that when one nucleotide site assumes the catalytic transition state conformation the other site cannot do so and suggesting that the two sites may alternate in catalysis.

Tidsskr Nor Laegeforen, 1995 Nov 10, 115(27), 3361 - 4
{Tuberculosis among AIDS patients in Ullev}l hospital}; Berild D; Records of patients with concomitant HIV infection and human tuberculosis were analysed . Nine out of 232 AIDS patients (4%) developed human tuberculosis with a preponderance (6/9) of immigrants from sub-Saharan Africa . In three patients the diagnosis was delayed because of atypical manifestations of the disease and lack of typical chest X-ray findings . Tuberculin skin tests were positive in only three patients, and became negative in one patient who developed two episodes of tuberculosis . All the patients who complied with the conventional triple anti-tuberculosis regimen responded well, and no multidrug-resistant tuberculosis was observed.

Biochem Pharmacol, 1995 Nov 9, 50(10), 1725 - 9
Localisation of the multidrug resistance-associated protein, MRP, in resistant large-cell lung tumour cells; Barrand MA et al.; The drug transport protein, P-glycoprotein, confers multidrug resistance (MDR) by expelling drugs across the cell surface . The structurally similar multidrug resistance-associated protein, or MRP, is also involved with drug efflux . In MDR variants of the human lung tumour cell line COR-L23 that overexpress MRP, there are also changes in intracellular drug distribution . To ascertain whether MRP could be involved in either process, experiments were performed to identify where MRP was located in these cells . Following separation of membranes by sucrose gradient centrifugation, MRP was found predominantly in the lighter membrane fractions containing plasma membrane enzyme activity . Immunofluorescent staining with a monoclonal antibody raised against MRP confirmed that MRP is present at the cell surface of these MDR lung tumour cells.

Biochem Pharmacol, 1995 Nov 9, 50(10), 1673 - 83
Susceptibility of idarubicin, daunorubicin, and their C-13 alcohol metabolites to transport-mediated multidrug resistance; Ross DD et al.; The intracellular pharmacokinetics and cytotoxicity of idarubicin (IDA), daunorubicin (DNR), and their corresponding C-13 alcohol metabolites, idarubicinol (IDAol) and daunorubicinol (DNRol), were studied in drug-sensitive HL-60/W human leukemia cells, and in two multidrug-resistant (MDR) sublines, HL-60/Vinc (overexpress P-glycoprotein, Pgp) and HL-60/Adr (overexpress multidrug resistance-associated protein, MRP) . Intracellular drug accumulation (1 micrograms/mL) and retention were measured by flow cytometry . Mean intracellular steady-state concentration (Css, fluorescence units/cell) and area under the intracellular drug concentration x time curve (AUC, Fl.U/cell.min) were calculated . Relative to the values for the respective drugs in HL-60/W cells, the Css and AUC of IDA were much higher than those of DNR in the MDR cell lines, with Css and AUC of IDAol intermediate between IDA and DNR . In the MDR cell lines, the MDR modulator cyclosporine A (CsA), in concentrations of 0.3 to 30 mumol/L, caused minimal effects on 3-hr IDA accumulation, intermediate enhancement of IDAol accumulation, and greatest enhancement of DNR accumulation . The MDR cell lines were much less resistant to IDA (3- to 16-fold) than to DNR (65- to 117-fold) . This difference was not the result of IDA being more potent than DNR, since the sensitivity of HL-60/W cells to IDA differed from their sensitivity to DNR by < 2-fold . The cellular pharmacokinetics and cytotoxicity of IDA in MDR human breast carcinoma cells MCF-7/AdrVp, which overexpress the putative MDR transporter P-95, were far superior to those of DNR, and were comparable to these parameters for IDA in parental MCF-7/W cells . These studies demonstrate that the cellular pharmacology and cytotoxicity of IDA in MDR cell lines that overexpress MRP, Pgp, or P-95 are more advantageous than those of DNR, suggesting that IDA is less susceptible to the transport-mediated MDR mechanism manifested . IDA is not completely invulnerable to MDR, however, since the MDR sublines studied did display a demonstrable level of resistance to IDA, compared with their drug-sensitive counterparts . IDAol, the major plasma metabolite of IDA, demonstrated behavior intermediate between the MDR-susceptible drug DNR and its parent compound, suggesting that its cytotoxic action is subject to transport-mediated cellular defenses.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1995 Nov 3, 270(44), 26639 - 48
Partial inhibition of multidrug resistance by safingol is independent of modulation of P-glycoprotein substrate activities and correlated with inhibition of protein kinase C; Sachs CW et al.; Safingol is a lysosphingolipid protein kinase C (PKC) inhibitor that competitively interacts at the regulatory phorbol binding domain of PKC . We investigated the effects of safingol on antineoplastic drug sensitivity and PKC activity of MCF-7 tumor cell lines . Safingol treatment of 32P-labeled MCF-7 WT and MCF-7 DOXR cells inhibited phosphorylation of the myristoylated alanine-rich protein kinase C substrate in both cell lines, suggesting inhibition of cellular PKC . However, only in MCF-7 DOXR cells did safingol treatment increase accumulation of {3H}vinblastine and enhance toxicity of Vinca alkaloids and anthracyclines . Drug accumulation changes in MCF-7 DOXR cells treated with safingol were accompanied by inhibition of basal and phorbol 12,13-dibutyrate-stimulated phosphorylation of P-glycoprotein (P-gp) . Expression of P-gp and levels of mdr1 message in MCF-7 DOXR cells were not altered by safingol treatment alone or in combination with vinblastine . Treatment of MCF-7 DOXR cell membranes with safingol did not inhibit {3H}vinblastine binding or {3H}azidopine photoaffinity labeling of P-gp . Furthermore, safingol did not stimulate P-gp ATPase activity in membranes prepared from MCF-7 DOXR cells . We conclude that enhanced drug accumulation and sensitivity in MCF-7 DOXR cells treated with safingol are correlated with inhibition of PKC rather than competitive interference with P-gp drug binding through direct interaction with P-glycoprotein.

J Biol Chem, 1995 Nov 3, 270(44), 26411 - 8
Association of sorcin with the cardiac ryanodine receptor; Meyers MB et al.; Sorcin is a 22-kDa calcium-binding protein initially identified in multidrug-resistant cells; however, its patterns of expression and function in normal tissues are unknown . Here we demonstrate that sorcin is widely distributed in rodent tissues, including the heart, where it was localized by immunoelectron microscopy to the sarcoplasmic reticulum . A > 500-kDa protein band immunoprecipitated from cardiac myocytes by sorcin antiserum was indistinguishable in size on gels from the 565-kDa ryanodine receptor/calcium release channel recognized by ryanodine receptor-specific antibody . Association of sorcin with a ryanodine receptor complex was confirmed by complementary co-immunoprecipitations of sorcin with the receptor antibody . Forced expression of sorcin in ryanodine receptor-negative Chinese hamster lung fibroblasts resulted in accumulation of the predicted 22-kDa protein as well as the unexpected appearance of ryanodine receptor protein . In contrast to the parental host fibroblasts, sorcin transfectants displayed a rapid and transient rise in intracellular calcium in response to caffeine, suggesting organization of the accumulated ryanodine receptor protein into functional calcium release channels . These data demonstrate an interaction between sorcin and the ryanodine receptor and suggest a role for sorcin in modulation of calcium release channel activity, perhaps by stabilizing the channel protein.

Pathol Res Pract, 1995 Nov, 191(11), 1122 - 32
Proliferative activity in breast carcinoma evaluated by BrdU and PCNA . Correlation with expression of p53, c-erbB-2, estrogen receptor and P-glycoprotein; Moriki T et al.; The proliferative activity in 35 cases of breast carcinoma was evaluated by bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA) and was compared with benign breast lesion . Overexpression of p53 and c-erbB-2 oncoprotein, presence of estrogen receptor (ER) and cellular localization of multidrug resistance gene product P-glycoprotein (P-gp) were immunohistochemically examined to investigate the relation with the proliferative activity and clinicopathologic characteristics . The mean BrdU labeling index (LI) was 12.6% and PCNA labeling rate (LR) was 33.5% in breast carcinomas, and good correlation was found between them . The proliferative activity of breast carcinomas was significantly higher than that of benign lesions . The BrdU LI correlated positively with tumor size, histologic grade, TNM stage and p53 immunoreactivity, and negatively with the presence of ER . PCNA LR correlated with histologic grade and expression of p53 . p53 protein was demonstrated in 43% of the breast carcinomas and correlated with proliferative activity . The extent of p53 immunoreactivity on carcinoma cells was also related to BrdU LI . c-erbB-2 oncoprotein was demonstrated in 51% of the breast carcinomas and correlated with histologic grade . ER was found in 34% of the breast carcinomas and correlated negatively with histologic grade, lymph node metastasis and TNM stage . P-gp was observed in 49% of the breast carcinomas and no correlation was found with clinicopathologic characteristics . None of the benign lesions expressed p53 protein, c-erbB-2 oncoprotein and P-gp . BrdU is a reliable standard and a more useful tool for the evaluation of proliferative activity of breast tumors . High proliferative activity, overexpression of p53 protein and the absence of ER are considered as a high grade malignancy of breast carcinoma . Expression of c-erbB-2 oncoprotein and P-gp may be related to malignant transformation of breast tumors.

Int J Urol, 1995 Nov, 2(5), 309 - 15
Comparison of multidrug resistance gene expression levels with malignant potentials and influence of chemotherapy in urothelial cancers; Kakehi Y et al.; BACKGROUND: We sought to determine how often P-glycoprotein is involved in the drug-resistance of urothelial cancer, and whether MDR1 gene expression is correlated with tumor grade, invasiveness, or metastasis . METHODS: Forty-two tumor specimens and two normal bladder mucosal samples obtained from 34 urothelial cancer patients were analyzed . Reverse-transcription and polymerase chain reaction were conducted . MDR1 mRNA levels were determined by measuring the relative ratio of the MDR1 to beta-2-microglobulin (b2 m) mRNA specific PCR products . RESULTS: The MDR1/b2 m in two normal urothelial samples were 0.044 and 0.045 . For untreated primary tumors, levels of MDR1 gene expression in 46% tumor samples were less than that of normal urothelium, while 27% showed expression levels with a MDR1/b2 m ratio more than 0.1 . There was no statistical correlation between MDR1 mRNA level and tumor grade, stage, or metastatic status . There was higher MDR1 gene expression in two lymph node metastasis specimens and almost equal expressions in two more . There was no significant difference in the mean MDR1/b2 m ratio between postchemotherapy and untreated tumors . A remarkable elevation of the MDR1 mRNA level (15 times greater than prechemotherapy) was found in one tumor; mRNA levels of the multidrug resistance-associated protein (MRP) gene, glutathione S-transferase pi (GST-pi) gene or DNA topoisomerase II (topo II) gene did not increase . CONCLUSIONS: It is still unclear whether the MDR1 gene expression in urothelial tumor cells is inducible by the current combination chemotherapy regimens . RT-PCR quantitation is useful for determining the expression level of MDR1 gene in urothelial cancer.

Pediatr Nurs, 1995 Nov-Dec, 21(6), 566 - 72
Tuberculosis and multi-drug resistant tuberculosis in children; Peloquin CA et al.; Infection and disease caused by Mycobacterium tuberculosis remain a hugh global problem, and are not well controlled in several areas within the United States . Co-infection with the human immunodeficiency virus (HIV) and immigration from areas with high rates of tuberculosis contribute to the problem in the United States . Organisms resistant to the two main drugs, isoniazid and rifampin, known as multidrug-resistant M . tuberculosis or MDR-TB, present serious therapeutic challenges . Strategies for the management of such cases are presented.

Anticancer Res, 1995 Nov-Dec, 15(6B), 2469 - 77
Adriamycin resistance modulation induced by lonidamine in human breast cancer cells; Zupi G et al.; The effect of Lonidamine (LND), an energolytic chemosensitizing agent, on the MDR (multidrug resistant) phenotype of a human breast cancer cell line (MCF-7) has been studied . The intracellular adriamycin (ADR) accumulation and distribution, the plasma membrane potential and the P170 glycoprotein phosphorylation, have been analysed after LND treatment . The analysis of the subcellular localisation of ADR in both wild type and resistant MCF-7 cells treated with ADR or ADR + LND revealed that LND induced an ADR intracellular redistribution in both cell lines . MCF-7 ADR resistant cells exposed to LND (50 micrograms/ml) showed a change in the electrical charges distribution across the plasma membrane and a time-dependent reduction of P170 phosphorylation (70% at 24 hr) . These effects were associated with a marked increase in intracellular ADR accumulation in resistant cells.

Anticancer Res, 1995 Nov-Dec, 15(6B), 2461 - 8
Immunocytochemical observation of multidrug resistance (MDR) p170 glycoprotein expression in human osteosarcoma cells . The clinical significance of MDR protein overexpression; Bodey B et al.; Resistance to several cytotoxic agents (MultiDrug Resistance MDR), including anthracyclines, vinca alkaloids and epipodophylline derivatives can occur in human osteosarcoma (OS) cells, detected by the overexpression of a 170 kD glycoprotein (p170), as a result of increased expression of the MDR gene (mdr1) . The p170 glycoprotein in normal cells is a membrane transport system protein and its quantitative increase results in increased drug efflux and decreased intracellular drug concentration . Normal renal epithelial cells express p170 as a function of their secretory duties therefore this human tissue was used as a positive tissue control in our immunocytochemical study . This partially retrospective immunocytochemical study was carried out on routine, 10% neutral formalin fixed, decalcified, paraffin embedded, tissue sections of 43 OSs, treated between 1981 and 1993 at the Orthopaedic Hospital of Los Angeles . The immunoperoxidase antigen detection protocol, submitted by Hsu et al (1981) was employed . The search for p170 was carried out with three newly developed monoclonal antibodies (MoABs) (JSB-I, C-219 and C-494, from Signet Laboratories, Dedham, MA 02026) . The initial expression of MDR was not detectable in seven OSs . 36/43 OSs expressed p170 on/in their cells . Heterogenous cellular microenvironment and various grades of differentiation features were also determined in the examined OSs . In 17/43 OS cases presence of intensive staining (probably overexpression) of p170 protein was registered . The 43 OSs exhibited different staining patterns with each MoAB . MoAB JSB-I reacted with a transmembranic antigen epitope . The long incubation time with C-219 resulted in heterogeneous cytoplasmic staining . MoAB C-494 also produced an intensive staining mainly localized on the cell membrane of the OS cells . These statistically significant immunocytochemical results suggest a direct correlation between the quantitative presence of p170 glycoprotein in human OS cells and the efficacy of the employed chemotherapy . Future observations employing the in situ hybridization technique will allow the quantitative measurement of the primary or secondary presence of MDR glycoprotein in human OS cells.

Surg Neurol, 1995 Nov, 44(5), 462 - 8; discussion 468-70
Investigation of chemoresistance-related genes mRNA expression for selecting anticancer agents in successful adjuvant chemotherapy for a case of recurrent glioblastoma; Nagane M et al.; BACKGROUND: Glioblastoma multiforme represents one of the most malignant forms of primary intracranial tumors, often intractable to multimodality of treatment including chemotherapy . The unsatisfactory results of chemotherapy are chiefly attributed to chemoresistance . Since various molecules that could confer drug resistance have been elucidated, screening of the amount of such molecules in the tumor cells could provide possibilities for predicting their chemoresistance beforehand and help select more effective drugs . METHODS: We present a 45-year-old woman with recurrent glioblastoma multiforme in the cerebellum and invading the brain stem, treated successfully by postoperative chemotherapy . In this patient, anticancer drugs were determined by measurements of mRNA expression of chemoresistance-related genes, such as O6-methylguanine-DNA methyltransferase (MGMT), mdr1, glutathione S-transferase (GST)-pi, and metallothionein (MT) in the resected tumor . RESULTS: Northern blot analysis demonstrated the moderate mRNA level of MGMT, a major molecule causing ACNU (1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride) resistance . On the other hand, expression levels of mdr1 which codes the P-glycoprotein responsible for multidrug resistance, and GST-pi, a detoxification enzyme, were low . Transcript of MT, another thiol containing molecule for cellular detoxification possibly associated with cisdiamminedichloroplatinum(II) (CDDP) resistance, was only faintly detectable . Postoperatively, the patient was treated initially with intravenous administration of ACNU and etoposide (VP16), resulting in a minor response of tumor regression . For maintenance therapy, we changed ACNU to CDDP according to the findings of the Northern blot analysis . Consequently, the residual tumor showed a marked response and almost disappeared after two courses of systemic chemotherapy with CDDP and VP16 . CONCLUSIONS: The successful tumor regression in this case suggests that Northern blot analysis on expression of these chemoresistance-related genes in tumor tissues could provide beneficial information for determination of optimal anticancer agents to improve the efficacy of chemotherapy.

Eur J Cell Biol, 1995 Nov, 68(3), 226 - 39
Nuclear immunolocalization of P-glycoprotein in multidrug-resistant cell lines showing similar mechanisms of doxorubicin distribution; Baldini N et al.; The MDR1 gene product P-glycoprotein is a plasma membrane efflux pump which is responsible for multiple drug resistance of cancer cells . Although the ability of multidrug-resistant cells to exclude drugs from the nucleus is a distinctive and possibly the main mechanism for resistance against a number of drugs, including doxorubicin, this phenomenon is not entirely understood . In this paper, the relationship between doxorubicin subcellular distribution and P-glycoprotein activity at different cell sites has been investigated by different techniques . Cytofluorometry and confocal microscopy were used to study doxorubicin subcellular distribution in U-2 OS human osteosarcoma cells and in the multidrug-resistant variant U-2 OS/DX580 . Stable levels of doxorubicin accumulation were found in the nuclei of sensitive cells, whereas the absence of detectable levels of drug in the nuclei of resistant cells could be attributed to an energy-dependent mechanism . Moreover, in resistant cells, inhibition of P-glycoprotein activity was able to induce drug accumulation in the nuclei of resistant cells and to achieve cytotoxic effects comparable to those observed in sensitive cells . Similar results were also found in isolated nuclei from U-2 OS/DX580 cells . The expression of P-glycoprotein in U-2 OS/DX580 and in two other multidrug-resistant cell lines (SW948-R-300 and LoVo-R-100) was investigated by confocal microscopy and immunoelectron microscopy, by using a panel of monoclonal antibodies directed against this protein . Higher levels of P-glycoprotein expression, not only in the plasma membrane and inside the cytoplasm, but also in the nucleus, were found in U-2 OS/DX580 and in LoVo-R-100 multidrug-resistant cells compared to their corresponding sensitive cells . SW948-R-300 cells, featuring increased amounts of MDR1 mRNA but lacking P-glycoprotein expression at the cell surface, showed a higher P-glycoprotein immunolabeling only in the nucleus and in the cytoplasm . The localization of P-glycoprotein in the nucleus of multidrug-resistant cells was confirmed also by studies on isolated nuclei and nuclear matrices, and by Western blot analysis on total cell and isolated nuclear extracts . These findings, suggesting the possible involvement of nuclear P-glycoprotein in the regulation of subcellular doxorubicin distribution in multidrug-resistant cells, open new insights on the mechanisms of P-glycoprotein-mediated resistance to anticancer drugs.

Leuk Lymphoma, 1995 Nov, 19(5-6), 431 - 6
Expression of MDR-1/P-glycoprotein in childhood acute megakaryoblastic leukemia cells; Ota S et al.; Multidrug resistance is a major clinical problem in chemotherapy of malignant disease . Acute megakaryoblastic leukemia (AMKL) is a rare form of childhood leukemia, and is often more resistant to many anticancer chemotherapeutic drugs compared to other types of childhood leukemia . There have been reports of the increased expression in hematologic malignancy of multidrug resistant (mdr-1) gene, which encodes for a transmembrane glycoprotein P-glycoprotein that acts as an efflux pump for structurally unrelated chemotherapeutic drugs . We investigated the malignant cells of 15 newly diagnosed childhood AMKL patients by immunocytochemical analysis and found P-glycoprotein expression in all samples from these patients . RNA prepared from five patients at the time of presentation confirmed the expression of mdr-1 specific message in all cases by Northern blot analysis . These results imply that malignant cells from all childhood AMKL might express the mdr-1/P-glycoprotein.

Clin Infect Dis, 1995 Nov, 21(5), 1245 - 52
Predictors and outcome of multidrug-resistant tuberculosis; Salomon N et al.; We identify early predictors of multidrug-resistant tuberculosis and describe improved clinical outcomes, including survival, for patients with human immunodeficiency virus (HIV)-related multidrug-resistant tuberculosis (MDR-TB) when they are prospectively identified and receive treatment under direct observation . Analysis by means of a Cox proportional hazards model revealed that failure to defervesce while receiving a standard four-drug antituberculous regimen was independently associated with multidrug resistance (P = .004) . When patients with HIV-related MDR-TB were prospectively identified and treated with at least two agents that were active in vitro, 100% bacteriologic conversion and improved survival (> or = 4 months for 88% of patients and > or = 1 year for 59% of patients) were observed . For patients with HIV-related tuberculosis, poorer survival was associated with a CD4+ lymphocyte count of < 25 mm3 (P = .03); multidrug resistance was not a predictor of poor outcome (P = .82) . These data suggest that patients with prolonged fever who are receiving antituberculous therapy may be an appropriate subgroup to target for broader empirical therapy . The findings also demonstrate that improved outcomes can be achieved with HIV-related MDR-TB when patients are prospectively identified and treated with agents that are active in vitro.

Clin Infect Dis, 1995 Nov, 21(5), 1238 - 44
Improved outcomes for patients with multidrug-resistant tuberculosis; Turett GS et al.; We conducted a retrospective study of patients with culture-confirmed multidrug-resistant tuberculosis (MDR-TB) at Bronx-Lebanon Hospital Center (South Bronx, NY) to determine what factors affected clinical and microbiological responses and survival . For the 38 patients with MDR-TB, reporting of first-line drug susceptibilities was relatively rapid (median time, 30 days) . Thirty-four patients (89%) were infected with human immunodeficiency virus (HIV), and initial and overall response rates were 59% and 50%, respectively; the median survival was 315 days; and 50% of these patients died of tuberculosis . Bivariate analysis revealed that the following factors had a positive impact on response and survival: receiving > or = 2 consecutive weeks of appropriate therapy with at least two drugs to which the isolate was susceptible in vitro; starting appropriate therapy within 4 weeks of the diagnosis; and having tuberculosis that was limited to the lungs . Multivariate analysis revealed that the only variable associated with response was receipt of appropriate therapy for > or = 2 consecutive weeks . In contrast to findings in the published literature, our results indicate the outcome of MDR-TB can be improved, particularly for severely immunosuppressed HIV-infected patients . Rapid reporting of susceptibilities and prompt initiation and continuation of appropriate antituberculous therapy improved response and survival.

Photochem Photobiol, 1995 Nov, 62(5), 875 - 81
Sites of photodamage in vivo and in vitro by a cationic porphyrin; Kessel D et al.; Localization and photodynamic efficacy of a monocationic porphyrin (MCP) were assessed using murine leukemia cells in culture . This sensitizer localized at surface membrane loci and catalyzed selective photodamage to membrane structures . Although both cationic and hydrophobic, this porphyrin was not recognized by the multidrug transporter, which excludes many cationic agents from cells that express multidrug resistance . Photodynamic studies with the murine radiation-induced fibrosarcoma tumor model indicated moderate photosensitization of neoplastic lesions in vivo at 3 h, but not at 24 h after sensitizer administration . Pharmacokinetic studies indicate that plasma levels, not tissue levels were the major determinant of photodynamic therapy (PDT) response . Consistent with this observation, vascular damage and disturbances of tissue perfusion followed PDT . These effects were more pronounced in tumor-bearing skin than in normal skin . The therapeutic response to MCP appeared to be related mainly to secondary, probably vascular, effects.

Jpn J Cancer Res, 1995 Nov, 86(11), 1112 - 8
Non-P-glycoprotein-mediated atypical multidrug resistance in a human bladder cancer cell line; Naito S et al.; A human bladder cancer cell line resistant to adriamycin (ADM), T24/ADM9 has been established in vitro by exposing T24 parent cells to progressively higher concentrations of the drug over a period of 12 months . The T24/ADM9 cells were found to be 9 times more resistant to ADM than the T24 parent, and showed various degrees of cross-resistance to an ADM derivative, vinea alkaloids and a DNA topoisomerase II (Topo II)-targeting agent, etoposide . No significant differences was observed in the cellular accumulation of ADM between the T24/ADM9 and T24 parent cells . A Northern blot analysis showed an overexpression of multidrug resistance-associated protein (MRP) mRNA, but no overexpression of multidrug resistance-1 (MDR1) mRNA was observed in the T24/ADM9 cells . A flow cytometric analysis showed that the MDR1 gene product, P-glycoprotein (Pgp), is not expressed on the T24/ADM9 cells . T24/ADM9 showed approximately the parental level of DNA Topo II catalytic activity . In Western blot and Northern blot analyses, however, the cellular level of DNA topo II was apparently much lower in T24/ADM9 than in the T24 parent . Thus, these results suggest that a decreased cellular level of DNA Topo II and an overexpression of MRP gene may be responsible for the expression of an MDR phenotype in the T24/ADM9 cells and that such non-Pgp-mediated, atypical MDR may develop in bladder cancer treated with chemotherapy including ADM.

Eur J Cancer, 1995 Nov, 31A(12), 1998 - 2002
Analysis of P-glycoprotein expression in osteosarcoma; Serra M et al.; Current treatment of high-grade osteosarcoma combines surgical removal of the lesion with chemotherapy . In this study we evaluated whether the expression of P-glycoprotein, a protein closely associated with multidrug resistance, may be helpful in identifying the patients whose tumours will be further resistant to specific agents . By using multidrug-resistant osteosarcoma cell lines as standards, the expression of P-glycoprotein was evaluated in 105 cases of primary and metastatic osteosarcoma by semiquantitative immunofluorescence . Overexpression of the protein was shown in 23% of primary and in 50% of metastatic lesions . In 38 cases, homogeneously treated and followed-up for at least 24 months, overexpression of P-glycoprotein appeared to be associated with a higher relapse rate and with a trend toward a worse outcome . These data support the role of P-glycoprotein in the response to chemotherapy and its involvement in the determination of the outcome of osteosarcoma patients.

Br J Haematol, 1995 Nov, 91(3), 652 - 7
Decreased potency of MDR-modulators under serum conditions determined by a functional assay; Ludescher C et al.; A variety of agents are capable of overcoming P-glycoprotein-mediated multidrug resistance (MDR) in vitro . However, the clinical potential of these compounds is often limited due to high plasma protein binding . We compared the efficacy of several MDR-reversing compounds in serum-free culture medium and under serum conditions by means of a functional assay . Using flow cytometry the efflux of the fluorescent dye rhodamine 123 (Rh123) was measured from normal peripheral blood CD8+ T-lymphocytes which express low levels of P-glycoprotein . Inhibition of Rh123 efflux by R-verapamil, dexnigludipine-HCl, cyclosporin A, SDZ PSC833 and the protein kinase C (PKC) inhibitor CGP 41251 was determined in serum-free medium and in serum at concentrations from 0.1 to 50 mumol/l . With the exception of SDZ PSC833 all MDR modulators showed an insufficient or suboptimal modulation of P-glycoprotein under serum conditions at concentrations achievable in vivo . The highest potency under serum conditions demonstrated SDZ PSC833: even at a concentration of 0.5 mumol/l a sufficient inhibitory effect was observed . Subsequently this approach was applied to patients suffering from B-cell chronic lymphocytic leukaemia (B-CLL; n = 3) and acute myeloid leukaemia (AML; n = 2) which were positive in the Rh123 efflux assay . As for normal CD8+ T-lymphocytes, much higher drug concentrations were required under serum conditions to effectively inhibit Rh123 efflux from the leukaemic cells . Thus the interpretation of results of clinical 'modulator' trials should consider the decreased bioavailability of MDR-reversing agents.

Thorax, 1995 Nov, 50(11), 1147 - 50
Mycobacterial infection in HIV seropositive and seronegative populations, 1987-93; Taylor IK et al.; BACKGROUND--Although the causes of the worldwide resurgence of tuberculosis are multifactorial, the HIV epidemic is believed to have had a central role . Control is further threatened by the emergence of multidrug-resistant tuberculosis . METHODS--A retrospective evaluation was undertaken of trends in pulmonary and extrapulmonary culture positive mycobacterial pathology, and the prevalence of drug-resistant tuberculosis in both HIV seropositive and, presumptively, HIV seronegative patients receiving their clinical care at St Mary's Hospital, London . Five hundred and thirty eight patients (188 of whom were known to be HIV seropositive) with positive mycobacterial isolates between January 1987 and March 1993 were identified from laboratory records . These were cross referenced with drug surveillance records . RESULTS--Overall, between 1987 and 1992 there was a progressive 3.5 fold increase in positive mycobacterial isolates and a 2.5 fold increase in patients with proven mycobacterial infection . This increase was greater within the HIV seropositive population . A total of 663 positive mycobacterial isolates was evaluated; the major pathogen identified was Mycobacterium tuberculosis (379 isolates, 57%) . Three hundred and fourteen patients were diagnosed as having M tuberculosis, 49 of whom were HIV seropositive . M tuberculosis was predominantly isolated from the lung . Of 358 positive cultures for M tuberculosis (68 HIV seropositive, 290 presumptively HIV seronegative), only 27 isolates (7.6%), almost exclusively derived from presumed HIV seronegative patients, were resistant to either isoniazid, rifampicin, or both drugs together . No increases in drug-resistant isolates were observed over this period . CONCLUSIONS--There has been a considerable increase in the incidence of tuberculosis in both HIV seronegative and seropositive populations during the study period . The emergence of drug-resistant tuberculosis was not observed.

Curr Opin Oncol, 1995 Nov, 7(6), 532 - 40
Multidrug resistance proteins and other drug transport-related resistance to natural product agents; Broxterman HJ et al.; The term multidrug resistance is defined in this article as cellular resistance to anticancer agents due to a decreased concentration of active drug at the target sites that is caused by increased metabolism or altered transport or routing of the active drug species . Resistance related to alterations in the drug targets or apoptotic pathways is not discussed . Until recently multidrug resistance was associated almost exclusively with p-glycoprotein (Pgp)-overexpression . However, other non-Pgp-related mechanisms have been tracked down . It has been shown that transfection of the gene that encodes a novel drug transport protein, the multidrug resistance protein, induces cross-resistance for many multidrug resistance drugs as well as active transport of daunorubicin from tumor cells . Surprisingly, it has also been found that multidrug resistance protein mediates transport of negatively charged species that are not classic multidrug resistance drugs, such as leukotriene C4 and other glutathione conjugates as well as negatively charged dyes . It was therefore suggested that multidrug resistance protein is identical with the multispecific organic anion transporter . The transport rate of several positively charged drugs (vincristine, rhodamine-123, daunorubicin) by multidrug resistance protein appeared to be dependent on the cellular glutathione levels . Multidrug resistance protein seems to be constitutively expressed in normal tissues at a low level with few tissues having higher expression . Multidrug resistance protein overexpression in in vitro-selected MDR cell lines occurs relatively frequently in lung cancer and leukemia cell lines and often precedes Pgp overexpression . Differential expression has been demonstrated in tumor samples, which suggests a role in resistance to chemotherapy in at least certain tumor types . Modulation studies of multidrug resistance protein activity are still scarce . Other non-Pgp, non-multidrug resistance protein multidrug resistance mechanisms probably exist but have not been identified at the molecular level as yet.

West J Med, 1995 Nov, 163(5), 441 - 5
Drug-resistant Mycobacterium tuberculosis in California, 1991 to 1992; Koo D et al.; To determine the proportion and distribution of drug-resistant Mycobacterium tuberculosis in California, we surveyed all California counties for drug-susceptibility test results for initial isolates from tuberculosis cases counted during the first quarters of 1991 and 1992 . Overall, drug-susceptibility test results were not available for 17% of isolates . Among isolates with available test results, the proportion with resistance to isoniazid averaged 8.7%, and the proportion with resistance to at least 2 drugs, multidrug resistance, averaged 5.9% during these two quarters . The proportion of isolates with drug resistance did not change substantially during these time periods . The proportion with combined isoniazid and rifampin resistance remained stable at about 1.1% . Among persons whose isolates were tested for drug resistance, those with a known previous diagnosis of tuberculosis (relative risk {RR} = 2.6; 95% confidence interval {CI}, 1.6 to 4.3; P < .01) and persons who were foreign born (RR = 1.7; 95% CI, 1.1 to 2.7; P = .014) were more likely to have isoniazid-resistant organisms . These statewide data suggest that the initial tuberculosis treatment regimen in California should include 4 antituberculosis drugs, as recommended by the American Thoracic Society and the Centers for Disease Control and Prevention for areas with a prevalence of isoniazid resistance of 4% or greater . The lack of test results for 1 in 6 patients with tuberculosis suggests the need for improved physician and laboratorian education to implement the recommendations that drug susceptibility be tested on all initial isolates.

Cancer Res, 1995 Nov 1, 55(21), 4837 - 43
Riboflavin-mediated photosensitization of Vinca alkaloids distorts drug sensitivity assays; Granzow C et al.; Poor reproducibility of cytotoxicity tests with Vinca alkaloids has frequently been reported . A commonly presumed light sensitivity of the drugs could not be confirmed . However, we found that they are photosensitized by riboflavin (vitamin B2), an obligatory component of cell culture media . Light of wavelengths below 500 nm triggered rapid photoreactions of riboflavin with vinblastine, vincristine, and vindesine in aqueous solutions . The photoreactions altered the absorption spectra of these alkaloids and yielded degradation products that could be separated by TLC . In cell cultures, both immediate and persisting, riboflavin-mediated photoreactivity could be distinguished . They preclude reliable determinations of sensitivity and resistance to Vinca alkaloids, as exemplified on chemosensitive and multidrug-resistant mouse ascites cells . In experiments involving photosensitization, the 50% inhibitory concentration values of sensitive and resistant cells were overlapping and fluctuated in the ranges from 3 to 30 nM and 15 to 360 nM vinblastine, respectively . Corresponding values from series of experiments protected from photosensitization were 1.02 +/- 0.22 nM and 18.5 +/- 3.42 nM . Hence, riboflavin-mediated photoreactions must be fully prevented in assays of cellular drug sensitivity . Procedures for eliminating immediate as well as persisting photoreactivity were established.

Mol Cell Biol, 1995 Nov, 15(11), 6100 - 8
12-O-tetradecanoylphorbol-13-acetate activation of the MDR1 promoter is mediated by EGR1; McCoy C et al.; P-glycoprotein, the product of the MDR1 gene (multidrug resistance gene 1), is an energy-dependent efflux pump associated with treatment failure in some hematopoietic malignancies . Its expression is regulated during normal hematopoietic differentiation, although its function in normal hematopoietic cells is unknown . To identify cellular factors that regulate the expression of MDR1 in hematopoietic cells, we characterized the cis- and trans-acting factors mediating 12-O-tetradecanoylphorbol-13-acetate (TPA) activation of the MDR1 promoter in K562 cells . Transient-transfection assays demonstrated that an MDR1 promoter construct containing nucleotides -69 to +20 conferred a TPA response equal to that of a construct containing nucleotides -434 to +105 . TPA induced EGR1 binding to the -69/+20 promoter sequences over a time course which correlated with increased MDR1 promoter activity and increased steady-state MDR1 RNA levels . The -69/+20 promoter region contains an overlapping SP1/EGR site . The TPA-responsive element was localized to the overlapping SP1/EGR site by using a synthetic reporter construct . A mutation in this site that inhibited EGR protein binding blocked the -69/+20 MDR1 promoter response to TPA . The expression of a dominant negative EGR protein also blocked the TPA response of the -69/+20 promoter construct . Finally, the expression of EGR1 was sufficient to activate a construct containing tandem MDR1 promoter SP1/EGR sites . These data suggest a role for EGR1 in modulating MDR1 promoter activity in hematopoietic cells.

Mol Cell Biol, 1995 Nov, 15(11), 5879 - 87
Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae; Egner R et al.; Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter . We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development . Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane . In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min . A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis . However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation . Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole.

J Natl Cancer Inst, 1995 Nov 1, 87(21), 1593 - 602
Decreased mutation rate for cellular resistance to doxorubicin and suppression of mdr1 gene activation by the cyclosporin PSC 833; Beketic-Oreskovic L et al.; BACKGROUND: Various mechanisms can contribute to cellular resistance to doxorubicin . These include expression of the multidrug transporter P-glycoprotein (product of the mdr1 gene {also known as PGY1}, Mrp (multidrug resistance-associated protein), the p110 major vault protein, altered glutathione metabolism, and altered levels or activity of topoisomerase II (Topo II) . We reported recently that single-step treatment of human MES-SA sarcoma cells with 40 nM doxorubicin resulted in selection of spontaneous mutants at a rate of 1.8 x 10(-6) per cell generation . All individually selected mutants manifested the multidrug-resistant phenotype, related to activation of the mdr1 gene . PURPOSE: Luria and Delbruck fluctuation analysis was performed with MES-SA cells to determine the mutation rate and the nature and mechanisms of resistance after single-step selection with doxorubicin in the presence of the cyclosporin PSC 833, a potent modulator of multidrug resistance . METHODS: Ten flasks were seeded with 2000 cells/flask and grown to confluent populations of approximately 8 x 10(6) cells . After reseeding in 96-well plates, the populations were treated with 40 nM doxorubicin and 2 microM PSC 833 for 3 weeks . Surviving colonies were scored, individually harvested, and propagated . The drug-resistant phenotype was assessed by the tetrazolium dye (MTT) cytotoxicity assay and by monitoring cellular glutathione content and radiolabeled drug accumulation . Coupled reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate mdr1, MRP, Topo II alpha, and Topo II beta gene expression . Topo II, P-glycoprotein, and p110 levels were examined by immunoblotting or immunocytochemistry . Topo II activity was assessed by decatenation of kinetoplast DNA, and etoposide-induced cleavable complex formation was studied by the potassium-sodium dodecyl sulfate precipitation assay . RESULTS: Mutations were detected at a rate of 2.5 x 10(-7) per cell generation . Analysis of variance indicates that spontaneous mutations, rather than changes in cellular function, conferred resistance to doxorubicin and PSC 833 . None of the isolated clones expressed mdr1 messenger RNA or P-glycoprotein, and none exhibited an increase in MRP expression . No alterations were found in cellular glutathione content, intracellular accumulations of daunorubicin and etoposide, levels of p110 protein, or levels of Topo II beta transcripts . However, a significant decrease in Topo II alpha messenger RNA and protein was found in all examined clones, as well as decreased Topo II catalytic activity and reduced cleavable complex formation in the presence of etoposide . CONCLUSIONS: PSC 833 co-selection reduced the mutation rate for doxorubicin-selected resistance by 10-fold and suppressed the emergence of mdr1 mutants . Survival of cells exposed to doxorubicin and PSC 833 occurs by selection of spontaneously arising mutants that exhibit altered Topo II alpha expression . IMPLICATIONS: Our results suggest that treatment with multidrug resistance modulators such as PSC 833 together with multidrug resistance-related cytotoxins may suppress the activation of mdr1 and prevent the emergence of resistant cancer cell clones with the multidrug-resistant phenotype.

Am J Pathol, 1995 Nov, 147(5), 1238 - 47
In situ mRNA hybridization technique for analysis of metastasis-related genes in human colon carcinoma cells; Kitadai Y et al.; The purpose of this study was to determine whether the expression level of several genes that regulate different steps of the metastatic process correlates with the metastatic potential of human colon carcinoma cells . The mRNA expression level for epidermal growth factor receptor (growth), basic fibroblast growth factor and interleukin-8 (angiogenesis), type IV collagenase (invasion), E-cadherin and carcinoembryonic antigen (adhesion), and the multidrug resistance gene mdr-1 (drug resistance) in the human KM12 colon carcinoma cell lines and clones with different metastatic potential was measured by Northern blot analysis and by in situ hybridization technique . Highly metastatic KM12SM and KM1214 cells growing in culture uniformly expressed high levels of epidermal growth factor receptor, basic fibroblast growth factor, and carcinoembryonic antigen mRNA, whereas cultures of low metastatic KM12C, clone 1, clone 3, and clone 6 cells displayed heterogeneous patterns of expression . KM12C (low metastatic) and KM12SM (highly metastatic) cells were implanted into the subcutis (ectopic) or the wall of the cecum (orthotopic) of nude mice . The mRNA expression level for epidermal growth factor receptor, basic fibroblast growth factor, interleukin-8, type IV collagenase, carcinoembryonic antigen, and mdr-1 was increased in the cecal wall tumors as compared with subcutaneous tumors or in vitro cultures . These data demonstrate a direct correlation between constitutive and inducible expression of several metastasis-related genes and the metastatic potential of human colon carcinoma cells.

Leukemia, 1995 Nov, 9(11), 1882 - 7
Discordant P-glycoprotein antigen expression and transport function in acute myeloid leukemia; Xie XY et al.; Expression of the multidrug resistance efflux pump P-glycoprotein (Pgp) was measured in a series of AML patients using two flow cytometry methods . Transport function was assessed by measuring the modulating effect of the Pgp inhibitor cyclosporin A (CsA) on the cellular accumulation of daunorubicin, and Pgp antigen expression by surface immunofluorescence using the MRK-16 antibody . Both methods showed a wide range of values for Pgp expression between individual patients, but in contrast to a series of cell lines expressing Pgp there was no correlation between antigen expression and transport function in the clinical samples . As previously reported for chronic lymphocytic leukemia (CLL), pretreatment with neuraminidase markedly improved MRK-16 staining in some cases, indicating that abnormal glycosylation can cause epitope masking in AML blasts . Because experience with cell lines shows that Pgp expression is a continuous variable which correlates with the level of drug resistance, rather than the 'positive' or 'negative' which are frequently reported by clinical flow cytometry laboratories, we used a calibration procedure to estimate the actual number of Pgp molecules expressed in the AML samples . Despite the additional refinements of neuraminidase treatment and antigen quantification, the correlation between Pgp antigen expression and daunorubicin accumulation remained extremely weak (r = 0.11; P = 0.63) . It is suggested that the assay for transport function can detect molecules that affect daunorubicin accumulation but are antigenically distinct from classical P-glycoprotein . Heterogeneity of multidrug resistance efflux pumps might in part explain the relatively weak prognostic significance of immunofluorescence detection of Pgp in AML patients.

Leukemia, 1995 Nov, 9(11), 1870 - 4
Expression of the multidrug resistance P glycoprotein in newly diagnosed adult acute lymphoblastic leukemia: absence of correlation with response to treatment; Wattel E et al.; We analyzed P glycoprotein (PGP) expression and its correlation with hematological parameters and outcome in 50 cases of newly diagnosed adult acute lymphoblastic leukemia (ALL) . PGP expression was evaluated by flow cytometry using MRK16 monoclonal antibody (MoAb) and/or immunocytochemistry on marrow slides, using JSB1 MoAb . Thirty-two of the 50 patients (64%) were PGP positive by at least one of the two methods, which gave concordant results in 15 of the 18 cases in which they were both used . No correlation between PGP expression and clinical and hematological parameters including WBC counts, immunophenotype and karyotype was seen, although there was a trend for more frequent CD34 expression in PGP-positive cases . All patients were treated with intensive chemotherapy . We found no difference in complete remission (CR) rate, actuarial disease-free survival and survival in PGP-positive and PGP-negative cases . Our findings suggest that the clinical significance of PGP expression is less clear in ALL than in AML . Wider use of functional techniques of evaluation of mdr1 gene expression, which assess the 'pumping' activity of PGP, and their correlation with quantitative analysis of mdr1 mRNA and protein, would probably improve knowledge of the role of PGP in ALL . Analysis of other mechanisms of drug resistance, especially multidrug resistance-associated protein (MRP) expression, would also be useful.

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