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),