Anticancer Res, 2000 Mar-Apr, 20(2B), 1061 - 7 P-glycoprotein expression is a marker for chemotherapy resistance and prognosis in advanced ovarian cancer; Baekelandt MM et al.; BACKGROUND: It was our aim to study the prevalence and possible prognostic and predictive significance of the expression of P-glycoprotein, a transmembrane transport protein related to classical multidrug resistance, in patients with advanced ovarian cancer . STUDY DESIGN: Tumor tissue from 73 previously untreated patients with FIGO stage 3 ovarian cancer was examined with immunohistochemistry for the expression of P-glycoprotein before and after chemotherapy . Response to 4 cycles of combination chemotherapy with cisplatin and epirubicin was assessed with second look laparotomy . The log rank test was used for univariate survival and the Cox proportional hazards regression model for multivariate survival analysis . RESULTS: P-glycoprotein expression was detected in 47% of untreated cases, and correlated with unfavourable prognostic factors such as advanced age, presence of ascites and larger residual disease deposits after primary surgery . P-glycoprotein negative cases responded significantly better to chemotherapy (P < .001) . In the multivariate survival analysis P-glycoprotein expression was an independent predictor of both overall (P = .045) and progression free (P = .006) survival . When P-glycoprotein expression and residual disease status were considered together, the patients could be divided in three clearly distinct prognostic groups (P = .0009) . CONCLUSION: P-glycoprotein expression is a predictor of response and survival in a uniformly treated and followed cohort of advanced ovarian cancer patients. Anticancer Res, 2000 Mar-Apr, 20(2A), 1029 - 31 Oncocalyxones A and C, 1,4-anthracenediones from Auxemma oncocalyx: comparison with anticancer 1,9-anthracenediones; Leyva A et al.; Oncocalyxones A and C are 1,4-anthracenediones isolated from Auxemma oncocalyx (Boraginaceae) that have been shown to be cytotoxic to tumor cells in vitro . The present study compared the cytotoxicity of these compounds with that of two conventional anticancer agents doxorubicin and mitoxantrone, both 1,9-anthracenediones, in a panel of human tumor cell lines . The effect on cell growth was examined using an MTT microtiter assay in two leukemia lines, five solid tumor lines of different histological origin, and two multidrug-resistant sublines of a lung tumor line . The oncocalyxones showed much lower potency than the 1,9-anthracenediones, but were similarly more cytotoxic to leukemia cells compared to solid tumor lines . However, in the multidrug-resistant cells with 10 to 500 times decreased sensitivity to doxorubicin, the cytotoxicity of oncocalyxones A and C was only modestly reduced by about twofold, 1,4-Anthracenediones may be a promising novel class of chemotherapeutic agents effective against multidrug resistant tumors. Anticancer Res, 2000 Mar-Apr, 20(2A), 987 - 96 Multiwavelength videomicrofluorometric study of cytotoxic effects of a marine peptide, didemnin B, on normal and MDR resistant CCRF-CEM cell lines; Rocchi E et al.; The development of multidrug resistance (MDR) in heterogeneous cell sensitive and resistant populations to a variety of clinically important cytotoxic drugs poses a major obstacle to cancer chemotherapy . Didemnin B, a marine cyclic depsipeptide, displays interesting biological properties: antiviral activity, inhibition of DNA, RNA and protein synthesis, initiation of apoptosis and ability to block the cell cycle . As very little is known about its mode of action, we studied the effect of increasing doses of Didemnin B on sensitive and resistant human leukemic lymphoblast cell lines . The fluorescence of living cells simultaneously stained with Hoechst 33,342, Rhodamine 123 and Nile Red, were analyzed in a multiparametric approach involving multiwavelength microfluorometry . High concentrations of Didemnin B induced, in the sensitive cell line, a very early decrease in the energetic state of the mitochondria that occurs before a significant decrease of nuclear DNA content, observed simultaneously on sensitive and resistant cells, that could be related to an apoptosis process . Furthermore low Didemnin doses (50 nM) affected CEM-WT and CEM VLB differently, while higher doses (200 nM-250 nM and over) affected the two cell lines in the same way . This indicated that, at these doses, the membranar Pgp has no effect on the mode of action of Didemnin, suggesting that Didemnin does not need to be internalized to be active. Anticancer Res, 2000 Mar-Apr, 20(2A), 861 - 7 Membrane associated antitumor effects of crocine-, ginsenoside- and cannabinoid derivates; Molnar J et al.; In the present work a systematic study was initiated with crocine, ginsenoside and cannabinoid derivatives on multidrug resistant mouse lymphoma cells, viral tumor antigen expression and some human leukocyte functions . Among saffron derivatives, crocin and picrocrocin, triglucosyl and diglucosyl crocetin were ineffective on the reversal of multidrug resistance of lymphoma cells . Ginsenoside increased drug accumulation and tumor antigen expression at 2.0-20.0 micrograms/mL . Some cannabinoid derivatives such as cannabinol, cannabispirol and cannabidiol increased drug accumulation, while cannabidiolic acid, delta-9-THC and tetrahydro-cannabidiolic acid reduced drug accumulation of the human mdr1-gene transfected mouse lymphoma cells . The reversal of multidrug resistance is the result of the inhibition of the efflux pump function in the tumor cells . Crocetin esters were less potent than crocin itself in the inhibition of EBV early antigen expression . However crocin and diglucosylcrocetin inhibited early tumor antigen expression of adenovirus infected cells, but triglucosylcrocetin was less effective at 0.01-1.0 microgram/mL . The crocin had no antiviral effect {on HSV-2 infected vero cells} up to 25 micrograms/mL concentration . Ginsenosides had a moderate inhibitory effect except ginsenoside Rb1 (was the less effective) on the drug efflux pump . Among the cannabinoid derivatives the cannabinol and cannabispirol increased drug accumulation, while cannabidiolic acid and delta-8-THC, delta-9-THC and tetrahydro-cannabinol reduced drug accumulation in multidrug resistant mouse lymphoma cells . It is interesting that ginsenosides had a chemical structure-dependent immunomodulating effect by enhancing the activity of NK-cells and ADCC activities. Anticancer Res, 2000 Mar-Apr, 20(2A), 799 - 808 Doxorubicin-gallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells; Wang F et al.; Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin (ADR) uptake as a result of increased efflux, increased intracellular sequestration, and decreased membrane permeability . In this study, we investigated whether conjugation of ADR to transferrin (Tf) could overcome MDR in breast cancer cells . The multidrug resistant MCF-7/ADR breast cancer cell line was over 1000-fold more resistant to ADR, than its parental MCF-7 cell line, as determined by 3{H}-thymidine assay . The MCF-7/ADR cell line also expressed both MDR1 and MRP genes, as detected by RT-PCR . The ADR was coupled using a glutaraldehyde technique to human transferrin saturated with either ferric chloride (Fe-Tf) or gallium nitrate (Ga-Tf) . These conjugates were tested for cytotoxicity on both MCF-7 and MCF-7/ADR cells after 6 days of incubation . The doxorubicin-gallium-transferrin conjugate (ADR-Ga-Tf) exhibited approximately the same inhibitory effect as ADR on MCF-7 cells with IC50s of 2.34 x 10(-3) microM and 1.42 x 10(-3) microM, respectively . However in MCF-7/ADR cells ADR-Ga-Tf reversed resistance to free ADR and decreased 100-fold the IC50 from 8.98 microM with free ADR to 9.52 x 10(-2) microM . ADR-Fe-Tf was 10-fold more inhibitory to MCF-7/ADR cells than free ADR . Compared to Ga-Tf, ADR-Ga-Tf was 500- and 3000-fold more inhibitory to MCF-7 and MCF-7/ADR cells, respectively . These results demonstrated that ADR-Ga-Tf reverses resistance to free ADR and Ga-Tf in MCF-7/ADR cells . The distribution of ADR in both cell lines was examined by fluorescence microscopy . It was noted that ADR mainly accumulated in the cytoplasm around the nucleus in MCF-7/ADR cells, but in both the cytoplasm and nucleus of MCF-7 cells . However the conjugate of ADR-Ga-Tf allowed ADR to accumulate in the cytoplasm and nucleus of both the MCF-7/ADR and MCF-7 cells . Further investigation of MDR1 and MRP genes expression by RT-PCR demonstrated that Ga-Tf decreased expression of the MRP more than the MDR1 gene . Therefore the reversal of resistance to ADR by the ADR-Ga-Tf conjugate is mediated by the transferrin receptor transmembrane transport mechanism, redistribution of ADR into the nucleus of ADR resistant MCF-7/ADR cells and inhibition of MRP gene expression. J Intern Med, 2000 May, 247(5), 521 - 34 Multidrug resistance in haematological malignancies; Sonneveld P; The development of refractory disease in acute myeloid or lymphoblastic leukaemias (AML, ALL) and multiple myeloma (MM) is frequently associated with the expression of one or several multidrug resistance (MDR) genes . MDR1, MRP1 and LRP have been identified as important adverse prognostic factors in AML, T-ALL and MM . Recently, it has become possible to reverse clinical multidrug resistance by blocking P-glycoprotein-mediated drug efflux . The potential relevance of these reversal agents of MDR and potential new approaches to treat refractory disease are discussed. Eur J Clin Invest, 2000 May, 30(5), 447 - 53 The 16p11 breakpoint in myxoid liposarcomas might affect the expression of the LRP gene on 16p11.2 encoding the multidrug resistance associated major vault protein; Plaat BE et al.; BACKGROUND: Chromosome breakage could influence the expression of genes . This has been noticed in specific cases of acute myeloid leukaemia, where the 16p13 breakpoint affects the expression of the multidrug resistance related protein (MRP) . Myxoid liposarcomas (LPS) are characterized by the t(12; 16)(q13; p11), which leads to the formation of a FUS-CHOP fusion transcript . This study investigates the relationship between the cytogenetically detected breakpoint 16p11 in myxoid LPS, the presence of the FUS-CHOP fusion transcript in nonmyxoid LPS and the expression of the lung resistance major vault protein (LRP) gene on 16p11.2 . MATERIALS AND METHODS: Of 16 cases with a diagnosis of a (possible) liposarcoma with an abnormal karyotype, fresh frozen tumour material was available for immunohistological detection of LRP . Cases without a cytogenetically detected t(12; 16)(q13; p11), were analyzed for the presence of a FUS-CHOP fusion transcript by RT-PCR . RESULTS: In all 9 myxoid LPS a t(12; 16)(q13; p11) was found and LRP expression was absent or low . In none of the remaining 7 cases was a FUS-CHOP fusion transcript found, and four tumours were LRP positive (P = 0 . 02) . LRP expression in myxoid LPS (mean: 1.3%) was lower (P = 0.07) than in the nonmyxoid tumours (mean: 35.7%) . CONCLUSIONS: These observations indicate a relation between the t(12; 16)(q13; p11), leading to a FUS-CHOP fusion transcript in myxoid LPS, and the low or absent expression of the LRP-gene located on 16p11.2. Biochem Pharmacol, 2000 Jul 1, 60(1), 83 - 90 Antisense inhibition of P-glycoprotein expression using peptide-oligonucleotide conjugates; Astriab-Fisher A et al.; Antisense oligonucleotides are potentially a powerful tool for the therapeutic manipulation of genes associated with cancer . However, pharmacological applications of oligonucleotides have been hindered by the inability to effectively deliver these compounds to their sites of action within cells . In this study, we have prepared peptide-oligonucleotide conjugates with the intent of improving intracellular delivery . The phosphorothioate oligonucleotide component of the conjugates was complementary to a site flanking the AUG of the message for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells . Two types of peptide-antisense oligonucleotide conjugates, but not mismatched control conjugates, provided substantial inhibition of cell surface expression of P-glycoprotein . Surprisingly, the peptide-oligonucleotide conjugates were more potent in the presence of serum than when used under serum-free conditions; this is in striking contrast to most other approaches for intracellular delivery of nucleic acids . Effective inhibition of P-glycoprotein expression was attained with submicromolar concentrations of antisense conjugates under serum-replete conditions . The combination of relatively modest molecular size and good efficacy in the presence of serum proteins suggests that peptide-antisense oligonucleotide conjugates may have significant promise for in vivo therapeutic applications. Zhongguo Yao Li Xue Bao, 1999 Jul, 20(7), 647 - 50 R-dl-verapamil downmodulates multidrug resistance of KBv200 cells to vincristine and doxorubicin; Fang G et al.; AIM: To study the attenuation of multidrug resistance (MDR) by R-dl-verapamil (R-Ver) and the acute animal toxicity of R-Ver, and to compare these results of R-Ver with the results of dl-verapamil (Ver) . METHODS: Cytotoxicity was determined by tetrazolium (MTT) assay . Cellular accumulation of doxorubicin (Dox) was measured by fluorescence spectrophotometry . Acute animal toxicity was tested by i.p . drug administration in BALB/c mice . RESULTS: R-Ver attenuated MDR of KBv200 cells to vincristine (VCR) and Dox . This attenuation ability was dose-related, and was also dependent on drug exposure time . R-Ver 1.25 mumol.L-1 increased the sensitivity of KBv200 cells to VCR (P < 0.01) with a 24-h period of drug exposure . R-Ver downmodulated MDR and increased cellular Dox accumulation of KBv200 cells as effectively as Ver, but possessed lower acute toxicity in BALB/c mice . While LD50 of Ver was 60 (49-73) mg.kg-1, LD50 of R-Ver was 166 (137-202) mg.kg-1 . CONCLUSION: R-Ver downmodulated the MDR to VCR and Dox at 1.25 mumol.L-1, and this effect on VCR can be realized with drug exposure duration of 24 h. J Cell Sci, 2000 Jun, 113 ( Pt 11), 2011 - 21 The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2); Litman T et al.; Mechanisms of drug resistance other than P-glycoprotein are of increasing interest as the list of newly identified members of the ABC transport family has grown . We sought to characterize the phenotype of the newly discovered ABC transporter encoded by the mitoxantrone resistance gene, MXR, also known as ABCP1 or BCRP . The pharmacodynamics of mitoxantrone and 12 other fluorescent drugs were evaluated by confocal microscopy in four multidrug-resistant human colon (S1) and breast (MCF-7) cancer cell lines . We utilized two sublines, MCF-7 AdVp3000 and S1-M1-80, and detected overexpression of MXR by PCR, immunoblot assay and immunohistochemistry . These MXR overexpressing sublines were compared to cell lines with P-glycoprotein- and MRP-mediated resistance . High levels of cross-resistance were observed for mitoxantrone, the anthracyclines, bisantrene and topotecan . Reduced levels of mitoxantrone, daunorubicin, bisantrene, topotecan, rhodamine 123 and prazosin were observed in the two sublines with high MXR expression . Neither the P-glycoprotein substrates vinblastine, paclitaxel, verapamil and calcein-AM, nor the MRP substrate calcein, were extruded from MCF-7 AdVp3000 and S1-M1-80 cells . Thus, the multidrug-resistant phenotype due to MXR expression is overlapping with, but distinct from, that due to P-glycoprotein . Further, cells that overexpress the MXR protein seem to be more resistant to mitoxantrone and topotecan than cells with P-glycoprotein-mediated multidrug resistance . Our studies suggest that the ABC half-transporter, MXR, is a potent, new mechanism for conferring multiple drug resistance . Definition of its mechanism of transport and its role in clinical oncology is required. Acta Neuropathol (Berl), 2000 May, 99(5), 555 - 62 Evidence for a constitutive, verapamil-sensitive, non-P-glycoprotein multidrug resistance phenotype in malignant glioma that is unaltered by radiochemotherapy in vivo; Rieger L et al.; Human malignant gliomas are commonly resistant to chemotherapy . Here, we examined the role of the multidrug resistance (mdr) mechanism in the chemo-resistance of these tumors, using a twofold approach: (i) by assessing a possible mdr phenotype before and after chronic drug exposure of glioma cells in vitro, and (ii) by assessing the modulation of expression of the mdr-associated P-glycoprotein (Pgp) using radiotherapy and serial cycles of chemotherapy in human glioblastoma patients in vivo . T98G, and to a lesser degree, LN-229 human malignant glioma cells exhibit a constitutive mdr phenotype as determined by the modulation of dye transport and by the augmentation of chemosensitivity by the mdr antagonist, verapamil . Thus, coexposure to verapamil enhances the cytotoxicity of vincristine, doxorubicin and VM26 in T98G cells and that of vincristine in LN-229 cells . Chronic exposure of the cells to low concentrations of vincristine and doxorubicin, but not VM26, topotecan or BCNU, moderately enhances the mdr-like phenotype, as assessed by drug expulsion assays . However, chronic exposure to increasing drug concentrations does not significantly alter the sensitivity to the respective drugs . These data are consistent with a constitutive, but not drug-inducible, mdr-like drug resistance in glioma cells in vitro . Immunocytochemical analysis of human malignant gliomas in vivo reveals that Pgp expression is more abundant in endothelial cells within the gliomas, than in the glioma cells proper . Importantly, Pgp expression is unaltered by radiochemotherapy, assessed by comparative immunocytochemistry of glioma specimens obtained serially before and after radiochemotherapy . We conclude that (i) glioma cells exhibit constitutive mdr-like drug resistance that is not significantly altered by chronic drug exposure in vitro; (ii) endothelial cells may play an important role in Pgp-mediated drug resistance of gliomas in vivo; (iii) radiotherapy and repeated chemotherapy cycles do not modulate Pgp expression in human malignant gliomas in vivo; (iv) there is preliminary evidence for a non-Pgp, verapamil-sensitive drug transport activity in glioma cells. Jpn J Cancer Res, 2000 Apr, 91(4), 439 - 45 Photodynamic inactivation with acridine orange on a multidrug-resistant mouse osteosarcoma cell line; Kusuzaki K et al.; Overcoming multidrug resistance (MDR) is an urgent issue to improve the prognosis of osteosarcoma patients . In this study, we undertook to clarify the effect of photodynamic therapy (PDT) with acridine orange (AO) on the MDR mouse osteosarcoma (MOS / ADR1) cell line, by comparing the outcome with the effect on a chemosensitive osteosarcoma (MOS) cell line . Cultured cells of MOS and MOS / ADR1 cell lines were exposed to AO at various concentrations for various times, followed by long- or short-term (10 or 1 min) illumination with blue light (466.5 nm) for excitation . Living cells were counted by means of the trypan blue exclusion test . The results showed that AO rapidly bound to DNA, RNA and lysosomes of living MOS and MOS / ADR1 cells and also that most tumor cells in both cell lines died rapidly (viability ratio to untreated cells: 1/1000) within 48 h under conditions of continuous or 15-min flash exposure to AO at concentrations above 1.0 microg/ml plus 10-min illumination with blue light . Even after flash exposure to AO at concentrations above 1.0 microg/ml plus 1-min illumination, the viability of MOS/ADR1 cells decreased to a viability ratio of less than 1/ 1000 within 72 h . Based on these results, we concluded that AO with photo-excitation has a strong cytocidal effect, not only on chemosensitive mouse osteosarcoma cells, but also on MDR mouse osteosarcoma cells . These results suggested that photodynamic therapy with AO may be a new approach to treating MDR human osteosarcomas. Mol Biochem Parasitol, 2000 Apr 30, 108(1), 13 - 23 The tyrosine-86 allele of the pfmdr1 gene of Plasmodium falciparum is associated with increased sensitivity to the anti-malarials mefloquine and artemisinin; Duraisingh MT et al.; Although chloroquine-resistance (CQR) in Plasmodium falciparum is increasing and resistance to other blood schizonticidal anti-malarials has been reported, the molecular basis remains unclear . In this study fresh field isolates were obtained from The Gambia, an area of emerging CQR and tested for sensitivity to the anti-malarial drugs mefloquine, halofantrine, artemisinin, dihydroartemisinin, chloroquine and quinine . Sequence polymorphisms in the pfmdr1 gene and size polymorphisms in the cg2 gene were assessed using PCR-based systems . A strong association was observed between the presence of the tyr-86 allele of pfmdr1 and increased sensitivity to mefloquine and halofantrine, as well as the structurally unrelated drugs artemisinin and dihydroartemisinin . A weaker association was found between the presence of tyr-86 and increased resistance to chloroquine and quinine . The cg2 Dd2-like omega repeat size polymorphism was associated with increased resistance to chloroquine and increased sensitivity to mefloquine and halofantrine . An intragenic association was also found between a polymorphism in the polyasparagine linker region of pfmdr1 and the tyr-86 allele, which may be due to genetic hitchhiking, indicative of recent selection by chloroquine . Our data support a hypothesis where the pfmdr1 gene confers a true multidrug resistance phenotype which is lost by mutation. Chem Biol, 2000 May, 7(5), 335 - 43 Novel gallium(III) complexes transported by MDR1 P-glycoprotein: potential PET imaging agents for probing P-glycoprotein-mediated transport activity in vivo; Sharma V et al.; BACKGROUND: Multidrug resistance (MDR) mediated by expression of MDR1 P-glycoprotein (Pgp) represents one of the best characterized barriers to chemotherapy in cancer patients . Positron emission tomography (PET) agents for analysis of Pgp-mediated drug transport activity in vivo would enable noninvasive assessment of chemotherapeutic regimens and MDR gene therapy . RESULTS: Candidate Schiff-base phenolic gallium(III) complexes were synthesized from their heptadentate precursors and gallium(III)acetylacetonate . Crystal structures demonstrated a hexacoordinated central gallium with overall trans-pseudo-octahedral geometry . Radiolabeled (67)Ga-complexes were obtained in high purity and screened in drug-sensitive (Pgp(-)) and MDR (Pgp(+)) tumor cells . Compared with control, lead compound 6 . demonstrated antagonist-reversible 55-fold lower accumulation in Pgp-expressing MDR cells . Futhermore, compared with wild-type control, quantitative pharmacokinetic analysis showed markedly increased penetration and retention of 6 . in brain and liver tissues of mdr1a/b((-/-)) gene disrupted mice, correctly mapping Pgp-mediated transport activity at the capillary blood-brain barrier and hepatocellular biliary cannalicular surface in vivo . CONCLUSIONS: These results indicate that gallium(III) complex 6 . is recognized by MDR1 Pgp as an avid transport substrate, thereby providing a useful scaffold to generate (68)Ga radiopharmaceuticals for molecular imaging of Pgp transport activity in tumors and tissues in vivo using PET. Am J Public Health, 2000 May, 90(5), 699 - 701 Helping the urban poor stay with antiretroviral HIV drug therapy; Bamberger JD et al.; Recent studies have documented dramatic decreases in opportunistic infections, hospitalizations, and mortality among HIV-infected persons, owing primarily to the advent of highly active antiretroviral medications . Unfortunately, not all segments of the population living with HIV benefit equally from treatment . In San Francisco, only about 30% of the HIV-infected urban poor take combination highly active antiretroviral medications, as compared with 88% of HIV-infected gay men . Practitioners who care for the urban poor are reluctant to prescribe these medications, fearing inadequate or inconsistent adherence to the complicated medical regimen . Persons typically must take 2 to 15 pills at a time, 2 to 3 times a day . Some of the medications require refrigeration, which may not be available to the homeless poor . Most homeless persons do not have food available to them on a consistent schedule . Therefore, they may have difficulty adhering to instructions to take medications only on an empty stomach or with food . Lack of a safe place to store medications may be an issue for some . In addition, many urban poor live with drug, alcohol, or mental health problems, which can interfere with taking medications as prescribed . Inconsistent adherence to medication regimens has serious consequences . Patients do not benefit fully from treatments, and they will become resistant to the medications in their regimen as well as to other medications in the same classes as those in their regimen . Development of resistance has implications for the broader public health, because inadvertent transmission of multidrug-resistant strains of HIV has been demonstrated . Concern that the urban poor will not adhere to highly active antiretroviral medication regimens has led to debate on the role of clinicians and public health officials in determining who can comply with these regimens . Rather than define the characteristics that would predict adherence to these regimens, the San Francisco Department of Public Health created a program to support adherence among those who may have the greatest difficulty complying with complicated highly active antiretroviral medication regimens . The program, dubbed the Action Point Adherence Project, was conceived through a community planning process in preparation for a city-wide summit on HIV/AIDS that took place in January 1998 . Action Point is funded by the city and the county of San Francisco . Now in its 10th month, the program continues to show promising evidence of improving clients' biological and social indicators. Biochem Biophys Res Commun, 2000 May 10, 271(2), 534 - 6 Vectors with hidden cloning sites; Welker E et al.; A general strategy is described for using the cleavage site of restriction enzymes in vectors for cloning regardless of how many sites the given enzymes have in the vector . The application of this method allows one to open any vector at its cloning site with protruding ends which can be compatible with almost every commercially available Class II restriction enzyme . By employing this method, the laborious construction of new vectors can be simplified considerably . This general strategy is based on the known ability of Class IIS restriction enzymes to cut any sequence located outside of their recognition site; the introduction of a linker containing recognition site(s) for Class IIS restriction enzyme(s), not present originally in the vector, gives rise to the possibility of opening the vector so as to produce overhangs of arbitrary sequence . In particular, when a symmetrical short sequence representing the protruding end of any Class II enzyme is situated at the cutting position of the Class IIS enzyme, cleavage with the Class IIS enzyme exposes the hitherto hidden, "unique" cloning site . This technique is demonstrated by cloning the cDNA of the multidrug resistance protein to an expression vector . NMR Biomed, 2000 Apr, 13(2), 92 - 101 Magnetic resonance spectroscopy of cellular lipid extracts from sensitive, resistant and reverting K562 cells and flow cytometry for investigating the P-glycoprotein function in resistance reversion; Le Moyec L et al.; The proton NMR spectra of K562 cells contain resonances of lipids . When these cells acquire multidrug resistance phenotype, the NMR lipid signals are modified and partially recovered when the resistance is reversed . The goals of the present study are to elucidate the mechanism of the resistance phenotype reversion and to investigate the possible origin of lipid signals detected in whole cells with proton NMR spectroscopy . Therefore, the K562 drug-sensitive cell line, its adriamycin resistant counterpart and two reverting derivates, obtained by verapamil treatment and long term culture in drug-free medium, were used in this study . The P-glycoprotein (P-gp) pump function was measured by flow cytometry and lipids were extracted to be analysed by proton and phosphorus spectroscopy . The phenotype reversion is due to the decrease of the P-gp function and an increased entrance of anthracycline drug when compared with the resistant cells . The spectra obtained on extracts showed no modification of the fatty acid composition and of the ratio of total cholesterol to fatty acid content . A different phospholipid composition in sensitive and resistant cells was found, but the reversion of resistance did not produce a recovery of these lipids . Thus, the lipid NMR spectra of extracts could not explain the spectral modifications observed on whole cells, in relation to acquiring and reverting drug resistance . These results are in favour of a different lipid organization or of localization within the cell . Int J Cancer, 2000 May 15, 86(4), 506 - 11 Alu-associated interstitial deletions and chromosomal re-arrangement in 2 human multidrug-resistant cell lines; Harada T et al.; Previous studies have shown that gene re-arrangements play a significant role in tumorigenesis . Gene re-arrangements involving the human multidrug resistance-1 (MDR1) gene have been identified as a mechanism for MDR1 over-expression in human malignant cells . In 2 multidrug-resistant human cancer sublines with high levels of MDR1 and P-glycoprotein (MCF7/TX400 and S48-3s/Adr10), hybrid mRNAs containing sequences from MDR1 and an unrelated gene have previously been identified . To characterize and determine the site of the re-arrangements resulting in generation of hybrid mRNAs, we first constructed a lambda phage library extending over a contiguous genomic region of 100 kb and containing the region upstream of MDR1 . In MCF7/TX400 cells, homologous recombination was observed involving an Alu repeat 80 kb upstream of the MDR1 gene, with a 79 bp intra-Alu deletion flanked by chi-like sequences at the re-arrangement junction . By contrast, non-homologous recombination was observed in S48-3s/Adr10 cells with Alu repeats near the junction sequence . While the specific features of the breakpoints appear to be different, Alu repeats might be involved in both gene re-arrangements . The gene re-arrangements at or near the Alu sequence should be regarded as potentially involved in the transcriptional activation of human MDR1 . Zhonghua Wai Ke Za Zhi, 1997 Nov, 35(11), 697 - 9 {Drug resistance and its mechanism of intrinsic drug-resistant cell line GRC-1}; Kong X et al.; In order to probe the characteristics of drug resistance and its mechanisms of renal cell carcinoma, drug-resistant spectrum of renal cell carcinoma cell line GRC-1 was detected by in vitro MTT colorimetric assay, the mechanism of drug resistance in GRC-1 was also studied by the methods of both immunocytochemistry assay and flow fluorescence cytometry . The results demonstrated that GRC-1 was cross-resistant to adriamycin, vincrinstine, etoposide and carboplatinium, both mdr1 gene product P-glycoprotein and GST-pi which was an isozyme of glutathione S-transferases were expressed in GRC-1 . The accumulation of net intracellular drugs of GRC-1 was less than that of drug sensitive breast cancer cell line MCF7, and the ability of pumping drugs out of cells was higher than that of MCF7 . The results suggested that there is an intrinsic multidrug resistance in GRC-1 cell line, and both P-glycoprotein and glutathione systems play a role in the development of drug resistance for GRC-1 . GRC-1 is an ideal target cell line for the study of drug resistance. Cochrane Database Syst Rev . 2000;(2):CD000256. Artemisinin derivatives for treating uncomplicated malaria; McIntosh HM et al.; BACKGROUND: Artemisinin derivatives are a relatively new group of drugs with antimalarial properties . As resistance to other antimalarial drugs continues to increase, artemisinin drugs may be useful alternatives . OBJECTIVES: The objective of this review was to assess the effects of artemisinin drugs for treating uncomplicated falciparum malaria . SEARCH STRATEGY: We searched the Cochrane Infectious Diseases Group trials register, the Cochrane Controlled Trials Register, Medline, Embase, Science Citation Index, Lilacs, African Index Medicus; conference abstracts and reference lists of relevant articles . We contacted organisations, researchers in the field and drug companies . SELECTION CRITERIA: Randomised and quasi-randomised trials of artemisinin derivatives, alone or in combination with other antimalarials, compared with standard antimalarial treatments, in adults or children with uncomplicated falciparum malaria . Only trials where treatment was given by mouth or suppository were included . Comparisons between different artemisinin derivatives and treatment regimens were also included . DATA COLLECTION AND ANALYSIS: Eligibility and trial quality were assessed and data were extracted independently by the two reviewers . MAIN RESULTS: Forty-one trials involving over 5000 patients were included . Variation in study design and quality made synthesis of the data problematic . Allocation concealment was adequate in only two trials . Most data were from areas of multidrug resistant falciparum malaria in South East Asia . Compared with standard antimalarial treatments, artemisinin drugs showed fast parasite clearance and high cure rates at follow-up, provided the duration of treatment with artemisinin drugs was adequate . Combination with mefloquine improved sustained parasite clearance and was effective in multidrug resistant areas . When doses were adequate, the combination shortened the duration of treatment . We found no evidence that artemisinin drugs are more harmful than standard treatment drugs over a typical trial period of 28 days . REVIEWER'S CONCLUSIONS: The evidence suggests that artemisinin drugs are effective and safe for treating uncomplicated malaria . There is no evidence from randomised trials that one artemisinin derivative is better than the others . In areas where there is mefloquine resistance, combination therapy with an artemisinin derivative appears to improve sustained parasite clearance compared with either drug alone. Biochem Pharmacol, 1999 May 1, 57(9), 1047 - 58 pH and drug resistance . II . Turnover of acidic vesicles and resistance to weakly basic chemotherapeutic drugs; Raghunand N et al.; Resistance to chemotherapeutic agents is a major cause of treatment failure in patients with cancer . The primary mechanism leading to a multidrug-resistant phenotype is assumed to be plasma-membrane localized overexpression of drug efflux transporters, such as P-glycoprotein (P-gp) . However, acidic intracellular organelles can also participate in resistance to chemotherapeutic drugs . In this study, we investigated, both experimentally and theoretically, the effect of acidic vesicle turnover on drug resistance . We have developed a general model to account for multiple mechanisms of resistance to weakly basic organic cations, e.g . anthracyclines and Vinca alkaloids . The model predicts that lower cytosolic concentrations of drugs can be achieved through a combination of high endosomal turnover rates, a low endosomal pH, and an alkaline-inside pH gradient between cytosol and the extracellular fluid . Measured values for these parameters have been inserted into the model . Computations using conservative values of all parameters indicate that turnover of acidic vesicles can be an important contributor to the drug-resistant phenotype, especially if vesicles contain an active uptake system, such as H+/cation exchange . Even conservative estimates of organic cation-proton antiport activity would be sufficient to make endosomal drug extrusion a potent mechanism of resistance to weakly basic drugs . The effectiveness of such a drug export mechanism would be comparable to drug extrusion via drug pumps such as P-gp . Thus, turnover of acidic vesicles can be an important factor in chemoresistance, especially in cells that do not overexpress plasma membrane-bound drug pumps like P-glycoprotein. Biochem Pharmacol, 1999 May 1, 57(9), 1037 - 46 pH and drug resistance . I . Functional expression of plasmalemmal V-type H+-ATPase in drug-resistant human breast carcinoma cell lines; Martinez-Zaguilan R et al.; A major obstacle for the effective treatment of cancer is the phenomenon of multidrug resistance (MDR) exhibited by many tumor cells . Many, but not all, MDR cells exhibit membrane-associated P-glycoprotein (P-gp), a drug efflux pump . However, most mechanisms of MDR are complex, employing P-gp in combination with other, ill-defined activities . Altered cytosolic pH (pHi) has been implicated to play a role in drug resistance . In the current study, we investigated mechanisms of pHi regulation in drug-sensitive (MCF-7/S) and drug-resistant human breast cancer cells . Of the drug-resistant lines, one contained P-gp (MCF-7/DOX; also referred to as MCF-7/D40) and one did not (MCF-7/MITOX) . The resting steady-state pHi was similar in the three cell lines . In addition, in all the cell lines, HCO3- slightly acidified pHi and increased the rates of pHi recovery after an acid load, indicating the presence of anion exchanger (AE) activity . These data indicate that neither Na+/H+ exchange nor AE is differentially expressed in these cell lines . The presence of plasma membrane vacuolar-type H+-ATPase (pmV-ATPase) activity in these cell lines was then investigated . In the absence of Na+ and HCO3-, MCF-7/S cells did not recover from acid loads, whereas MCF-7/MITOX and MCF-7/DOX cells did . Furthermore, recovery of pHi was inhibited by bafilomycin A1 and NBD-Cl, potent V-ATPase inhibitors . Attempts to localize V-ATPase immunocytochemically at the plasma membranes of these cells were unsuccessful, indicating that V-ATPase is not statically resident at the plasma membrane . Consistent with this was the observation that release of endosomally trapped dextran was more rapid in the drug-resistant, compared with the drug-sensitive cells . Furthermore, the drug-resistant cells entrapped doxorubicin into intracellular vesicles whereas the drug-sensitive cells did not . Hence, it is hypothesized that the measured pmV-ATPase activity in the drug-resistant cells is a consequence of rapid endomembrane turnover . The potential impact of this behavior on drug resistance is examined in a companion manuscript. Biochem J, 2000 May 15, 348 Pt 1, 183 - 8 Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide; Dombrowski F et al.; Immunohistochemical studies suggest that canalicular secretion via multidrug resistance protein 2 (Mrp2), a conjugate export pump encoded by the Mrp2 gene, is regulated by rapid transporter retrieval from/insertion into the canalicular membrane . The present study was undertaken in order to investigate this suggestion by means of immunogold electron microscopy . Therefore the effects of lipopolysaccharide (LPS) and osmolarity on Mrp2 localization were studied following immunogold labelling in the perfused rat liver by quantitative electron microscopy and morphometric analyses, and by confocal laser scanning microscopy . Mrp2 activity was assessed in the isolated perfused rat liver by measuring the excretion of dinitrophenyl-S-glutathione as a substrate of Mrp2 . Both LPS and hyperosmolarity resulted in a statistically significant decrease in immunogold-labelled Mrp2 in the canalicular membrane and canalicular villi, and an increase in labelling in the pericanalicular cytoplasm . Canalicular morphometric parameters were unchanged under these conditions compared with controls . Under hyperosmolar perfusion Mrp2, but not the canalicular protein dipeptidylpeptidase IV, was found inside the cells, as shown by double immunofluorescence and confocal laser scanning microscopy . The findings suggest a selective retrieval of Mrp2 from the canalicular membrane under the influence of hyperosmolarity and LPS, whereas canalicular morphology remains unchanged. Aquat Toxicol, 2000 Apr 1, 48(4), 357 - 389 Multixenobiotic resistance as a cellular defense mechanism in aquatic organisms; Bard SM; Multixenobiotic resistance in aquatic organisms exposed to natural toxins or anthropogenic contaminants is a phenomenon analogous to multidrug resistance in mammalian tumor cell lines tolerant of anti-cancer drugs . Multidrug resistance is commonly due to the elevated expression of transmembrane P-glycoproteins (P-gp) which actively transport a wide variety of structurally and functionally diverse compounds . The purpose of this review is to place aquatic ecotoxicological data in context of the larger multidrug resistance field of study . Information on P-glycoproteins structure, mechanism of transport, and substrate specificity gained through traditional mammalian and cell culture models is examined in conjunction with recent work on aquatic species exposed to xenobiotics both in the field and in the laboratory . The physiological function of P-glycoproteins is explored through studies of gene knockout models and expression patterns in normal tissues and tumors . The effect of xenobiotic exposures on P-gp activity and protein titer is examined in wild and captive populations of aquatic invertebrates and vertebrates . Substrate overlap and evidence of co-expression of phase I detoxification enzymes (e.g . cytochromes P450) and P-gp are presented . The role of P-gp chemosensitizers as environmental pollutants and the ecotoxicological consequences of P-gp inhibition are highlighted . The overwhelming evidence suggests that P-glycoproteins provide aquatic organisms with resistance to a wide range of natural and anthropogenic toxins. Virology, 2000 May 10, 270(2), 310 - 6 Multidrug resistance genotypes (insertions in the beta3-beta4 finger subdomain and MDR mutations) of HIV-1 reverse transcriptase from extensively treated patients: incidence and association with other resistance mutations; Tamalet C et al.; Multiple nucleoside resistance involves specific mutational patterns of the HIV-1 pol gene that are independent of the classic mutations conferring resistance to individual dideoxynucleosides . These include a cluster of five mutations in the reverse-transcriptase (RT) coding region (A62V, V75I, F77L, F116Y, and Q151M) generally referred to as multidrug resistance (MDR) mutations, and insertions of one or several amino acid residues between codons 67 and 70 of RT, a flexible region joining two antiparrallel beta sheets (beta3-beta4 insertions) . The objectives of this study were (i) to determine the prevalence of multidrug resistance genotypes (MDR mutations and beta3-beta4 insertions) in a cohort of 632 patients who were extensively pretreated with anti-HIV drugs and not responding to their current antiretroviral therapy, and (ii) to analyze the association of multidrug resistance genotypes with other resistance mutations in the RT and protease genes . Among viruses sequenced from these patients, 15 (2.4%) of them contained an insertion and 2 (0.3%) contained a deletion in the beta3-beta4 finger subdomain of RT . In 9 cases, the insertion was associated with a D67S, G, or E mutation . In addition, we identified 13 (2.1%) viruses harboring specific MDR mutations (mainly Q151M and/or A62V, V75I, F116Y) . Interestingly, the A62V mutation was found in 6 of the 15 strains with an insertion, whereas the other MDR mutations were not observed in insertion mutant strains . Especially high levels of resistance to zidovudine were observed for viruses with a beta3-beta4 insertion in the background of A62V, L210W, and T215Y . Otherwise, MDR mutations and beta3-beta4 insertions were found in association with the classic mutations conferring resistance to zidovudine, lamivudine, nonnucleoside RT inhibitors, and protease inhibitors, according to treatment history . Finally, we observed a genome with a deletion of codon 70 associated with a Q151M MDR mutation . These data suggest that the emergence of HIV-1 multidrug resistance, which may occur in various genetic contexts, poses a challenging problem in formulating treatment strategies . Mol Microbiol, 2000 Apr, 36(2), 402 - 13 Hyperactive forms of the Pdr1p transcription factor fail to respond to positive regulation by the hsp70 protein Pdr13p; Hallstrom TC et al.; Multidrug resistance in Saccharomyces cerevisiae is commonly associated with the overproduction of ATP-binding cassette transporter proteins such as Pdr5p or Yor1p . The Cys6-Zn(II)2 cluster-containing transcription factors Pdr1p and Pdr3p are key regulators of expression of these pleiotropic drug resistance (PDR) loci . Previous experiments have demonstrated that the Hsp70 protein encoded by the PDR13 gene is a positive regulator of Pdr1p function . We have examined the mechanism underlying the control of Pdr1p by Pdr13p . Expression of deletion, insertion and amino acid substitution mutant variants of Pdr1p suggest that the centre region of the transcription factor is the target for Pdr13p-mediated positive regulation . Immunological and fusion protein analyses demonstrate that Pdr13p is located in the cytoplasm, while Pdr1p is found in the nucleus . Biochemical fractionation experiments indicate that Pdr13p is associated with a high-molecular-weight complex and suggest the association of some fraction of Pdr13p with ribosomes. Br J Haematol, 2000 Mar, 108(4), 703 - 9 P-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP) expression in acute promyelocytic leukaemia; Michieli M et al.; We analysed the expression of three drug transporter proteins {p-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP1)} involved in anthracycline resistance that are frequently overexpressed in poor-risk adult acute non-lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution . Cellular daunorubicin accumulation was also evaluated . At onset, no case had PGP or MRP1 expression that exceeded that of non-multidrug-resistant (MDR) cell lines . Only one case showed LRP overexpression . No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission . In the onset vs . first relapse, only one patient showed an increased (threefold) PGP expression at relapse . At second relapse, three out of four patients showed a PGP expression two- to threefold higher than baseline values . These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline . It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse . The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse . In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses. Br J Clin Pharmacol, 2000 May, 49(5), 437 - 44 In vitro sensitivity of Plasmodium falciparum and clinical response to lumefantrine (benflumetol) and artemether; Tanariya P et al.; AIMS: To assess the sensitivity of 103 Plasmodium falciparum isolates to a combination of lumefantrine (benflumetol) and artemether (CGP 56697), with the objective of determining a correlation between in vitro drug sensitivity and therapeutic outcome . METHODS: Patients suffered from uncomplicated falciparum malaria and came from areas of Thailand affected by multidrug resistance . CGP 56697 was given in the form of tablets containing 20 mg artemether and 120 mg lumefantrine . The standard dose regimen, 4 doses of 4 tablets over 48 h, was compared with two lower dose regimens (4 x 2 tablets and 3 x 4 tablets) . RESULTS: The parasites showed high resistance to chloroquine, fairly advanced resistance to mefloquine and compromised sensitivity to quinine . Sensitivity to artemisinin and lumefantrine prior to treatment was similar in all treatment groups . The 4 x 4 tablet regimen was more effective than the other regimens in coping with infections with relatively low sensitivity to artemisinin and/or lumefantrine . The EC90 for artemisinin is an important determinant of treatment success . Parasite density at the start of treatment was identified as another critical predictor of treatment outcome . CONCLUSIONS: The results indicate that parasite exposure to the drugs may have been inadequate and/or too short in the cases of treatment failure, particularly marked in the lower dose regimens . This could probably be remedied by expanding the dose regimen in areas affected by multidrug resistance and in the case of relatively high parasitaemia. J Clin Invest, 2000 May, 105(9), 1261 - 7 Regulation of expression of the multidrug resistance protein MRP1 by p53 in human prostate cancer cells; Sullivan GF et al.; The expression of several drug-resistance genes, including MRP and p53, increases with advancing stage of human prostate cancer . Altered transcription could account for the genotypic alterations associated with prostate cancer progression, and it was recently reported that the promoter of MRP1 is activated in the presence of mutant p53 . To determine whether there is a relationship between p53 status and the expression of MRP1, a human, temperature-sensitive p53 mutant (tsp Val(138)) was transfected into LNCaP human prostate cancer cells . In the transfected cell line (LVCaP), the wild-type p53 produced growth arrest at the G1/S interface of the cell cycle, inhibited colony formation, and induced p21(waf1/cip1) . Temperature shifting to 38 degrees C (p53 mutant) produced a time-dependent increase in expression of MRP1 . This change in MRP1 expression was also seen in isogenic cell lines in which p53 was inactivated by human papilloma virus (HPV)16E6 protein or by a dominant-negative mutant . Functional assays revealed a decrease in drug accumulation and drug sensitivity associated with mutant p53 and increased MRP1 expression . These results provide the first mechanistic link between expression of MRP1 and mutation of p53 in human prostate cancer and support recent clinical associations . Furthermore, these data suggest a mechanism tying accumulation of p53 mutations to the multidrug resistance phenotype seen in this disease. J Hematother Stem Cell Res, 1999 Oct, 8(5), 503 - 14 Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs; Omori F et al.; Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance . The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients . We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR . This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml . Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide . Southern blot analyses indicated that most clones had only one proviral integration . Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones . Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase) . Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6 . PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted . To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide . All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR . Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer. Yeast, 2000 Apr, 16(6), 561 - 71 The products of YCF1 and YLL015w (BPT1) cooperate for the ATP-dependent vacuolar transport of unconjugated bilirubin in Saccharomyces cerevisiae; Petrovic S et al.; Since bilirubin-like pigments are present in the environment as degradation products of heme-containing proteins, yeast could have developed a detoxifying system to transport these compounds into their vacuoles . Vacuoles from Saccharomyces cerevisiae showed an ATP-dependent, saturative transport of unconjugated bilirubin (UCB) that was reduced by 60% and 40% in YCF1 and YLL015w-deleted cells, respectively; the double deletant showed no UCB uptake . Conversely, the transport of bile acids (taurocholate) was comparable in wild and deleted stains . These data identify YCF1 and YLL015w, named BPT1 (Bile Pigment Transporter), as the genes responsible for ATP-dependent UCB transport in yeast . Since YCF1 and YLL015w are rather homologous with multidrug resistant proteins (MRPs), they also suggest the involvement of this class of transporters in the ATP-dependent transport of unconjugated bilirubin . J Clin Microbiol, 2000 May, 38(5), 1901 - 8 Technical assessment of the affymetrix yeast expression GeneChip YE6100 platform in a heterologous model of genes that confer resistance to antimalarial drugs in yeast; Nau ME et al.; The advent of high-density gene array technology has revolutionized approaches to drug design, development, and characterization . At the laboratory level, the efficient, consistent, and dependable exploitation of this complex technology requires the stringent standardization of protocols and data analysis platforms . The Affymetrix YE6100 expression GeneChip platform was evaluated for its performance in the analysis of both global (6,000 yeast genes) and targeted (three pleiotropic multidrug resistance genes of the ATP binding cassette transporter family) gene expression in a heterologous yeast model system in the presence and absence of the antimalarial drug chloroquine . Critical to the generation of consistent data from this platform are issues involving the preparation of the specimen, use of appropriate controls, accurate assessment of experiment variance, strict adherence to optimized enzymatic and hybridization protocols, and use of sophisticated bioinformatics tools for data analysis. J Org Chem, 2000 Apr 21, 65(8), 2479 - 83 Total synthesis of ningalin B utilizing a heterocyclic azadiene Diels-Alder reaction and discovery of a new class of potent multidrug resistant (MDR) reversal agents; Boger DL et al.; A concise, efficient approach to the total synthesis of ningalin B (1) based on a heterocyclic azadiene Diels-Alder strategy (1,2,4,5-tetrazine-->1,2,-diazine-->pyrrole) ideally suited for construction of the densely functionalized pyrrole core found in the natural product is detailed . Examination of the natural product and a number of synthetic intermediates revealed that while lacking inherent cytotoxic activity, many reverse the multidrug-resistant (MDR) phenotype, resensitizing a human colon cancer cell line (HCT116/VM46) to vinblastine and doxorubicin at lower doses than the prototypical agent verapamil. Eur J Cancer, 2000 May, 36(7), 881 - 8 Incidence of P-glycoprotein overexpression and multidrug resistance (MDR) reversal in adult soft tissue sarcoma; Coley HM et al.; Multidrug resistance (MDR) is a widespread problem in the treatment of neoplastic diseases and may limit the effectiveness of treatment of adult soft tissue sarcomas (STS) . We examined the levels of expression of the MDR marker P-glycoprotein (Pgp) in fresh, surgical material and matched paraffin-embedded tissue using MRK-16 and JSB-1 monoclonal antibodies . Using fresh tumour material in short-term culture an assessment of doxorubicin sensitivity (MTT assay) and MDR modulation using PSC-833 in daunorubicin (DNR) accumulation experiments (FACS analysis) was carried out . 44 patients were studied at various disease stages with a mean follow-up duration of 487 days (range: 45-1095 days) . Immunocytochemistry and immunohistochemistry showed 62% and 58%, respectively, of STS samples were positive for Pgp . Patients showing negative Pgp expression had a median survival of 544 days versus 431 days for Pgp-positive patients (P=0.311), with disease-free survival medians of 508 and 355 days, respectively (P=0.203) . In vitro doxorubicin sensitivity was not informative in this respect and there was no apparent relationship between this and Pgp expression . Eleven out of 29 samples evaluated for MDR modulation showed enhanced tumour cell DNR accumulation . However, the effects of PSC-833 on drug accumulation in clinical material were modest compared with those seen for MDR cell lines, with a maximum of only 20% enhancement . Moreover, there was no relationship between the extent of PSC-833 effects on accumulation and the levels of Pgp seen in the STS samples . Nevertheless, we show evidence that a proportion of cases of STS express moderate to high levels of Pgp . There may be a role for MDR modulating agents in association with doxorubicin in the treatment of these tumours, either in the adjuvant setting or at first relapse. Mod Pathol, 2000 Apr, 13(4), 407 - 13 Effects of multidrug resistance gene expression in acute erythroleukemia; Mazzella FM et al.; Acute erythroleukemia is a relatively rare disorder of a multilineal nature . Patients with this type of leukemia traditionally have been treated with a standard myeloid protocol, with a wide variation in prognosis between M6a, which has a similar prognosis to acute myelogenous leukemias, and M6b, with an extremely poor outcome despite aggressive therapy . Forty-eight archival cases of acute erythroleukemia, subtypes M6a (the traditional FAB-M6), M6b (pure erythroleukemia), and M6c (>30% myeloblasts and >30% pronormoblasts by FAB exclusion criteria), were evaluated for multidrug resistance gene (MDR-1) status . Findings were correlated with clinical course and karyotypes . Immunohistochemical stain for the protein product of MDR-1, P-glycoprotein, was variably positive in 11 of 23 patients with M6a, as well as in all of the patients with M6b (strongly positive) and M6c (weakly positive) . P-glycoprotein expression positively correlated with unfavorable cytogenetic aberrations, poor response to chemotherapeutic agents, and short survival . Most significant was that P-glycoprotein expression demonstrated a negative additive effect on response to treatment and prognosis with unfavorable cytogenetic anomalies . P-glycoprotein expression and multiple cytogenetic anomalies most probably contribute to the resistance to chemotherapy and poor survival characteristic of the patients with M6b (mean survival, 3.15 +/- 4.2 mo) and M6c (mean survival, 10.5 +/- 12.7 mo) . Because patients with M6b and M6c have increased numbers of pronormoblasts in their bone marrow and past chemotherapeutic attempts have failed, chemotherapy directed at these cells is appropriate . Additional therapy directed toward the MDR-1 gene and its protein product seems indicated from our findings. J Clin Oncol, 2000 May, 18(9), 1867 - 75 Mitoxantrone, etoposide, and cyclosporine therapy in pediatric patients with recurrent or refractory acute myeloid leukemia; Dahl GV et al.; PURPOSE: To determine the remission rate and toxicity of mitoxantrone, etoposide, and cyclosporine (MEC) therapy, multidrug resistance-1 (MDR1) status, and steady-state cyclosporine (CSA) levels in children with relapsed and/or refractory acute myeloid leukemia . PATIENTS AND METHODS: MEC therapy consisted of mitoxantrone 6 mg/m(2)/d for 5 days, etoposide 60 mg/m(2)/d for 5 days, and CSA 10 mg/kg for 2 hours followed by 30 mg/kg/d as a continuous infusion for 98 hours . Because of pharmacokinetic interactions, drug doses were decreased to 60% of those found to be effective without coadministration of CSA . MDR1 expression was evaluated by reverse transcriptase polymerase chain reaction, flow cytometry, and the ability of CSA at 2.5 micromol/L to increase intracellular accumulation of (3)H-daunomycin in blasts from bone marrow specimens . RESULTS: The remission rate was 35% (n = 23 of 66) . Overall, 35% of patients (n = 23) achieved complete remission (CR), 12% of patients (n = 8) achieved partial remission, and 9% of patients (n = 6) died of infection . Exposure to CSA levels of greater than 2,400 ng/mL was achieved in 95% of patients (n = 56 of 59) . Toxicities included infection, cardiotoxicity, myelosuppression, stomatitis, and reversible increases in serum creatinine and bilirubin . In most who had relapsed while receiving therapy or whose induction therapy had failed, response was not significantly different for MDR1-positive and MDR1-negative patients . CONCLUSION: Serum levels of CSA capable of reversing multidrug resistance are achievable in children with acceptable toxicity . The CR rate of 35% achieved in this study is comparable to previously reported results using standard doses of mitoxantrone and etoposide . The use of CSA may have improved the response rate for the MDR1-positive patients so that it was not different from that for the MDR1-negative patients. Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 209 - 16 Modulation of multidrug resistance in human leukemia cells with mdr1-targeted antisense oligonucleotides using variable treatment schedules; Dassow H et al.; OBJECTIVE: The purpose of the current study was to characterize the effect of chimeric AS-ODNs encapsulated with cationic lipids on MDR in human leukemia cells and to determine if this modification of the ODN alone or in combination with the cationic lipid might offer advantages over classical ODN treatment with free unmodulated or phosphorothiolated AS-ODNs . Furthermore, we extended the antisense method to the use of AS-ODNs in the parental drug-sensitive leukemia cells which express mdr1-mRNA at a relative low level and lack P170 expression to evaluate the effectiveness of prophylactic AS-ODN treatment . METHODS: The effect of a 4-day AS-ODN treatment in drug-resistant human leukemia cells which exhibit the classic MDR phenotype at a moderate level was examined . Twenty-four hours after the last ODN administration the cells were analyzed for mdr1-mRNA (quantitative RT-PCR) and P170 expression (FCM), for R123 accumulation/efflux capacity (FCM) and for sensitivity to vincristine (MTT) . In the parental drug-sensitive CCRF-CEM cells the mdr1-mRNA expression was assessed 24, 48 and 72 h after AS-ODN treatment administered as free phosphorothioate or conjugated with DMRIE-C . RESULTS: Cationic lipids produced a clear increase in cellular ODN uptake but also caused an increase in variability of uptake rates (30% vs . 10% variability after free phosphorothioates) . Both AS-ODNs inhibit P170 expression whereby the antisense effect of the chimeric ODN seems to be stronger compared to the phosphorothioate (30% vs . 22% MRK16 staining) . Consistent with the inhibition of P170 expression, an increased sensitivity to vincristine was observed . In parental drug-sensitive cells, AS-ODN treatment caused nearly complete inhibition of mdr1-mRNA expression (5% of control) . CONCLUSION: The data demonstrate that it is nearly impossible to achieve a complete reversal of the MDR phenotype in drug-resistant cells using AS-ODNs . A more promising approach seems to be the prophylactic treatment with AS-ODNs. Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 196 - 203 Resistance modulation in CHO cells by R-verapamil and bile salts is associated with physical and chemical changes in the cell membrane; Dolderer JH et al.; OBJECTIVES: Changes in multidrug resistance by resistance modifiers such as R-verapamil cause changes in fluidity of the cell membrane . The extent to which these changes involve structural alterations in membrane lipids has been investigated in CHO cells . METHODS: Sensitive (AUXB1) and resistant (CH(R)C5) chinese hamster ovary cells (CHO) were grown in culture . Incubations were carried out with R-verapamil (0-10 microM) or the membrane perturbing agents tauro-cheno-deoxycholate (0-1.6 mM, TCDC) and tauro-urso-deoxycholate (0-3.5mM, TUDC) . Cell membrane fluidity was determined by electron-paramagnetic resonance spectroscopy and membrane lipids by HPLC and TLC . RESULTS: The resistant CH(R)C5 subline had a higher cell membrane order (lower fluidity, S = 0.7234) in the interface region of the cell membrane than sensitive AUXB1 cells (S = 0.6984) determined using EPR . The MDR-modulator R-verapamil and TCDC, but not TUDC, lowered cell membrane order in a concentration-dependent manner and increased membrane fluidity of the resistant CH(R)C5 subline . TCDC and R-verapamil were without effect on the cell membrane fluidity of AUXB1 cells . These changes were accompanied by alterations in the fatty acid composition of the plasma membrane . Untreated sensitive AUXB1 cells had higher levels of unsaturated fatty acids than resistant CH(R)C5 cells . In CH(R)C5 cells, R-verapamil increased the content of poly-unsaturated fatty acids and TCDC, but not TUDC, increased the content of mono-unsaturated fatty acids . CONCLUSIONS: The results demonstrate that resistance modifiers such as verapamil may influence cytostatic drug action by producing structural changes to lipid domains in the plasma membrane. Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 187 - 95 Diverse effects of P-glycoprotein inhibitory agents on human leukemia cells expressing the multidrug resistance protein (MRP); Lehne G et al.; Multidrug resistance (MDR) to cancer chemotherapy is frequently associated with decreased drug accumulation in cancer cells due to drug expulsion by multidrug transporters such as P-glycoprotein (Pgp) and multidrug resistance protein (MRP) . The novel resistance modifying agents PSC 833, 280-446, and LY 335979 are primarily targeted at inhibition of Pgp, and their MRP inhibitory potential is largely unknown . OBJECTIVE: In the present study we addressed the effect of these agents on MRP-derived drug resistance . MATERIALS: Drug-resistant human leukemia cells with Pgp+/MRP- (KG1a/200, K562/150) and Pgp-/MRP+ (HL60/130) phenotypes were maintained in suspension cultures for experimental studies of drug accumulation and drug sensitization by Pgp inhibitors . METHODS: Intracellular accumulation of the fluorescent anthracycline daunorubicin was measured by flow cytometry and fluorescence detection . Daunorubicin dose-response curves were generated by non-linear regression of electronically measured cell counts of 72- - 96-h cultures . The half-maximal growth inhibitory dose (GI50) was used as measure of growth inhibition . RESULTS: All MDR phenotypes studied exercised significant resistance to daunorubicin . PSC 833, 280-446 and LY335979 were equal in sensitizing Pgp+/MRP- cells to daunorubicin-induced growth inhibition (p < 0.0001) . The Pgp-/MRP+ cells responded to PSC 833 and 280-446 by increased accumulation of daunorubicin (p = 0.0022 and p = 0.0005, respectively) and sensitization to the drug (p = 0.0009 and p = 0.0007, respectively) . Conversely, LY335979 did not affect accumulation of daunorubicin in Pgp-/MRP+ cells nor sensitize these cells to daunorubicin . CONCLUSION: Pgp inhibitory agents have differential effects on MRP-derived drug resistance which could be exploited in treatment of multidrug resistance in cancer patients. Int J Clin Pharmacol Ther, 2000 Apr, 38(4), 180 - 6 Simultaneous measurement of cellular P-glycoprotein content and function by multiparametric flow-cytometry; Muller MR et al.; OBJECTIVE: A multiparametric approach was applied to simultaneously determine expression and function of the drug efflux pump P-glycoprotein (PGP) in multidrug-resistant (MDR) human leukemic lymphoblast cell lines and isolated leukemic blasts using flow-cytometry in a patient with acute myeloid leukemia (AML) . METHODS: The antigen was measured by staining PGP using the monoclonal antibody 4e3 which does not inhibit the function of PGP . The 4e3 antibody binds to an external epitope of PGP and can therefore be used for staining living cells . Drug transport, mediated by PGP, was determined simultaneously by measuring rhodamine 123 (rho123) efflux . The MDR cell lines, CEM/VLB10-2 and CEM/VBL100 are 10-fold and 270-fold resistant to vinblastine (VBL), respectively, compared to the human PGP-negative parent cell line CEM/WT and they express different amounts of PGP . Initially, living cells were stained using the 4e3 antibody and a secondary antibody labeled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA) . Cells were then incubated for 60 min with rho123 (10 microM) and analyzed for rhodamine and AMCA-derived fluorescence . The decrease in rho123 fluorescence was determined after a further period of 30 min . RESULTS: CEM/VLB100 cells expressed larger amounts of PGP, and rho123 fluorescence after 30 min was 85% lower than the parent cell line . PGP expression and rho123 efflux were also detected in CEM/VLB10-2 cells which display a low degree of resistance, thus reflecting the high sensitivity of this method . PGP-expressing blasts and moderate rho123 efflux were also observed in a specimen derived from a patient with clinically resistant acute myeloid leukemia (AML) . CONCLUSION: A multiparametric approach using flow-cytometry allows the reliable and sensitive measurement of both PGP expression and function simultaneously in single cells. Acta Clin Belg, 2000 Jan-Feb, 55(1), 34 - 6 Multidrug-resistant tuberculosis spondylitis; Cherifi S et al.; We report a case of multidrug-resistant spinal tuberculosis complicated by epiduritis and paraspinal abscess in a 68-year-old black woman . Multidrug-resistant tuberculous spondylitis is still rare in Belgium . Two others cases were reported from 1992 to 1997 . The optimal therapy is not standardized and the mandatory duration of treatment is not known . Clinical presentation, radiological findings, and treatment are presented . The need for prompt diagnosis and optimal therapy is emphasized. Curr Opin Pulm Med, 2000 May, 6(3), 193 - 7 Advances in the management of tuberculosis: clinical trials and beyond; Grange JM et al.; Modern short-course treatment for tuberculosis is highly effective and cost-effective, yet the disease remains a leading cause of suffering and death . The problem has been exacerbated in recent years by the human immunodeficiency virus (HIV) pandemic and the increasing prevalence of multidrug-resistant tuberculosis . Improvements in diagnosis, vaccination, chemoprophylaxis, and therapy are thus urgently needed . Molecular techniques are facilitating the development of rapid and sensitive diagnostic tests and the rational approach to the production of new vaccines . New forms of treatment are being investigated and there is also considerable emphasis on optimizing the deployment of the available treatment regimens . This has resulted in the World Health Organization's five-point directly observed therapy, short course (DOTS) strategy and proposed modifications (DOTS-plus) for the management of multidrug-resistant (MDR) tuberculosis . Despite these advances, it is becoming abundantly clear that the failure to control tuberculosis is a direct consequence of the gross inequities in the distribution of wealth and health care provision worldwide, which do not allow for putting advances in the management of tuberculosis into practice . The control of tuberculosis will therefore require attention to justice and human rights as well as greatly increased technical and financial support from the developed nations. Curr Opin Pulm Med, 2000 May, 6(3), 174 - 9 Dynamics and control of the global tuberculosis epidemic; Bleed D et al.; Studies of disease burden have reaffirmed that tuberculosis is among the top 10 causes of death in the world . The tuberculosis epidemic in most countries could eventually be brought under control by implementing the World Health Organization's (WHO) directly observed therapy, short course (DOTS) strategy, although tuberculosis linked to human immunodeficiency virus (HIV) in Africa and multidrug-resistant tuberculosis (MDR-TB) in the former Soviet Union urgently demand adaptations and extensions of DOTS . Most high-incidence countries have achieved treatment success rates approaching the WHO 85% target in pilot projects . In the long term, we may have better diagnostics, drugs, and vaccines to control the disease; for the next 5 years, the central problem in global tuberculosis control is to expand DOTS coverage in high-incidence countries . Improved case finding and diagnosis, coupled with best-practice short-course chemotherapy, could quickly and dramatically cut the number of years of healthy life lost due to tuberculosis, especially by preventing death. Leuk Res, 2000 Jun, 24(6), 535 - 41 In vitro leukemia cell models of Ara-C resistance; Funato T et al.; Cells of the human leukemia line K562 were continuously exposed to cytosine arabinoside (Ara-C) at increasing concentrations for 3 months . The resulting cell line, termed K562/AC, showed 48-fold resistance to Ara-C, compared with the parental K562 cells . The sensitivities of K562/AC to adriamycin (ADR), vincristine (VCR) and etoposide (VP16) were similar to those of parental K562 . Gene analysis revealed that this cell line lacked expression of the deoxycytidine kinase (dCK) gene, which was evident in Ara-C-sensitive cells . As in K562 cells, multidrug resistance (MDR-1) and multidrug resistance protein (MRP) genes were not expressed in K562/AC . We also established an in vitro model of Ara-C resistance using phosphorothioate antisense oligonucleotides to dCK (dCK-AS) . Treatment of K562 with dCK-AS caused decreased dCK expression and 6- to 10-fold increases in resistance to Ara-C, compared with that in cells treated with sense oligonucleotides to dCK (dCK-S) or in non-transfected cells . The cells described here may contribute to the study of a novel mechanism associated with Ara-C resistance, in which reduced dCK activity may play an important role. J Biol Chem, 2000 Jul 7, 275(27), 20280 - 7 Allosteric interactions between the two non-equivalent nucleotide binding domains of multidrug resistance protein MRP1; Hou Y et al.; Membrane transporters of the adenine nucleotide binding cassette (ABC) superfamily utilize two either identical or homologous nucleotide binding domains (NBDs) . Although the hydrolysis of ATP by these domains is believed to drive transport of solute, it is unknown why two rather than a single NBD is required . In the well studied P-glycoprotein multidrug transporter, the two appear to be functionally equivalent, and a strongly supported model proposes that ATP hydrolysis occurs alternately at each NBD (Senior, A . E., al-Shawi, M . K., and Urbatsch, I . L . (1995) FEBS Lett 377, 285-289) . To assess how applicable this model may be to other ABC transporters, we have examined adenine nucleotide interactions with the multidrug resistance protein, MRP1, a member of a different ABC family that transports conjugated organic anions and in which sequences of the two NBDs are much less similar than in P-glycoprotein . Photoaffinity labeling experiments with 8-azido-ATP, which strongly supports transport revealed ATP binding exclusively at NBD1 and ADP trapping predominantly at NBD2 . Despite this apparent asymmetry in the two domains, they are entirely interdependent as substitution of key lysine residues in the Walker A motif of either impaired both ATP binding and ADP trapping . Furthermore, the interaction of ADP at NBD2 appears to allosterically enhance the binding of ATP at NBD1 . Glutathione, which supports drug transport by the protein, does not enhance ATP binding but stimulates the trapping of ADP . Thus MRP1 may employ a more complex mechanism of coupling ATP utilization to the export of agents from cells than P-glycoprotein. Cancer Lett, 2000 May 29, 153(1-2), 95 - 100 Expression of mediated P-glycoprotein multidrug resistance related to Tc-99m MIBI scintimammography results; Sun SS et al.; We prospectively studied a total of 24 patients with breast cancer to evaluate the relationship between the degree of accumulation of technetium-99m sestamibi (Tc-99m MIBI) and p-glycoprotein (Pgp) expression in tumor tissues . All 24 patients underwent Tc-99m MIBI scintimammography before surgery or biopsy . Immunohistochemical studies were performed on multiple non-consecutive sections of the same tumor using a Pgp specific monoclonal antibody, JSB-1 . Planar images were started 10 min after injection of Tc-99m MIBI . Tumor to background (T/B) ratios calculated from the planar images were correlated with Pgp expression as determined by immunohistochemical studies . The T/B ratios were significantly lower for tumors in eight patients with positive Pgp expression (Group 1) than in 16 patients with negative expression (Group 2) (1.40+/-0.11 and 2.76+/-0.60, P<0 . 05) . Our data confirmed that Tc-99m MIBI scintimammography is useful for determination of the presence of multidrug resistance due to Pgp expression in patients with breast cancer. Blood, 2000 May 1, 95(9), 2897 - 904 P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin-ceramide apoptotic pathway; Pallis M et al.; P-glycoprotein (pgp), which is the product of the MDR1 (multidrug resistance-1) gene, has an established role as a mediator of cytotoxic drug resistance in acute myeloid leukemia (AML) . To study the role of pgp in mediating apoptosis resistance in AML cells deprived of serum and growth factors, apoptosis was quantified by flow cytometry using uptake of the dye 7-amino-actinomycin D (7-AAD) alongside low forward scatter . In pgp+ve primary AML samples, there was a significant increase in apoptosis in the presence of the pgp-specific antibody UIC2 (mean increase: 58%; range: 11%-95%; P < . 05) . Likewise, apoptosis in growth factor-deprived TF1 cells cultured for 30 hours increased 2.5-fold in the presence of 25 microg/mL UIC2 . The pgp reversal agent PSC-833 (1 micromol/L) augmented in vitro apoptosis by a median of 52% in pgp+ve patient samples and to a comparable degree in 6 pgp-ve samples . To determine whether the sphingomyelin-ceramide (SM-ceramide) pathway of apoptosis occurs in AML blasts in response to cytotoxic drugs, cells were incubated with daunorubicin at the patient-specific IC(30) (the concentration of daunorubicin that caused apoptotic cell death in 30% of cells) in the presence of the ceramide synthase inhibitor fumonisin B1, which inhibited apoptosis by 18%-81% (median: 40%) . Exogenous SM failed to augment apoptosis induced by growth factor withdrawal in pgp+ve TF1 cells and was significantly more effective at augmenting apoptosis in pgp-ve patient blasts (median increase in cell death: 33%; range: 19%-88%) than in pgp+ve samples (median: 7%; range: 0%-27%; P =.028) . Cellular accumulation of exogenous SM was associated with apoptosis and also occurred in nonapoptotic patient cells treated with PSC-833 . However, this effect was not seen following treatment with the UIC2 antibody . These results indicate that pgp is able to exert a protective effect on AML cell viability and that this is associated with a reduced effect of exogenous SM on apoptosis . The pgp reversal agent PSC-833 acts, at least in part, by a pgp independent mechanism to alter SM distribution and to augment apoptosis induced in AML cells by serum and growth factor withdrawal . (Blood . 2000;95:2897-2904) J Assoc Physicians India, 1999 Sep, 47(9), 883 - 5 An open study to evaluate the efficacy of artemether in severe falciparum malaria; Sharma P et al.; An open clinical trial was conducted in 30 patients of severe falciparum malaria with heavy parasitaemia (parasitized erythrocytes above 5%) . Artemether (methyl ether of dihydroartemisinin-active principle isolated from Chinese plant Qinghaosu) was administered as 80 mg intramuscular injection twice on first day and then single dose of 80 mg intramuscular on 2nd to 5th day . The trial could be completed in 28 patients and two patients expired . In our observation falciparum malaria affected the young adults in their most productive period of life i.e . 25-44 yrs . All patients became afebrile by the 4th day with fever clearance time approximately 31.92 +/- 15.30 hr . Twenty-five patients (83.33%) became parasite free by 5th day with mean parasite clearance time approximately 47.04 +/- 19.95 hr . Deranged liver function and renal profile was observed in 63% and 50% patients respectively . Two patients, who died had very high degree of parasitaemia (50% and 16%) with cerebral malaria . One died due to multiorgan failure and other due to massive hematemesis and shock . The type of response achieved by artemether therapy was analysed as per WHO criteria suggested for chloroquine resistance . S response was observed in 25 patients (cure rate 83.33%) . Two patients (6.66%) patients showed R II response, one patient (3.33%) showed R III response and R I response was not observed in any patient . No significant side effects were noted . This pilot study demonstrated that intramuscular artemether is a useful addition to antimalarial drugs in this era of multidrug resistant P . falciparum malaria showing high clinical potency with virtually no side effect. J Biol Chem, 2000 Apr 28, 275(17), 13098 - 108 Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1; Gao M et al.; Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics . The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates . It is not known whether the two NBDs of MRP1 are functionally equivalent . To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-{(32)P}ATP and to trap 8-azido-{(32)P}ADP in the presence of orthovanadate . We found that NBD1 was preferentially labeled with 8-azido-{(32)P}ATP, while trapping of 8-azido-{(32)P}ADP occurred predominantly at NBD2 . Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH(2)-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide . Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2 . Both mutations decreased leukotriene C(4) (LTC(4)) transport by approximately 70% . Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC(4) transport activity without affecting labeling of NBD1 with 8-azido-{(32)P}ATP. J Virol, 2000 May, 74(10), 4621 - 33 Identification of a key target sequence to block human immunodeficiency virus type 1 replication within the gag-pol transframe domain; Sei S et al.; Although the full sequence of the human immunodeficiency virus type 1 (HIV-1) genome has been known for more than a decade, effective genetic antivirals have yet to be developed . Here we show that, of 22 regions examined, one highly conserved sequence (ACTCTTTGGCAACGA) near the 3' end of the HIV-1 gag-pol transframe region, encoding viral protease residues 4 to 8 and a C-terminal Vpr-binding motif of p6(Gag) protein in two different reading frames, can be successfully targeted by an antisense peptide nucleic acid oligomer named PNA(PR2) . A disrupted translation of gag-pol mRNA induced at the PNA(PR2)-annealing site resulted in a decreased synthesis of Pr160(Gag-Pol) polyprotein, hence the viral protease, a predominant expression of Pr55(Gag) devoid of a fully functional p6(Gag) protein, and the excessive intracellular cleavage of Gag precursor proteins, hindering the processes of virion assembly . Treatment with PNA(PR2) abolished virion production by up to 99% in chronically HIV-1-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates with the multidrug-resistant phenotype . This particular segment of the gag-pol transframe gene appears to offer a distinctive advantage over other regions in invading viral structural genes and restraining HIV-1 replication in infected cells and may potentially be exploited as a novel antiviral genetic target. Joint Bone Spine, 2000 Jan, 67(1), 40 - 8 Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders . Part II: Increased P-glycoprotein activity in lymphocytes from systemic lupus erythematosus patients might affect steroid requirements for disease control; Diaz-Borjon A et al.; BACKGROUND: Over-expression of the membrane glycoprotein called P-glycoprotein has been widely observed in a variety of both normal and neoplastic cells . P-glycoprotein is a pump molecule that transports hydrophobic drugs (including steroids) and toxins outside the cells, thus inhibiting their therapeutic or toxic effects . The gene encoding P-glycoprotein is named multidrug resistance-1 (MDR-1) . OBJECTIVE: To evaluate the functional activity of P-glycoprotein in lymphocytes and monocytes from patients with systemic lupus erythematosus . METHODS: 30 systemic lupus erythematosus patients and 20 healthy controls were studied . Peripheral blood mononuclear cells isolated by gradient centrifugation were incubated in the presence of daunorubicin (a fluorescent drug extruded by P-glycoprotein) at 37 degrees C or 4 degrees C for 30 min . P-glycoprotein activity was then analyzed using flow cytometry . Results were expressed as the percentage of lymphocytes or monocytes with high P-glycoprotein activity (i.e., low fluorescence) . RESULTS: Mean fluorescence values for lymphocytes and monocytes were comparable between patients and healthy controls . However, because our method allowed to measure P-glycoprotein function at the single-cell level, we were able to show that the mean percentage of lymphocytes with high P-glycoprotein activity was increased in the patients (11.51% +/- 14.3%) as compared to the healthy controls (0.71% +/- 0.57%) (P < 0.05) . Moreover, P-glycoprotein activity was lower in the patients in clinical remission than in those with active disease . CONCLUSIONS: Our results suggest that P-glycoprotein function might affect glucocorticoid requirements in systemic lupus erythematosus. Joint Bone Spine, 2000 Jan, 67(1), 30 - 9 Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders . Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid arthritis patients might influence disease outcome; Llorente L et al.; BACKGROUND: Multidrug resistance (MDR) is characterized by overexpression of P-glycoprotein, a pump molecule that decreases intracellular drug concentrations by increasing drug efflux from cells . OBJECTIVE: To look for correlations between clinical status and P-glycoprotein activity and/or TNF-alpha mRNA levels in patients with rheumatoid arthritis . METHODS: Sixteen patients were studied . Based on response to therapy, eight were refractory and eight nonrefractory to treatment . Findings were compared to those in 24 healthy controls . Flow cytometry was used to evaluate P-glycoprotein activity in peripheral blood mononuclear cells isolated by gradient centrifugation and incubated with the P-glycoprotein substrate daunorubicin . TNF-alpha mRNA levels were determined using quantitative PCR . RESULTS: Patients with rheumatoid arthritis showed an increased number of lymphocytes with high P-glycoprotein activity (p = 0.0001) as compared to the normal controls . P-glycoprotein activity was higher in the refractory than in the non-refractory patient subgroup (p = 0.006) . Also, TNF-alpha mRNA levels were markedly higher in the refractory subgroup than in the nonrefractory subgroup, and were undetectable in the normal controls . CONCLUSIONS: Enhanced P-glycoprotein activity may be closely related to an unfavorable clinical course and a poor response to treatment . Increased TNF-alpha expression and chronic exposure to various drugs, including glucocorticoids, may contribute to increase P-glycoprotein activity . Both high P-glycoprotein activity and excessive amounts of TNF-alpha seem associated with poor outcome in rheumatoid arthritis. Nucl Med Biol, 2000 Feb, 27(2), 135 - 41 Assessment of the in vitro and in vivo properties of a (99m)Tc-labeled inhibitor of the multidrug resistant gene product P-glycoprotein; Bergmann R et al.; Overexpression of P-glycoprotein (Pgp), which is present in the plasma membrane of various tumor cells and in several normal cell types, contributes to the multidrug resistance (MDR) phenotype of many human cancers . As a prerequisite for therapy, the expression of Pgp must be studied . The available clinical radiopharmaceuticals for studying the expression of Pgp include the lipophilic (99m)Tc cations (sestamibi, tetrofosmin) as well as {(99m)Tc}Q57, {(99m)Tc}Q58, and {(99m)Tc}Q63 . Here we describe the in vitro and in vivo properties of the structurally different complex (3-thiapentane-1, 5-dithiolato){{N-(3-phenylpropyl)-N-2(3-quinazoline-2, 4-dionyl)-ethyl}amino-ethylthiolato inverted question mark oxotechnetium(V) ((99/99m)Tc1) as a potential inhibitor of Pgp . (99)Tc1 enhances the net cell accumulation of Pgp substrates {(3)H}vinblastine, {(3)H}vincristine, {(3)H}colchicine, {(99m)Tc}sestamibi, and {(99m)Tc}tetrofosmin in rat brain endothelial cells (RBE4), an immortalized endothelial cell line that expresses Pgp . In addition, the cell accumulation of (99m)Tc1 could be increased by verapamil and reserpine, which are known Pgp inhibitors . A multitracer approach was used to study the side effects of (99)Tc1 on cell metabolism . The cells were simultaneously incubated with {(99m)Tc}sestamibi, 2-{(18)F}fluoro-2-deoxyglucose ({(18)F}FDG), and various (3)H-labeled tracers . Two-dimensional scatter plots of {(99m)Tc}sestamibi uptake/{(18)F}FDG uptake show typical changes of known Pgp inhibitors including (99)Tc1 . The effects of (99)Tc1 on the in vivo distribution of {(99m)Tc}sestamibi and {(18)F}FDG in rats also are comparable with the effects of verapamil, an established Pgp inhibitor and calcium channel blocker . We conclude that (99/99m)Tc1 is a transport substrate and a potential inhibitor of Pgp . Our approach may be useful in the design of further radiotracers with specificity to Pgp. Int J Antimicrob Agents, 2000 Apr, 14(3), 193 - 201 Chemistry and biological activity of new 3-benzazepines; Kawase M et al.; This review summarizes our experiments investigating structure-activity relationships of 3-benzazepines . Three 7, 8-dihydroxy-3-benzazepines {7-9} were cytotoxic to human promyelotic leukaemia HL-60 cells . Compound {9} showed the highest cytotoxicity and the activity was twice as high as that of dopamine (DA, {11}) . Three active compounds {7-9} produced radicals, whereas other less potent benzazepines {1-6, 10} did not produce radicals . Furthermore, cytotoxic 3-benzazepines {7-9} also enhanced the decay of ascorbic acid in rat brain homogenate . Two 7,8-dimethoxy-3-benzazepines {5, 10} were able to form a complex with the replicative form of plasmid DNA . The multidrug resistance (MDR) P-glycoprotein (Pgp) efflux pump of mouse lymphoma cells was inhibited by three compounds {5, 8, 10} . Compound {8} has the highest activity in MDR reversal and is two times more potent than verapamil . Three cytotoxic 3-benzazepines {7-9} showed inhibitory effects against reverse transcriptase (RT) of Moloney leukemia. Int J Antimicrob Agents, 2000 Apr, 14(3), 173 - 6 Phenothiazines: an alternative to conventional therapy for the initial management of suspected multidrug resistant tuberculosis . A call for studies; Amaral L et al.; Increased frequency of multidrug resistant strains of Mycobacterium tuberculosis results from inappropriate treatment and lack of patient compliance . The Center for Disease Control/American Thoracic Society (CDC/ATS) guidelines issued for the management of newly diagnosed cases of tuberculosis (TB) will not be totally effective regardless of adherence to the guidelines and patient cooperation . The long interim period between the diagnosis of TB and confirmation of antibiotic susceptibility contributes to the infection rate . Consequently, the use of an adjuvant that is known to inhibit all encountered multidrug resistant strains of M . tuberculosis may be helpful until antibiotic susceptibility is known . Phenothiazines such as chlorpromazine, methdilazine and thioridazine are effective against strains of M . tuberculosis in vitro and in vivo . It is recommended that studies be designed and conducted for the purpose of managing new cases of TB that emanate from areas known to harbour multidrug resistant strains of M . tuberculosis, with phenothiazines as adjuvants to the regimen recommended by the CDC/ATS guidelines until antibiotic susceptibility is defined . Because the normal maximum period for obtaining conventional antibiotic susceptibility results is less than 7 or 8 weeks, the probability of serious side effects from the use of a phenothiazine is remote. J Pharmacol Exp Ther, 2000 May, 293(2), 530 - 8 Verapamil stimulates glutathione transport by the 190-kDa multidrug resistance protein 1 (MRP1); Loe DW et al.; Multidrug resistance in tumor cells is often associated with reduced drug accumulation resulting from increased expression of the 190-kDa multidrug resistance protein 1 (MRP1) or the 170-kDa P-glycoprotein . However, unlike P-glycoprotein, MRP1 is a primary active transporter of many conjugated organic anions, including the cysteinyl leukotriene LTC(4) . Moreover, agents such as verapamil that reverse P-glycoprotein-mediated resistance are often poorly, or not at all, effective in MRP1-overexpressing cells . In the present study, we investigated the effects of verapamil on MRP1-mediated transport processes . We found that verapamil inhibited LTC(4) transport into inside-out membrane vesicles prepared from MRP1-transfected cells in a competitive manner, but only in the presence of reduced glutathione (GSH) or its nonreducing S-methyl derivative . In the presence of 1 mM GSH, the apparent K(i) for verapamil was 1.2 microM, and in the presence of 100 microM verapamil, the apparent K(i) for GSH was 77 microM . Verapamil itself was not transported by MRP1 in either intact cells or membrane vesicles . However, verapamil strongly stimulated MRP1-mediated GSH uptake by membrane vesicles in a concentration-dependent and osmotically sensitive manner that was inhibitable by MRP1-specific monoclonal antibodies . In the presence of 100 microM verapamil, the apparent K(m) and V(max) for GSH uptake were 83 microM and 55 pmol mg(-1) min(-1), respectively . It is proposed that the variable ability of verapamil to modulate MRP1-mediated resistance in different cell lines may be more closely linked to its effect on the GSH status of the cells than on its ability to inhibit the MRP1 transporter itself. Drug Metab Dispos, 2000 May, 28(5), 522 - 8 In vitro flow cytometry method to quantitatively assess inhibitors of P-glycoprotein; Wang EJ et al.; P-glycoprotein (Pgp)-mediated drug efflux is a major factor contributing to the variance of absorption and distribution of many drugs . A simple and reliable in vitro method to identify inhibitors of Pgp helps to prevent the potential of drug interactions . Using daunorubicin as a fluorescent marker and vanadate as a positive control compound, a functional flow cytometry method for assessing the ability of a drug to inhibit Pgp-mediated drug efflux from CR1R12 multidrug-resistant cells has been evaluated . Quantitation of the relative fluorescence was used to compare potency of individual inhibitors . Known Pgp inhibitors, such as cyclosporin A, nicardipine, verapamil, quinidine, terfenadine, tamoxifen, and vinblastine were demonstrated to inhibit the Pgp-mediated efflux of daunorubicin . Cyclosporin A and terfenadine were the most potent inhibitors among the compounds tested . Tetraphenylphosphonium and alpha-tocopherol had little inhibitory effect . Progesterone produced significant inhibition at relatively high concentrations . This study demonstrated that this in vitro flow cytometry method is a simple, sensitive, and quantitative tool to assess the capacity of a drug to inhibit Pgp transporters, and is useful for screening or identifying inhibitors of Pgp as well as evaluation of potential for drug interactions. Southeast Asian J Trop Med Public Health, 1998 Dec, 29(4), 696 - 701 A clinical trial of combination of artesunate and mefloquine in the treatment of acute uncomplicated falciparum malaria: a short and practical regimen; Wilairatana P 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 fall in patient compliance not only lowers cure rates but also predisposes to the further spread of drug-resistance . Sequential treatment with artesunate given over 5 days followed by mefloquine produced 100% cure rates in previous study, but might not be a suitable regimen for field treatment . We conducted a clinical trial of a combination of artesunate and mefloquine given twice daily for 2 days in 150 patients with acute uncomplicated falciparum malaria . The dose of artesunate (200 mg) and mefloquine (312.5 mg) were given simultaneously in a separate package . All patients were admitted to a hospital in Bangkok for 28 days to exclude re-infection and monitor the possible adverse effects . One hundred and thirty patients completed the study with 28 days follow up . Twenty patients (13%) left the hospital prior to completion of follow-up for reasons unrelated to their treatment . Cure rate was 97% (126/130) . There were no RII or RIII failures and all four patients with treatment failures were of the RI type . The mean parasite clearance time and fever clearance time were 46.4 and 42.5 hours, respectively . All patients were tolerated the combination drugs well and there were no serious toxic adverse reactions . The results indicate that combination of artesunate and mefloquine given twice daily for 2 days is effective and well tolerated in patients with acute, uncomplicated falciparum malaria and suitable as an alternative treatment for multidrug resistant falciparum malaria. Southeast Asian J Trop Med Public Health, 1998 Dec, 29(4), 669 - 84 Application of geographical information systems to co-analysis of disease and economic resources: dengue and malaria in Thailand; Indaratna K et al.; Two vector-borne communicable diseases, malaria and dengue, are among a number of diseases of particular importance in relation to economic development in Southeast Asia and thus need to be assessed in relation to economic parameters in the region . Geographical Information Systems (GIS) provide one means of comparing disease and resource data versus time and place, to facilitate rapid visualization by planners and administrators . Given that Thailand is a global epicenter of multidrug resistant falciparum malaria and of dengue hemorrhagic fever, both of which are mosquito-borne, application of GIS methods to these two diseases gives opportunity for comparison of resource needs and allocation in relation to disease epidemiologic patterns . This study examined per capita gross provincial product (GPPpc) and health care resources in relation to geographic distribution of malaria and dengue in Thailand . The two diseases vary greatly in overall seasonal patterns and in relation to provincial economic status, and present differing demands on resource utilization: planned integration of control of malaria and dengue could utilize such analyses in relation to resource sharing and consideration of allocative efficiency . The concentration of malaria (and to a lesser extent dengue) along international border areas underscores the desirability of multi-country coordination of disease management and control programs . Because socio-economic and disease data are collected by quite different means and in different time frames, there are some limitations to the dynamic interpolation of these two broad data sets, but useful inferences can be drawn from this approach for application to overall planning, at both national and multi-country levels. Antimicrob Agents Chemother, 2000 May, 44(5), 1328 - 32 Susceptibility to PNU-140690 (Tipranavir) of human immunodeficiency virus type 1 isolates derived from patients with multidrug resistance to other protease inhibitors; Rusconi S et al.; In our study we examined the anti-human immunodeficiency virus type 1 (anti-HIV-1) activity of a novel HIV-1 protease inhibitor, PNU-140690 (tipranavir), against patient-derived isolates resistant to multiple other protease inhibitors (PIs) . The aim of our experiments was to investigate the genotypes and the in vitro phenotypes of drug resistance of PNU-140690 . We carried out drug susceptibility tests with peripheral blood mononuclear cells and a fixed amount of infectious virus (1,000 50% tissue culture infective doses) to determine the 50% inhibitory concentration (IC(50)) and IC(90), PCR assays for the detection of drug resistance mutations in RNA in plasma, and direct sequencing of PCR products . Phenotypic resistance to PIs was invariably related to genotypic mutations . The substitutions among the amino acid residues of the protease included L10I, K20R, L24I, M36I, N37D, G48V, I54V, L63P, I64V, A71V, V77I, V82A, I84V, and L90M . Isolates from all of the patients had developed a maximal degree of resistance to indinavir, ritonavir, and nelfinavir (IC(50)s, >0.1 microM) . We also compared these mutations with the amino acid changes previously described in association with in vivo tipranavir administration . The mutations included the following: I15V, E35D, N37D, R41K, D60E, and A71T . Infections with IIIB, 14aPre, and N70 were inhibited by an average drug IC(90) of 0.18 +/- 0.02 microM in multiple experiments . The average mean +/- standard error of mean IC(90) for the entire group of multidrug-resistant isolates derived from the mean values for two culture wells with p24 antigen supernatant appeared to be 0.619 +/- 0.055 microM (range, 0.31 to 0.86 microM) . Tipranavir retained a sustained antiviral activity against PI-MDR clinical isolates and might be useful in combination regimens with other antiretroviral agents for patients who have already failed other PI-containing therapies. Anticancer Res, 2000 Jan-Feb, 20(1A), 373 - 7 3,5-diacetyl-1,4-dihydropyridines: synthesis and MDR reversal in tumor cells; Shah A et al.; Eleven 4-phenyl-3,5-diacetyl-1,4-dihydropyridines (AcDHPs) {G1-11} substituted at the phenyl ring were synthesized and compared for their cytotoxic activity and multidrug resistance (MDR)-reversing activity in in vitro assay systems . Among them, compound {G7} showed the highest cytotoxic activity against human promyelocytic leukemia HL-60 and human squamous cell carcinoma HSC-2 cells . However, no compounds tested produced radicals at pH 7.4-12.5 . The activity of P-glycoprotein (Pgp) responsible for MDR in tumor cells was reduced by compounds {G2, 3, 6, 5, 8, 1, 11}, verapamil {VP} and nifedipine {NP} . However, compounds {G4, 7, 10} were hardly active while G9 did not show a MDR reversing effect at 2.0-20.0 micrograms/mL . These data show a relationship between chemical structures and MDR-reversing effect on tumor cells. Anticancer Res, 2000 Jan-Feb, 20(1A), 139 - 50 Cytosine arabinoside (ara-C) resistance confers cross-resistance or collateral sensitivity to other classes of anti-leukemic drugs; Martin-Aragon S et al.; The major limitation of treatment with antimetabolite drugs is that they produce resistant clones both in vitro and in patients who either do not respond to treatment or relapse soon after response has been documented . To better understand the phenomenon of cross-resistance, we developed seven CEM/ara-C-resistant leukemic clones from the CEM/0 (wt) cell line . These clones ranged from 4- to 3.5 x 10(8)-fold more resistant to ara-C than the wt CEM/0 cell line . Using this model, we determined IC50 concentrations to several chemotherapeutic agents and gamma radiation, and we also studied pro- (p53) and anti-apoptotic (bcl-2) proteins, as well as P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) . The cell viability assays showed that these clones were cross-resistant to 6-thioguanine (6-TG) or 6-mercaptoguanosine (6-TGuo) from 1.1- to 8.8-fold with ara-C; cross-resistance to vincristine (VCR) was from 200- to 1 x 10(4)-fold with ara-C . Taxotere (TXR) showed cross-resistance with ara-C from 1.39- to 3.03 x 10(3)-fold; dexamethasone (DEX) also showed a significant degree of cross-resistance from 27.4- to 3.87 x 10(7)-fold . Gamma radiation treatments from 0.77 Gy to 12.3 Gy showed a radiation dose-dependent cross-resistance with ara-C from 1.43- to 2.93-fold . Idarubicin was collaterally sensitive with ara-C from 4.6- to 1 x 10(9)-fold in these cell lines . The CEM/ara-C/G resistant cell line was 3-fold more sensitive to 6-TG or VCR than CEM/0 (wt), and 5-fold more sensitive to 6-TGuo . This cell clone expressed p53 and did not overexpress bcl-2 protein . All of the cell lines studied, CEM/0 (wt) and the ara-C resistant clones, showed functional p53 protein . The cell treatment with 0.1, 1 and 10 microM ara-C for 48 hours showed increased p53 protein expression in most of these lines . No increase in bcl-2 protein expression was seen in the wt cell line after ara-C treatment for 48 hours . Three cell lines resistant to ara-C (CEM/ara-C/B, CEM/ara-C/D and CEM/ara-C/I) showed an important increased expression of bcl-2 protein after treatment with 1 microM ara-C, but not after 10 microM . This alteration may lead to resistance to apoptosis and enhanced cell survival . The ratio of bcl-2 to p53 was increased significantly in these three clones, thus favoring an anti-apoptotic drive . All of the cell lines examined were negative for MRP expression and only two, CEM/ara-C/B and CEM/ara-C/J, were positive for MRP functional activity . However, three ara-C resistant cell clones, CEM/ara-C/7A, CEM/ara-C/B and CEM/ara-C/G, were positive for P-gp expression and functional activity . It is apparent that selection for ara-C resistance confers cross-resistance to many other classes of drugs and gamma radiation, probably due to bcl-2 protein overexpression or P-gp and MRP expression, as independent mechanisms. Lancet . 2000 Apr 8;355(9211):1246. Warsaw conference on emerging infections in central and eastern Europe; Balinska MA; PIP: On March 28-29, 2000, epidemiologists and microbiologists convened in Warsaw, Poland, to discuss emerging, re-emerging, and drug-resistant infections in central and eastern Europe . Delegates were from the Czech Republic, Greece, Hungary, Latvia, Poland, Romania, Russia, and Yugoslavia, with the exception of the US and the UK and other worst affected countries in central-eastern Europe . It has been documented that the cause of diphtheria epidemics in several countries in the mid-1990s resulted from the breakdown of vaccination campaigns following social dislocation . Currently, diphtheria morbidity has declined through targeted vaccination programs, although fundamental socioeconomic problems continue to threaten public health . Other infectious diseases raised during the conference were the evolution of tuberculosis and HIV/AIDS, which remains largely unpredictable . In addition, the growing incidence of multidrug-resistant tuberculosis is documented in several countries . Among the major problems in tuberculosis control include late diagnosis, nonexistent or erratic drug supplies and unreliable reporting . The syringe ecosystem in both healthcare settings and injecting drug users were singled out as the vectors of sharp increase in HIV/AIDS infection . Throughout the conference, the recurrent theme was on the overriding importance of providing specialized training and the building up of networks of public health specialists . Minerva Ginecol, 1999 Dec, 51(12), 463 - 70 {Expression and prognostic value of the drug resistance markers P-gp, Mrp1, Mrp2, and Lrp in ovarian carcinoma}; Katsaros D et al.; BACKGROUND: Intrinsic and/or acquired chemoresistance is the major obstacle to overcome in the treatment of patients with ovarian carcinoma . The aim of the present study was to investigate the prognostic value of drug resistance associated proteins P-glycoprotein (P-gp), multidrug resistance related protein (Mrp1), canalicular multispecific organic anion trans-porter (c-MOAT or Mrp2) and lung resistance protein (Lrp) in ovarian carcinoma . METHODS: Expression of P-gp, Mrp1, Mrp2 and Lrp was determined by immunohistochemistry of frozen tissue sections of 115 ovarian carcinoma patients and associated to clinico-pathological factors, response to chemotherapy and (progression free) survival . RESULTS: Expression of P-gp was observed in 20 out of 115 (17%), Mrp1 in 51 out of 115 (44%), Mrp2 in 19 out of 115 (16%) and Lrp in 85 out 115 (74%) tumors . Expression of Mrp1 was related to Mrp2 (p < 0.0001) and P-gp (p < 0.001) expression, while Lrp expression was more frequently observed in patients with stage I/II versus stage III/IV tumors (p < 0.01), grade I/II versus III tumors (p < 0.05) and residual tumor < 2 cm versus > 2 cm after laparotomy (p < 0.05) . Lower stage (p < 0.001), small residual tumor after first laparotomy (p < 0.001) and lower differentiation grade (p < 0.05) were related to longer (progression free) survival . P-gp, Mrp1, Mrp2, and Lrp expression was neither related to response to first line chemotherapy (59 evaluable patients) nor to (progression free) survival (all patients) . On multivariate analysis only stage and residual tumor after first laparotomy were independent prognostic factors for (progression free) survival . CONCLUSIONS: In ovarian carcinoma Mrp1 expression is associated with Mrp2 and P-gp expression, while Lrp expression is associated with favorable clinicopathological characteristics . Assessment of P-gp, Mrp1, Mrp2 or Lrp does not allow prediction of response to chemotherapy or (progression free) survival in ovarian carcinoma. Cancer Gene Ther, 2000 Mar, 7(3), 466 - 75 A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1; Stuart DD et al.; The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo . Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung . We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle . The coated cationic liposomes had an average diameter of 188 nm and entrapped 85-95% of the asODN . The biodistribution and pharmacokinetics of an 18-mer 125I-labeled phosphorothioate ODN formulated by this met