Int J Tuberc Lung Dis, 1998 Jun, 2(6), 490 - 8 Drug-resistant tuberculosis in the Dominican Republic: results of a nationwide survey; Espinal MA et al.; SETTING: The Dominican Republic . OBJECTIVE: To assess the extent of drug-resistant tuberculosis (TB) following the guidelines of the World Health Organization (WHO)/International Union Against Tuberculosis and Lung Disease (IUATLD) new global surveillance project on drug resistance in TB . METHODS: Using a multi-step proportional weighted approach, a sample of 688 sequential cases of smear positive pulmonary TB diagnosed between April 1994 and April 1995 was studied in six of the country's eight health regions . Pre-treatment sputum samples were cultured on Loewenstein-Jensen medium and drug susceptibility tests were performed using the economic variant of the proportion method . RESULTS: Of 420 cases with drug susceptibility results, resistance to one or more drugs was observed in 43.8%; resistance was found in 52.1% of 117 TB cases with a history of previous antituberculosis treatment and in 40.6% of 303 new TB cases . In five of the six health regions surveyed, > or = 41% of strains were resistant to one or more drugs . Multidrug resistance (MDR) to isoniazid and rifampicin with or without resistance to other drugs was found in 43 (10.2%) of 420 cases, including 6.6% of new TB cases . In five of the six health regions > or = 8% of strains were classified as MDR . Independent predictors of MDR-TB included being in the age group 25 to 44 years (odds ratio {OR} = 4.2, 95% confidence interval {Cl} 1.5, 11.6; P = 0.005), being aged 45 years and over (OR = 4.5, 95% CI 1.4, 14.4; P = 0.009), and having a prior history of TB (OR = 3.7, 95% CI 1.9, 7.4; P = 0.0001) . CONCLUSION: The proportion of Mycobacterium tuberculosis strains resistant to one or more anti-TB drugs in the Dominican Republic is among the highest observed world-wide . The severity of the problem urgently requires the full implementation of TB control strategies endorsed by the WHO and the IUATID, which include political commitment to a National TB Program, case detection utilizing sputum-smear microscopy, directly observed treatment, regular drug supply, and standardised recording and reporting systems . Also, the sale of TB drugs in the private market should be controlled. Int J Tuberc Lung Dis, 1998 Jun, 2(6), 484 - 9 Multidrug-resistant tuberculosis in the Florence province from 1992 to 1995; Nutini S et al.; SETTING: Epidemiological data on the frequency of drug-resistant tuberculosis is not available in Italy . OBJECTIVES: Evaluation of the rate of multidrug-resistant tuberculosis in the Province of Florence, Italy . DESIGN: Retrospective analysis of all sensitivity tests performed with the Bactec method on initial mycobacterial isolates, from 1 January 1992 to 31 December 1995, in the Province of Florence . RESULTS: The following rates of resistance were found in the 433 samples tested: isoniazid + rifampicin 2.5%, at least one drug 13.8%, isoniazid 10.6%, rifampicin 3.6%, streptomycin 3.6%, pyrazinamide 1.7% and ethambutol 0.6% . Resistance was higher in foreign-born individuals from high prevalence countries than in the Italian-born population, whereas resistance to streptomycin was more frequent in the latter . The yearly rates of resistance showed no significant variation in the period examined . Clinical data were available in 231 patients: the rate of resistance to at least one drug and to isoniazid + rifampicin were 10.8% and 0%, respectively, in never treated patients, and 28.5% and 7.1%, respectively, in previously treated patients . CONCLUSION: These data show higher multidrug resistance rates than those found in other European countries such as England and Wales, France and Switzerland . This result suggests the need to establish official guidelines for the correct treatment of tuberculosis in Italy, in order to prevent the onset of drug resistance, and to establish a national surveillance system for mycobacterial resistance. Clin Cancer Res, 1998 Jun, 4(6), 1563 - 6 Potential interactions between antitubulin agents and temperature: implications for modulation of multidrug resistance; Dumontet C et al.; We analyzed the effect of high temperature (a 1-h incubation at 43 degrees C) on the accumulation and cytotoxicity of vinblastine and docetaxel in two model cell lines, K562 and MESSA, and their multidrug resistance (MDR) counterparts, K562/R7 and MESSA/Dx5 . High temperature increased the amount of intracellular vinblastine and docetaxel significantly in MESSA cell and, to a much lesser extent, in K562 cells . MDR-positive cells retained a profound drug accumulation defect at 43 degrees C . Hyperthermia enhanced the cytotoxic effect of vinblastine (but not docetaxel) in both K562 and MESSA cells, but not in the MDR-positive variants . PSC833, a potent modulator of P-glycoprotein, induced high levels of drug accumulation in the two MDR-positive cell lines at both 37 degrees C and at 43 degrees C . PSC833 also significantly reduced the resistance levels of the two MDR-positive lines at both 37 degrees C and at 43 degrees C . The effect of hyperthermia on drug accumulation thus seems to depend on the cell line, whereas the effect on cytotoxicity depends on the type of compound . The MDR phenotype remains a therapeutic obstacle at 43 degrees C but is accessible to modulation. Clin Cancer Res, 1998 Jun, 4(6), 1393 - 403 The expression of drug resistance gene products during the progression of human prostate cancer; Sullivan GF et al.; Prostate cancer progresses from a localized disease to a widely disseminated malignancy . Each step along this progression pathway involves multiple genetic alterations that impart a survival advantage to the tumor cell over its normal counterparts and may confer resistance to therapy . Because metastatic prostate cancer is one of the most therapy-resistant human neoplasms, we studied the expression of certain molecular determinants of drug resistance in the context of tumor progression . Paraffin-embedded formalin-fixed resected prostates were chosen based on Gleason grade and surgical stage . Immunohistochemistry was used to detect the expression of multidrug resistance protein (MRP), topoisomerase II alpha, p53, glutathione S-transferase pi, Bcl-2, and P-glycoprotein in these specimens . We found that all of the proteins were expressed in resected prostate except for P-glycoprotein . The expression of MRP, topoisomerase II alpha, p53, and Bcl-2 increased with the Gleason grade . In addition, the expression of MRP, topoisomerase II alpha, and p53 increased with the surgical stage . In contrast, the glutathione S-transferase pi and Bcl-2 expression decreased with the increasing surgical stage . Stage was the strongest indicator of protein expression . These results suggest that drug resistance gene products are expressed in prostate cancer at the time of surgical resection . Thus, although the emergence of the "pan-resistance" phenotype in prostate cancer may partly be a function of the selection pressure exerted by therapeutic interventions, certain determinants of chemoresistance may be caused by genetic changes accompanying tumorigenesis. Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7024 - 9 The drug efflux protein, P-glycoprotein, additionally protects drug-resistant tumor cells from multiple forms of caspase-dependent apoptosis; Smyth MJ et al.; Multidrug resistance mediated by the drug efflux protein, P-glycoprotein (P-gp), is one mechanism that tumor cells use to escape death induced by chemotherapeutic agents . However, the mechanism by which P-gp confers resistance to a large variety of structurally diverse molecules has remained elusive . In this study, classical multidrug resistant human CEM and K562 tumor cell lines expressing high levels of P-gp were less sensitive to multiple forms of caspase-dependent cell death, including that mediated by cytotoxic drugs and ligation of Fas . The DNA fragmentation and membrane damage inflicted by these stimuli were defined as caspase dependent by various soluble peptide fluoromethylketone caspase inhibitors . Inhibition of P-gp function by the anti-P-gp mAb MRK-16 or verapamil could reverse resistance to these forms of cell death . Inhibition of P-gp function also enhanced drug or Fas-mediated activation of caspase-3 in drug-resistant CEM cells . By contrast, caspase-independent cell death events in the same cells, including those mediated by pore-forming proteins or intact NK cells, were not affected by P-gp expression . These observations suggest that, in addition to effluxing drugs, P-gp may play a specific role in regulating some caspase-dependent apoptotic pathways. J Pharmacol Exp Ther, 1998 Jun, 285(3), 1260 - 5 Characterization of efflux transport of organic anions in a mouse brain capillary endothelial cell line; Kusuhara H et al.; Cumulative evidence suggests that several organic anions are actively effluxed from the brain to the blood across the blood-brain barrier (BBB) . We examined the possibility of the presence of primary active transporters for organic anions (multidrug resistance associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT)) on the BBB by measuring the ATP-dependent uptake of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and leukotriene C4 (LTC4) into membrane vesicles prepared from a cell line derived from mouse brain capillary endothelial cells (MBEC4) . The ATP-dependent uptake of DNP-SG into the membrane vesicles was osmotically sensitive and was also supported by GTP, but not by AMP or ADP . An ATPase inhibitor, vanadate, blocked the ATP-dependent uptake of DNP-SG . The ATP-dependent uptake process was saturable, with Km values of 0.56 and 0.22 microM, and Vmax values of 5.5 and 27.5 pmol/min/mg protein for DNP-SG and LTC4, respectively . Northern and Western blot analyses showed the expression of murine MRP but not cMOAT in MBEC4 cells . Western blot analysis of the rat cerebral endothelial cells indicated the expression of protein(s) that is detectable with MRPr1, an antibody against MRP . These results, together with previous findings that both DNP-SG and LTC4 are good ligands for MRP, suggest that MRP is responsible for the unidirectional, energy-dependent efflux of organic anions from the brain into the circulating blood across the BBB. Mol Pharmacol, 1998 Jun, 53(6), 1068 - 75 Hepatic expression of multidrug resistance-associated protein-like proteins maintained in eisai hyperbilirubinemic rats; Hirohashi T et al.; The biliary excretion of several organic anions is mediated by the canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats such as Eisai hyperbilirubinemic rats (EHBR) . In addition, using a kinetic study with isolated canalicular membrane vesicles, we recently suggested the presence of ATP-dependent organic anion transporter(s) other than cMOAT in EHBR {Pharm Res (NY) 12:1746-1755 (1995); J Pharmacol Exp Ther 282:866-872 (1997)} . The aim of this study is to provide a molecular basis for the presence of multiplicity in the biliary excretion of organic anions in rats . Based on the homology with human multidrug resistance-associated protein (hMRP), two cDNA fragments encoding the carboxyl-terminal ATP-binding cassette region were amplified by reverse transcription-polymerase chain reaction from EHBR liver . These fragments exhibited approximately 70% amino acid identity with hMRP and rat cMOAT;, therefore, they were designated MRP-like proteins (MLP-1 and MLP-2) . The cloned full length cDNA of MLP-1 and -2 from the Sprague-Dawley (SD) rat liver and colon cDNA library was composed of 1502 and 1523 amino acids, respectively, had the characteristics of ATP-binding cassette transporters, and exhibited homology with hMRP and rat cMOAT . Northern blot analysis indicated that MLP-1 is expressed predominantly in the liver in both SD rats and EHBR, whereas hepatic expression of MLP-2 was observed only in EHBR . In addition, MLP-2 was markedly induced by ligation of the bile duct in SD rat liver . In both SD rats and EHBR, MLP-2 was expressed predominantly in the duodenum, jejunum, and colon . These findings suggest that MLP-1 and MLP-2 might be novel members of the MRP family responsible for the excretion of organic anions from these epithelial cells, and that MLP-2 is an inducible one. Mol Pharmacol, 1998 Jun, 53(6), 1062 - 7 Adenosine triphosphate-dependent transport of anionic conjugates by the rabbit multidrug resistance-associated protein Mrp2 expressed in insect cells; van Aubel RA et al.; The multidrug resistance-associated protein Mrp2 is expressed in liver, kidney, and small intestine and mediates ATP-dependent transport of conjugated organic anions across the apical membrane of epithelial cells . We recently cloned a rabbit cDNA encoding a protein that on basis of highest amino acid homology and tissue distribution was considered to be the rabbit homolog of rat Mrp2 . To investigate whether rabbit Mrp2 mediates ATP-dependent transport similar to rat Mrp2, we expressed rabbit Mrp2 in Spodoptera frugiperda (Sf9) cells using recombinant baculovirus . Mrp2 was expressed as an underglycosylated protein in Sf9 cells and to a higher level compared with rabbit liver and renal proximal tubules . Both 17beta-estradiol-17-beta-D-glucuronide ({3H}E217betaG, 50 nM) and {3H}leukotriene C4 (3 nM) were taken up by Sf9-Mrp2 membrane vesicles in an ATP-dependent fashion . Uptake of {3H}E217betaG was dependent on the osmolarity of the medium and saturable for ATP (Km = 623 microM) . Leukotriene C4, MK571, phenolphthalein glucuronide, and fluorescein-methotrexate were good inhibitors of {3H}E217betaG transport . The inhibitory potency of cyclosporin A and methotrexate was moderate, whereas fluorescein, alpha-naphthyl-beta-D-glucuronide, and p-nitrophenyl-beta-D-glucuronide did not inhibit transport . In conclusion, we show direct ATP-dependent transport by recombinant rabbit Mrp2 and provide new data on Mrp2 inhibitor specificity. J Cell Sci, 1998 Apr, 111 ( Pt 8), 1137 - 45 Cyclic AMP stimulates sorting of the canalicular organic anion transporter (Mrp2/cMoat) to the apical domain in hepatocyte couplets; Roelofsen H et al.; The canalicular membrane of rat hepatocytes contains an ATP-dependent multispecific organic anion transporter, also named multidrug resistance protein 2, that is responsible for the biliary secretion of several amphiphilic organic anions . This transport function is markedly diminished in mutant rats that lack the transport protein . To assess the role of vesicle traffic in the regulation of canalicular organic anion transport, we have examined the redistribution of the transporter to the canalicular membrane and the effect of cAMP on this process in isolated hepatocyte couplets, which retain secretory polarity . The partial disruption of cell-cell contact, due to the isolation procedure, leaves the couplet with both remnant apical membranes, as a source of apical proteins, and an intact apical domain and lumen, to which these proteins are targeted . The changes in distribution of the transporter were correlated to the apical excretion of a fluorescent substrate, glutathione-methylfluorescein . The data obtained in this study show that the transport protein, endocytosed from apical membrane remnants, first is redistributed along the basolateral plasma membrane . Then it is transcytosed to the remaining apical pole in a microtubule-dependent fashion, followed by the fusion of transporter-containing vesicles with the apical membrane . The cAMP analog dibutyrylcAMP stimulates all three steps, resulting in increased apically located transport protein, glutathione-methylfluorescein transport activity and apical membrane circumference . These findings indicate that the organic anion transport capacity of the apical membrane in hepatocyte couplets is regulated by cAMP-stimulated sorting of the multidrug resistance protein 2 to the apical membrane . The relevance of this phenomenon for the intact liver is discussed. Anticancer Drugs, 1998 Mar, 9(3), 263 - 71 Antitumor activity of KW-2170, a novel pyrazoloacridone derivative; Ashizawa T et al.; 5-(3-Aminopropyl)amino-7,10-dihydroxy-2-(2-hydroxethyl)-aminoethyl -6H-pyrazolo{4,5,1-de}acridin-6-one dihydroxy-chloride (KW-2170), a novel derivative of pyrazoloacridone, was selected and evaluated for its antitumor activity and toxicity in mice . KW-2170 exhibited antitumor activity superior to adriamycin (ADM) against Sarcoma 180, breast carcinoma MM102 and fibrosarcoma Meth A inoculated s.c . in mice . Its therapeutic index (LD10/ED50) was higher than that of ADM on two murine carcinoma models, MM102 and Meth A . KW-2170 showed significant antitumor activity against 17 human tumor xenografts of a total of 24 tumors tested and the total tumor response rate by treatment with KW-2170 was significantly higher than that by ADM (70.8 versus 58.3%) . In particular, human lung carcinoma was highly sensitive to KW-2170, and a marked tumor regression was observed on Lu-65 and Lu-99 human lung carcinoma xenograft models . Ovary and pancreas carcinomas were also sensitive to the drug . Additionally, its therapeutic index was also high on these human carcinoma models in comparison with that of ADM . The best antitumor efficacy of KW-2170 was observed by a weekly treatment schedule followed by a single treatment schedule and a successive administration schedule also tended to be toxic to the hosts . KW-2170 exhibited very low cross-resistance against four lines of multidrug resistant tumors expressing high levels of P-glycoprotein, and the drug showed significant antitumor activity against ADM-resistant human ovary carcinoma A2780/ADM and against nasopharynx carcinoma KB-A1 xenografts which were not sensitive to ADM . These results indicate that KW-2170 has a very potent antitumor activity and is feasible as a new antitumor drug against ADM-refractory solid tumors in clinics. Anticancer Drugs, 1998 Mar, 9(3), 255 - 61 The bisbenzylisoquinoline alkaloids, tetrandine and fangchinoline, enhance the cytotoxicity of multidrug resistance-related drugs via modulation of P-glycoprotein; Choi SU et al.; The occurrence of resistance to chemotherapeutic drugs is a major problem for successful cancer treatment and reducing drug accumulation by P-glycoprotein (P-gp) is one of the major mechanisms of multidrug resistance (MDR) . The present study was performed to evaluate the MDR-reversal abilities of two bisbenzylisoquinoline alkaloids, tetrandine (TET) and fangchinoline (FAN), compared with verapamil (VER), a well-known P-gp modulator . TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) reduced the paclitaxel (TAX) concentration required to achieve 50% inhibition of cell growth (EC50) to HCT15 (P-gp-positive) cells about 3100-, 1900- and 410-fold, and these compounds also reduced the EC50 value of actinomycin D (AMD) about 36.0-, 45.9- and 18.2-fold in the cells, respectively . Meanwhile, TET, FAN and VER had no effect on the cytotoxicity of the drugs to SK-OV-3 (P-gp-negative) cells . On the other hand, TET (3.0 microM), FAN (3.0 microM) and VER (10.0 microM) similarly enhanced the accumulation rates of rhodamine 123, a well known P-gp substrate, in HCT15 cells (200-250%) . After efflux for 2 h with fresh medium, TET and FAN also enhanced the residual rate of rhodamine 123 about 5.0- and 2.6-fold in comparison with control, respectively . TET, FAN and VER could not affect the accumulation and residual rate of rhodamine 123 in SK-OV-3 cells . From the result, we conclude that TET and FAN enhanced the cytotoxicity of MDR-related drugs via modulation of P-gp. Semin Oncol, 1998 Apr, 25(2 Suppl 6), 58 - 61 Megestrol acetate as a biomodulator; Chang AY; Megestrol acetate is a synthetic analog of progesterone . In general, megestrol acetate exerts its progesterone-like hormonal effect by binding to the progesterone receptor . It has been recognized that megestrol acetate can increase weight and improve some aspects of quality of life in cancer patients and in patients with the acquired immunodeficiency syndrome . This effect occurs in patients with or without concurrent chemotherapy or radiotherapy, through an as yet unknown mechanism . Recently, megestrol acetate has been shown to reverse, at least partially, multidrug resistance to doxorubicin and/or vincristine in cancer cell lines . This potentially beneficial effect has not yet been studied in clinical trials . This biological activity is thought to be mediated through the unique binding of megestrol acetate to p-glycoprotein . At least in vitro megestrol acetate can also enhance the cytotoxic effect of these two chemotherapeutic agents in some MDR-nonexpressing cell lines . These findings suggest that megestrol acetate deserves further preclinical and clinical studies to evaluate its potential role of enhancing cytotoxicity of chemotherapy and improving the quality of life of cancer patients. Antimicrob Agents Chemother, 1998 Jun, 42(6), 1524 - 6 Contribution of beta-lactamases to beta-lactam susceptibilities of susceptible and multidrug-resistant Mycobacterium tuberculosis clinical isolates; Segura C et al.; The beta-lactamases in 154 clinical Mycobacterium tuberculosis strains were studied . Susceptibilities to beta-lactam antibiotics, their combination with clavulanate (2:1), and two fluoroquinolones were determined in 24 M . tuberculosis strains susceptible to antimycobacterial drugs and in nine multiresistant strains . All 154 M . tuberculosis isolates showed a single chromosomal beta-lactamase pattern (pI 4.9 and 5.1) . M . tuberculosis beta-lactamase hydrolyzes cefotaxime with a maximum rate of 22.5 +/- 2.19 IU/liter (strain 1382) . Neither amoxicillin, carbenicillin, cefotaxime, ceftriaxone, nor aztreonam was active alone . Except for aztreonam, beta-lactam combinations with clavulanate produced better antimycobacterial activity. Antimicrob Agents Chemother, 1998 Jun, 42(6), 1375 - 81 Recombinant expression and characterization of the major beta-lactamase of Mycobacterium tuberculosis; Voladri RK et al.; New antibiotic regimens are needed for the treatment of multidrug-resistant tuberculosis . Mycobacterium tuberculosis has a thick peptidoglycan layer, and the penicillin-binding proteins involved in its biosynthesis are inhibited by clinically relevant concentrations of beta-lactam antibiotics . beta-Lactamase production appears to be the major mechanism by which M . tuberculosis expresses beta-lactam resistance . beta-Lactamases from the broth supernatant of 3- to 4-week-old cultures of M . tuberculosis H37Ra were partially purified by sequential gel filtration chromatography and chromatofocusing . Three peaks of beta-lactamase activity with pI values of 5.1, 4.9, and 4.5, respectively, and which accounted for 10, 78, and 12% of the total postchromatofocusing beta-lactamase activity, respectively, were identified . The beta-lactamases with pI values of 5.1 and 4.9 were kinetically indistinguishable and exhibited predominant penicillinase activity . In contrast, the beta-lactamase with a pI value of 4.5 showed relatively greater cephalosporinase activity . An open reading frame in cosmid Y49 of the DNA library of M . tuberculosis H37Rv with homology to known class A beta-lactamases was amplified from chromosomal DNA of M . tuberculosis H37Ra by PCR and was overexpressed in Escherichia coli . The recombinant enzyme was kinetically similar to the pI 5.1 and 4.9 enzymes purified directly from M . tuberculosis . It exhibited predominant penicillinase activity and was especially active against azlocillin . It was inhibited by clavulanic acid and m-aminophenylboronic acid but not by EDTA . We conclude that the major beta-lactamase of M . tuberculosis is a class A beta-lactamase with predominant penicillinase activity . A second, minor beta-lactamase with relatively greater cephalosporinase activity is also present. Eur J Cancer, 1998 Jan, 34(1), 168 - 74 In vitro modulation of doxorubicin and docetaxel antitumoral activity by methyl-beta-cyclodextrin; Grosse PY et al.; Methyl-beta-cyclodextrin (MEBCD) was investigated for its effect on the antitumoral activity of various antineoplastic agents (doxorubicin (DOX), docetaxel (DXL), 5-fluorouracil (5-FU) and cisplatin (CDDP)) in three different human parental sensitive cancer cell lines (K562 S, MCF7 S and A2780 S) and their multidrug resistant variant sublines (K562 R, MCF7 R and A2780 R) . At non-cytotoxic concentrations, MEBCD was able to increase significantly DOX and DXL cytotoxic activity in all the cell lines tested . The sensitisation ratios (IC50 drug control/IC50 drug-MEBCD treated) ranged from 3l1 to 14.3 . Moreover, intracellular DOX accumulation, determined by high-performance liquid chromatography, was also increased when cells were treated with MEBCD combined with DOX (approximately 2-3 fold) . The effects of MEBCD in resistant sublines were greater than in their parental sensitive cell lines . Other experiments demonstrated that the action of the MEBCD was independent of DOX . These data provided a basis for the potential therapeutic application of MEBCD in cancer therapy. Eur J Cancer, 1998 Jan, 34(1), 81 - 6 Expression of the multidrug resistance protein (MRP) in squamous cell carcinoma of the oesophagus and response to pre-operative chemotherapy; Nooter K et al.; One of the major problems in the treatment of squamous cell carcinoma of the oesophagus (ESCC) is the unresponsiveness to cytotoxic drugs . So far, the mechanisms underlying the intrinsic drug resistance of ESCC remain unclear . The aim of this study was to determine the role of the newly recognised drug resistance protein, the multidrug resistance protein (MRP), in ESCC drug resistance . Tumour biopsies from ESCCs were analysed by RNase protection assay (RPA) as well as by immunohistochemistry (IHC) for the presence of MRP mRNA or protein, respectively . The ESCC samples were obtained from patients participating in a prospective randomised clinical phase III trial, evaluating pre-operative chemotherapy (cisplatin and etoposide) followed by surgery versus surgery alone in patients with operable ESCC . For most patients, tumour biopsies taken at diagnosis by endoscopy as well as surgically resected primary tumours were available . Of 58 ESCC patients enrolled, 28 received chemotherapy before surgical resection of their tumours, and 30 were treated with surgery alone . 12 patients (3 complete and 9 partial responses; 43%) showed a major response after chemotherapy, 10 patients (36%) had stable disease (SD), and 6 (21%) progressive disease (PD) . On 14 surgically resected, untreated, primary ESCCs, the IHC scores correlated with the MRP mRNA levels, quantitated by RPA (multiple testing, P < 0.01) . MRP expression was detected by IHC in the vast majority (52/58; 90%) of the diagnostic biopsies . MRP expression did not differ significantly between CR + PR, and patients with SD or PD . In addition, multivariate analysis by logistic regression did not show any effect of tumour cell differentiation or UICC tumour stage on the outcome of pre-operative chemotherapy in relation to MRP expression . However, a difference became apparent (Sign-test, P < 0.05) for higher MRP expression in tumours from patients with PR or SD, when comparing MRP levels in paired tumour samples before and after chemotherapy, suggesting that chemotherapy selected for drug-resistant cell clones. J Exp Med, 1998 May 18, 187(10), 1583 - 98 Defective acidification in human breast tumor cells and implications for chemotherapy; Altan N et al.; Multidrug resistance (MDR) is a significant problem in the treatment of cancer . Chemotherapeutic drugs distribute through the cyto- and nucleoplasm of drug-sensitive cells but are excluded from the nucleus in drug-resistant cells, concentrating in cytoplasmic organelles . Weak base chemotherapeutic drugs (e.g., anthracyclines and vinca alkaloids) should concentrate in acidic organelles . This report presents a quantification of the pH for identified compartments of the MCF-7 human breast tumor cell line and demonstrates that (a) the chemotherapeutic Adriamycin concentrates in acidified organelles of drug-resistant but not drug-sensitive cells; (b) the lysosomes and recycling endosomes are not acidified in drug-sensitive cells; (c) the cytosol of drug-sensitive cells is 0.4 pH units more acidic than the cytosol of resistant cells; and (d) disrupting the acidification of the organelles of resistant cells with monensin, bafilomycin A1, or concanamycin A is sufficient to change the Adriamycin distribution to that found in drug-sensitive cells, rendering the cell vulnerable once again to chemotherapy . These results suggest that acidification of organelles is causally related to drug resistance and is consistent with the hypothesis that sequestration of drugs in acidic organelles and subsequent extrusion from the cell through the secretory pathways contribute to chemotherapeutic resistance. Biochemistry, 1998 May 5, 37(18), 6503 - 12 Proximity of the nucleotide binding domains of the P-glycoprotein multidrug transporter to the membrane surface: a resonance energy transfer study; Liu R et al.; Very little structural information is available for P-glycoprotein (Pgp), which has been implicated in the multidrug resistance of human tumors because of its ability to act as an ATP-driven efflux pump for hydrophobic compounds . Highly purified Pgp has been labeled on two cysteine residues with the fluorescence probe NBD-Cl (7-chloro-4-nitro-2,1,3-benzoxadiazole) . We show that NBD labels the same cysteine residues as MIANS {2-(4-maleimidoanilino)naphthalene-6-sulfonic acid}; they are located within the Walker A motif of the nucleotide binding domain, close to the site where ATP binds . NBD- and MIANS-labeled Pgps were reconstituted by detergent dilution into phospholipid vesicles containing increasing mole fractions of rhodamine- or NBD-labeled phosphatidylethanolamine (PE), respectively . The fluorescence of the NBD-Pgp and MIANS-Pgp donors was quenched in a concentration-dependent manner by the rhodamine-PE and NBD-PE acceptors . Using two different methods to analyze Forster resonance energy transfer, the distance of the Pgp-bound probes from the lipid-water interfacial region of the bilayer was estimated to be 31-35 A . This distance is compatible with the low-resolution structure of Pgp determined by electron microscopy, and indicates that the nucleotide binding domains lie close to the membrane surface . The experimental data fitted very well to theoretical quench curves for a single protein-bound fluor, suggesting that the two nucleotide binding domains are located equidistant from the bilayer . Following the addition of ATP to MIANS-Pgp, the NBD-PE quench curve no longer conformed to the models . These results imply that Pgp interacts differently with PE when it is in the ATP-bound form. Rapid Commun Mass Spectrom, 1998, 12(10), 620 - 4 Cellular uptake profile of paclitaxel using liquid chromatography tandem mass spectrometry; Kerns EH et al.; A new method for studying cellular uptake has been developed . This method is based on selected reaction monitoring liquid chromatography tandem mass spectrometry analysis of preparations from cell culture . The limit of detection for paclitaxel was approximately 0.1 microM intracellular concentration . This method has been utilized to study the uptake of paclitaxel and an analog (BMS-190616) in normal and multidrug resistant (MDR) cell lines . Paclitaxel and the analog, that had been noted to overcome MDR in animal models, were incubated with normal cells (HCT116) and MDR cells (HCT116(VM)46) at therapeutic concentrations . Intracellular drug concentrations were assayed at intervals from 0 to 1.0 h . Results show that paclitaxel accumulates to a level 12 times greater and BMS-190616 to a level 5 times greater in the normal cells as compared to MDR cells suggesting that paclitaxel is more sensitive to MDR than the analog . Furthermore, the steady state level of BMS-190616 was 4 fold greater than paclitaxel in the MDR cell line suggesting that at least part of this compound's increased therapeutic effect can be attributed to processes of uptake and efflux at the cellular level . These data show that the method is rapid, sensitive and presents a unique advantage over traditional radioisotopic methods in that it can readily be employed on a range of analogs without any additional synthetic effort. Emerg Infect Dis, 1998 Apr-Jun, 4(2), 195 - 209 Multidrug-resistant Mycobacterium tuberculosis: molecular perspectives; Rattan A et al.; Multidrug-resistant strains of Mycobacterium tuberculosis seriously threaten tuberculosis (TB) control and prevention efforts . Molecular studies of the mechanism of action of antitubercular drugs have elucidated the genetic basis of drug resistance in M . tuberculosis . Drug resistance in M . tuberculosis is attributed primarily to the accumulation of mutations in the drug target genes; these mutations lead either to an altered target (e.g., RNA polymerase and catalase-peroxidase in rifampicin and isoniazid resistance, respectively) or to a change in titration of the drug (e.g., InhA in isoniazid resistance) . Development of specific mechanism-based inhibitors and techniques to rapidly detect multidrug resistance will require further studies addressing the drug and drug-target interaction. J Cell Biochem, 1998 Jun 15, 69(4), 463 - 9 Upregulation of P-glycoprotein in rat hepatoma rho(o) cells: implications for drug-DNA interactions; Pillay V et al.; Rat hepatoma cells lacking mitochondrial DNA (rho(o) cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin) . The rho(o) cells were 45-fold less sensitive to Adriamycin than the parental rho+ cells containing mitochondrial DNA . Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the rho(o) cells . This was indicated by confocal microscopy where rho+ cells exhibited thirteenfold higher cellular levels of Adriamycin than rho(o) cells . Upregulation (tenfold) of P-glycoprotein in rho(o) cells was also confirmed by Northern dot blot analysis . Since the MDR phenotype is present in rho(o) cells and upregulation of P-glycoprotein is maintained in these cells, rho(o) cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution. Nucl Med Biol, 1998 Apr, 25(3), 225 - 32 A novel areneisonitrile Tc complex inhibits the transport activity of MDR P-glycoprotein; Rao VV et al.; P-glycoprotein (Pgp), the product of the multidrug resistance (MDR1) gene, has been an important cancer target for development of MDR modulators that act to inhibit Pgp efflux transport activity . From a series of novel substituted areneisonitrile analogues of Tc-sestamibi, a known Pgp transport substrate, emerged the hexakis(3,4,5-trimethoxyphenylisonitrile)Tc(I) complex (Tc-TMPI) as a potential modulator of Pgp . Tracer 99mTc-TMPI showed net cellular accumulation in inverse proportion to expression of Pgp and enhancement upon addition of classic MDR modulators . At pharmacological concentrations, the carrier-added 94Tc-TMPI complex showed potent inhibition of Pgp-mediated 99mTc-sestamibi transport (EC50, 1.1 +/- 0.2 microM) and displacement of a Pgp-specific photolabel in a concentration-dependent manner . We conclude that 99Tc-TMPI directly inhibited Pgp transport activity and serves as a convenient template for development of nonradioactive Re(I) analogues as novel MDR modulators. Leuk Res, 1998 Mar, 22(3), 249 - 56 Multidrug-resistant acute leukemia cells are responsive to prolonged exposure of daunorubicin: implications for liposome-encapsulated daunorubicin; Verdonck LF et al.; We examined the cytotoxic effects of free daunorubicin (DNR) and liposome-encapsulated DNR on multidrug-resistant (MDR1) leukemia cells of patients with acute leukemias who had failed primary induction treatment that included DNR . This was analyzed ex-vivo with DNR concentrations and exposure times that normally can be achieved in-vivo for both drugs with induction treatment . The leukemic blasts of patients both with drug-resistant AML and drug-resistant ALL were, ex-vivo, very sensitive to DNR concentrations and exposure times that can be achieved in-vivo by liposome-encapsulated DNR . However, under identical conditions, free DNR and liposome-encapsulated DNR had a similar cytotoxic profile, arguing against a unique mechanism of cytotoxicity by the liposomal constructure . These data suggest that liposome-encapsulated DNR may be preferable to free DNR for the treatment of acute leukemias. Cancer Lett, 1998 May 15, 127(1-2), 107 - 12 Alpha-tocopherol antagonizes the multidrug-resistance-reversal activity of cyclosporin A, verapamil, GF120918, clofazimine and B669; Van Rensburg CE et al.; The effects of the membrane-stabilizing agent, alpha-tocopherol (25 microg/ml), on the chemosensitizing interactions of cyclosporin A (5 microg/ml), verapamil (2 microg/ml), clofazimine (1 microg/ml), B669 (0.5 microg/ml) and GF120918 (0.015 microg/ml) with a P-glycoprotein-expressing human lung cancer cell line (H69/LX4) have been investigated in vitro . In an assay of cell proliferation, all the chemosensitizing agents restored the sensitivity of H69/LX4 cells to doxorubicin and vinblastine . The inclusion of alpha-tocopherol (25 microg/ml) antagonized the multidrug-resistance (MDR)-modifying activity of all five chemosensitizing agents, effectively preventing restoration of sensitivity to both doxorubicin and vinblastine in H69/LX4 cells. Pharm Res, 1998 May, 15(5), 706 - 11 Decreased expression and activity of P-glycoprotein in rat liver during acute inflammation; Piquette-Miller M et al.; PURPOSE: Drug disposition is often altered in inflammatory disease . Although the influence of inflammation on hepatic drug metabolism and protein binding has been well studied, its impact on drug transport has largely been overlooked . The multidrug resistance (MDR) gene product, P-glycoprotein (P-gp) is involved in the active secretion of a large variety of drugs . Our goal was to ascertain the influence of acute inflammation (AI) on the expression and functional activity of P-gp . METHODS: AI was induced in rats through turpentine or lipopolysaccharide (LPS) administration . Expression of P-gp in liver was detected at the level of protein on Western blots using the monoclonal antibody C-219 and at the level of mRNA using an RNase protection assay . P-gp mediated transport activity was assessed by measuring the verapamil-inhibitable efflux of rhodamine 123 (R123) in freshly isolated hepatocytes . RESULTS: Turpentine-induced AI significantly decreased the hepatic protein expression of P-gp isoforms by 50-70% and caused a significant 45-65% reduction in the P-gp mediated efflux of R123 . Diminished mRNA levels of all three MDR isoforms were seen . LPS-induced AI similarly resulted in significantly reduced levels and activity of P-gp in liver . Although differences in the constitutive levels of P-gp were seen between male and female rats, the influence of AI on P-gp expression and activity was not gender specific . CONCLUSIONS: Experimentally-induced inflammation decreases the in vivo expression and activity of P-gp in liver . This is the first evidence that expression of P-gp is modulated in response to experimentally-induced inflammation. Cancer Chemother Pharmacol, 1998, 42(1), 9 - 16 Reversal of multidrug resistance by a liposome-MDR1 ribozyme complex; Masuda Y et al.; PURPOSE: Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy . We examined whether cationic liposome-mediated transfer of a ribozyme could reverse MDR . METHODS: A ribozyme which cleaved codon 196 of MDR1 mRNA was constructed from synthetic oligonucleotides . The MDR1 ribozyme was mixed with N-(1-(2,3-dileoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (DOTAP) to form a liposomal complex . The complex was used to treat two P-glycoprotein-producing MDR cell lines: MCF-7/R human breast cancer cells resistant to doxorubicin and MOLT-3/TMQ800 human ALL cells resistant to trimetrexate (TMQ) . In order to investigate the differential sensitivity of these two cell lines to the liposome-ribozyme complex, cellular pharmacological studies including phase-contrast and confocal microscopic studies were performed . RESULTS: Treatment with the liposome-ribozyme complex resulted in reversal of vincristine (VCR) resistance in MCF-7/R cells, but not in MOLT-3/TMQ800 cells . In MCF-7/R cells the treatment resulted in decreases in MDR1 mRNA expression and P-glycoprotein production, whereas no changes in these parameters were seen in MOLT-3/TMQ800 cells . Phase-contrast microscopy revealed that in MCF-7/R cells treatment with DOTAP led to the formation of cytoplasmic vacuoles, and treatment with latex beads resulted in the development of a shiny material in the cytoplasm . In contrast, in MOLT-3/TMQ800 cells hardly any morphological changes occurred . Confocal microscopic imaging showed cytoplasmic fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads . In MOLT-3/TMQ800 cells no fluorescence was detected . Treatment with cytochalasin B abolished fluorescence in MCF-7/R cells after treatment with DOTAP/FITC-dextran or FITC-conjugated latex beads . These studies show that MCF-7/R cells have high endocytotic activity whereas MOLT-3/TMQ800 cells have little activity . CONCLUSIONS: Endocytotic activity was correlated with the success of cationic liposome-mediated transfer of MDR1 ribozyme . Determination of endocytotic activity of target tumor cells may be predictive of efficacy of liposome-mediated gene transfer. Planta Med, 1998 May, 64(4), 335 - 8 Mode of action of torilin in multidrug-resistant cancer cell lines; Kim SE et al.; The mechanism of action of multidrug-resistance reversing activity of torilin was studied . In vitro experiments for the accumulation and efflux of vinblastine clearly indicated that MDR reversing effects of torilin would directly be associated with the increase of the intracellular accumulation of anticancer drugs by blocking the drug efflux . Furthermore, torilin increased the membrane ATPase activity from KB-V1 cells, suggesting that torilin might function by inhibiting drug transport mediated by P-glycoprotein. Planta Med, 1998 May, 64(4), 332 - 4 Torilin, a sesquiterpene from Torilis japonica, reverses multidrug-resistance in cancer cells; Kim SE et al.; A sesquiterpene compound reversing multidrug-resistance in cancer cells was isolated from the fruits of Torilis japonica and the structure was identified as torilin . Torilin potentiated the cytotoxicities of adriamycin, vinblastine, taxol and colchicine against multidrug-resistant KB-V1 and MCF7/ADR cells. Cell Physiol Biochem, 1998, 8(3), 138 - 50 pH regulation in sensitive and multidrug resistant Ehrlich ascites tumor cells; Litman T et al.; Maintenance and regulation of intracellular pH (pHi) was studied in wild-type Ehrlich ascites tumor cells (EHR2) and five progressively daunorubicin-resistant, P-glycoprotein (P-gp)-expressing strains, the maximally resistant of which is EHR2/1.3 . Steady-state pHi was similar in cells expressing different amounts of P-gp, in the absence and presence of glucose . In EHR2/1.3, glucose-induced acidification was reduced, and proton efflux was increased, compared to the wild-type EHR2, differences which were not caused by increased activity of a Na+/H+ exchanger in the resistant cells . Comparing all six cell lines, no evidence was found for a correlation between the amount of P-gp in the membrane and pHi regulation, which was also unaffected by P-gp modulators . However, a correlation was seen between relative resistance/daunorubicin accumulation and acid extrusion rate, which is likely to be due to aspects of development of drug resistance other than P-gp. Anticancer Res, 1998 Mar-Apr, 18(2A), 1159 - 66 Cytoplasmic localization of anthracycline antitumor drugs conjugated with reduced glutathione: a possible correlation with multidrug resistance mechanisms; Serafino A et al.; The accumulation of adriamycin (ADR), daunomycin (DAUNO) and their glutathione (GSH)-conjugates, recently obtained by anaerobic reaction of the parent anthracyclines with reduced GSH, was examined in drug-sensitive and multidrug resistant (MDR) cell lines using confocal laser scanning microscopy . In all drug-sensitive lines used (TVM-A12 and TVM-A197 human melanoma cells, K562 lymphoblastoid cells and MCF-7 human breast cancer cells) ADR and DAUNO were mostly located in the nuclei, while their GSH-conjugates were found only in the cytoplasm, predominantly in the Golgi region . On the contrary, in MDR MCF-7/Dox cells, both conjugated and non conjugated anthracyclines gave fluorescence only in the cytoplasm, mostly in the Golgi region, the intensity of the fluorescence being stronger in cells pretreated with verapamil . Viability assay showed that the GSH-conjugate are significantly less cytotoxic than the parent anthracyclines in sensitive cells and showed the same scarce cytotoxicity in MDR MCF-7/Dox cells . These results demonstrate that conjugation of anthracycline antitumor drugs with GSH prevents their access to the nucleus and decreases their cytotoxicity . Furthermore, the observations on MCF-7/Dox suggest that GSH-conjugation of anthracycline might occur in resistant cells and can be in part responsible for the MDR in breast cancer cells. Anticancer Res, 1998 Mar-Apr, 18(2A), 1091 - 7 Multiwavelength videomicrofluorometric study of some human leukemic lymphoblasts: effect of adriamycin on some biological parameter; 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 . The MDR phenotype is characterized by a decrease the intracellular drug accumulation and by an overexpression of the MDR1 gene which encodes the membrane protein, P-glycoprotein (Pgp) . To evaluate the MDR phenotype, rationale investigations of the cytotoxic processes and effect,s of Adriamycin (ADR) were done to obtain information on individual cells . Such information could be obtained through a multiparametric approach involving multiwavelength microfluorometry and numerical image analysis on single living cells . To achieve this, cells should be simultaneously stained with Hoechst 33342 (nuclear staining), Rhodamine 123 (mitochondria staining) and Nile Red (cell contour delineation) . Changes in the biological parameters accessible from R123, Ho33342 and C-SNARF-1/AM (probe used for the pHi measurements) labelling were found more informative than changes in morphological parameters for the discrimination of sensitive and resistant cells . Furthermore, this approach allows the discrimination between two resistant cell lines expressing different mechanisms of resistance. Anticancer Res, 1998 Mar-Apr, 18(2A), 1005 - 10 Chromosome mediated gene transfer of drug resistance to mitoxantrone; Hazlehurst LA et al.; The anthracenedione, mitoxantrone, frequently selects for a unique drug resistance phenotype that is not mediated by either MDR 1, MRP, or altered DNA topoisomerase II . In this study, we demonstrate that mitoxantrone resistance is likely to be multifactorial with at least one resistance mechanism being the result of a dominant genetic event . This finding was demonstrated by conducting chromosome transfer experiments from human breast cancer cell lines that were either sensitive (MCF7/S) or resistant to mitoxantrone (MCF7/Mitox) . Chromosomes transferred from MCF7/Mitox cells into CHO-K1 cells resulted in the isolation of multiple clones resistant to mitoxantrone . In contrast, chromosomes transferred from the drug sensitive MCF7/S, parent cell line did not confer drug resistance in the rodent CHO-K1 recipient cell line . Both Alu-PCR analysis and Southern blot analysis demonstrated human DNA in the CHO-K1 cells receiving chromosomes from the MCF7/Mitox cells . Unlike the MCF7/Mitox cell line, the drug resistant, CHO-K1 chromosome transferrant clones did not have a decrease in total drug accumulation . We conclude that chromosome transfer from the MCF7/Mitox cell line into CHO-K1 cells, confers a non-transport mediated mechanism of drug resistance that is a dominant genetic event . These studies provide evidence of the genetic multifactorial nature of multidrug resistance in cells selected with mitoxantrone in-vitro. Anticancer Res, 1998 Mar-Apr, 18(2A), 739 - 42 Avoidance of doxorubicin resistance in osteosarcoma cells using a new quinoline derivative, MS-209; Takeshita H et al.; P-glycoprotein (Pgp), a membrane drug efflux pump, is thought to be responsible for the observed drug resistance in osteosarcoma . We have recently developed Pgp-positive, multidrug resistant (MDR) murine osteosarcoma cell lines, which may be suitable models for the study of drug resistance in osteosarcoma . In this study, we investigated the effect of a newly synthesized quinoline compound, MS-209, on the reversal of doxorubicin (DOX) resistance in these cell lines . Three different types of resistance modifying agents (RMAs) as well as MS-209 were studied . These included the calcium channel blocker verapamil, and the immunosuppressive agents cyclosporin A and FK506 . The reversal effects of the RMAs on DOX resistance were assessed by the MTT assay . In the absence of RMAs, the MDR osteosarcoma cells were 20-fold more resistant to DOX than the parental cells . When MS-209 was added at a final concentration of 0.1 to 3 microM to the MDR cells, 3-to 74-fold sensitization was observed . A complete reversal (37-fold sensitization) of the resistance was obtained at 1 microM MS-209 . This concentration of MS-209 was 3-, 8- and 28-fold more effective than the same concentration of FK506, verapamil and cyclosporin A, respectively . These results indicate that MS-209 may be a more effective RMA, and that DOX resistance in osteosarcoma cells could be reversed by comparatively low doses of MS-209. Anticancer Res, 1998 Mar-Apr, 18(2A), 701 - 5 Expression analysis of protein kinase C isozymes and multidrug resistance associated genes in ovarian cancer cells; Beck JF et al.; We recently demonstrated a correlation between the expression levels of the PKC eta isozyme and the MDR1 or MRP genes in blasts from AML patients, and in primary breast cancers . In order to extend these findings we analysed ovarian cancer cells from 14 ascites aspirates from 8 patients using a cDNA-PCR approach . 5 patients were examined in follow up studies . 4 out of these 5 patients received continuous chemotherapy . The relative increases in MDR1, MRP, LRP or PKC eta mRNA expression levels were monitored . In one of these patients combined significant increase in MDR1, MRP, LRP and PKC was seen . One follow up sample was obtained after chemotherapy was discontinued . In this case significant relative decreases of MDR1, LRP and PKC eta mRNA expression levels were found . Furthermore, a significant positive correlation was determined for the relative mRNA expression levels of MRP and PKC eta . These results point to a multifactorial emergence of MDR in this type of tumor with a possible involvement of the PKC eta isozyme. Ann Dermatol Venereol, 1996, 123(9), 574 - 6 {Aggressive cutaneous T-cell lymphoma associated with the presence of Epstein-Barr virus . 2 cases}; Mouly F et al.; INTRODUCTION: The factors of prognosis of the cutaneous T-cell lymphomas are less well known as those of the B-cell lymphomas and the role of the Epstein-Barr virus (EBV) is not yet definitively evaluated . CASE REPORTS: Two male patients aged 62 and 82 years had a mycosis fungoides with a lethal outcome . The first patient had mutilating facial tumors; the RNA m of EBV and the genome of EBV were demonstrated in the diseased skin . The second patient had an erythrodermic course with enlarged peripheral lymph nodes and circulating Sezary's cells; the genome of EBV was demonstrated by PCR in the diseased skin . DISCUSSION: The role of the EBV has already been demonstrated in peripheral aggressive T-cell lymphomas . In the mycosis fungoides, the EBV is associated with the lesions in 0 to 32 p . cent according to the published series . EBV associated T-cell lymphomas have a poor survival rate and the EBV infection may be associated with the expression of the multidrug resistant gene-1 (MDR-1) and the risk of a terminal hemophagocytosis . In our both patients the presence of the EBV in the lymphocytes of the skin lesions is also an argument in favour of the pathogenic role of the virus. Gene Ther, 1998 Mar, 5(3), 403 - 8 Feasibility of multidrug resistance (MDR-1) gene transfer in patients undergoing high-dose therapy and peripheral blood stem cell transplantation for lymphoma; Devereux S et al.; We have performed a pilot study of MDR-1 gene transfer in patients receiving CD34-selected peripheral blood stem cell (PBSC) transplant for lymphoma . To ensure minimum engraftment thresholds and facilitate CD34 purification, mobilisation of > 2 x 10(6) CD34 cells/kg was a condition for recruitment . Of 11 patients counselled for study entry, only five achieved this target in a single apheresis . In three consenting patients, purified CD34 cells were exposed to A12M1 MDR-1 retroviral supernatant for 6 h, cryopreserved then thawed and readministered following ablative chemotherapy . No delay in engraftment was observed, although one patient received additional back-up cells . Gene transfer was demonstrated by polymerase chain reaction (PCR) for vector-derived MDR-1 cDNA sequence in all cases . Analysis of peripheral blood and bone marrow cells after transplant has, however, shown no evidence of in vivo gene transfer with a follow-up of 12, 15 and 18 months . The effect of MDR-1 substrate drugs has not yet been tested as all patients remain in clinical and radiological remission of their lymphoma . These results confirm the difficulty of achieving in vivo gene transfer in human haemopoietic cells and indicate major logistical constraints in PBSC mobilisation in patients with relapsed and resistant disease in whom initial studies are appropriate. Ann Trop Med Parasitol, 1998 Jan, 92(1), 31 - 6 Plasma concentrations of artemether and its major plasma metabolite, dihydroartemisinin, following a 5-day regimen of oral artemether, in patients with uncomplicated falciparum malaria; Karbwang J et al.; Plasma concentrations of artemether and its active plasma metabolite (dihydroartemisinin) were measured in 49 male, Thai patients with acute, uncomplicated, multidrug-resistant, Plasmodium falciparum malaria, following their treatment with oral artemether (300 mg on the first day, then 100 mg daily for another 4 days) . Four patients recrudesced (on days 19-22) . After the first dose, artemether became undetectable in < or = 18 h and this drug was also undetectable in samples collected immediately before each dose . Although dihydroartemisinin followed similar trends, three patients had detectable plasma concentrations of this metabolite 24 h after the first dose (i.e . immediately before the second dose) . Median (range) values for plasma concentrations of dihydroartemisinin 6 h {354 (150-751) v . 196 (178-220) ng/ml} and 12 h {158 (25-420) v . 54 (25-115) ng/ml} after the initial dose, estimated antimalarial activities (calculated as dihydroartemisinin equivalents) 6 h {331 (78.2-644.1) v . 23 (183.3-270) nmol/litre} and 12 h {98.3 (10-192.2) v . 56.7 (9.8-59.4) nmol/litre} after the initial dose, and the corresponding 'areas under the curves' (AUC) {3684 (1562-8216) v . 834 (1401-2030) ng.h/ml} were all significantly higher in the patients with sensitive responses than in those who recrudesced. N Engl J Med, 1998 Jun 4, 338(23), 1641 - 9 Global surveillance for antituberculosis-drug resistance, 1994-1997 . World Health Organization-International Union against Tuberculosis and Lung Disease Working Group on Anti-Tuberculosis Drug Resistance Surveillance; Pablos-Mendez A et al.; BACKGROUND: Drug-resistant tuberculosis threatens efforts to control the disease . This report describes the prevalence of resistance to four first-line drugs in 35 countries participating in the World Health Organization-International Union against Tuberculosis and Lung Disease Global Project on Anti-Tuberculosis Drug Resistance Surveillance between 1994 and 1997 . METHODS: The data are from cross-sectional surveys and surveillance reports . Participating countries followed guidelines to ensure the use of representative samples, accurate histories of treatment, standardized laboratory methods, and common definitions . A network of reference laboratories provided quality assurance . The median number of patients studied in each country or region was 555 (range, 59 to 14,344) . RESULTS: Among patients with no prior treatment, a median of 9.9 percent of Mycobacterium tuberculosis strains were resistant to at least one drug (range, 2 to 41 percent); resistance to isoniazid (7.3 percent) or streptomycin (6.5 percent) was more common than resistance to rifampin (1.8 percent) or ethambutol (1.0 percent) . The prevalence of primary multidrug resistance was 1.4 percent (range, 0 to 14.4 percent) . Among patients with histories of treatment for one month or more {corrected}, the prevalence of resistance to any of the four drugs was 36.0 percent (range, 5.3 to 100 percent), and the prevalence of multidrug resistance was 13 percent (range, 0 to 54 percent) . The overall prevalences were 12.6 percent for resistance to any of the four drugs {corrected} (range, 2.3 to 42.4 percent) and 2.2 percent for multidrug resistance (range, 0 to 22.1 percent) . Particularly high prevalences of multidrug resistance were found in the former Soviet Union, Asia, the Dominican Republic, and Argentina . CONCLUSIONS: Resistance to antituberculosis drugs was found in all 35 countries and regions surveyed, suggesting that it is a global problem. BMJ, 1998 May 9, 316(7142), 1423 - 5 Drug resistant tuberculosis in prisons in Azerbaijan: case study; Coninx R et al.; OBJECTIVES: To document the existence of drug resistance in a tuberculosis treatment programme that adheres strictly to the DOTS principles (directly observed treatment, short course) and to determine the extent of drug resistance in a prison setting in one of the republics of the former Soviet Union . DESIGN: Case study . SETTING: Central Penitentiary Hospital in Baku, the referral centre for tuberculosis patients from all prisons in Azerbaijan . SUBJECTS: Prisoners with tuberculosis: 28 selected patients not responding clinically or bacteriologically to the standard treatment (group 1) and 38 consecutive patients at admission to the programme (group 2) . MAIN OUTCOME MEASURES: Drug resistance of Mycobacterium tuberculosis strains grown from sputum . RESULTS: All the non-responding patients (group 1) had strains resistant to at least one drug . 25 (89%) of the non-responding patients and nine (24%) of the consecutive patients had M tuberculosis strains resistant to both rifampicin and isoniazid . A further 17 patients in group 2 had strains resistant to one or more first line drugs . CONCLUSIONS: Drug resistant M tuberculosis strains are common in prisons in Azerbaijan . Tuberculosis problems tend to be worse in prisons, but prisoners and former prisoners may have an important role in the transmission of tuberculosis, particularly of drug resistant forms, in the community . National programmes to control tuberculosis will have to take into account and address the problems in prisons to ensure their successPIP: Tuberculosis is a significant health problem in Azerbaijan . In prisons, this problem is compounded by overcrowding, poor general health, a high representation of risk groups, late case finding, and incomplete treatments . The present study investigated the extent of drug resistance at the Central Penitentiary Hospital in Baku--the country's only treatment center for prisoners with tuberculosis . This International Committee of the Red Cross program, established in 1995, uses the directly observed treatment, short course (DOTS) strategy . Sputum samples were collected from two groups of prisoners: 1) 28 patients who failed to respond, clinically or bacteriologically, after a minimum of 8 weeks to the treatment regimen recommended by the World Health Organization and 2) 38 patients consecutively enrolled over a 4-week period from whom sputum was taken before the start of treatment . Mycobacterium tuberculosis was isolated from all 66 sputum specimens . In the first group, 25 strains (98%) were multidrug resistant (to rifampicin and isoniazid) . Such resistance occurred in all new cases and 14 (82%) of the 17 failure or relapse cases . In the second group, 9 strains (24%) were multidrug resistant and only 12 (32%) were fully susceptible . This resistance was found in 3 strains (15%) among the 20 new cases and in 6 strains (33%) among the 18 cases of treatment failure or relapse . These findings suggest that prisoners may have an important future role in the transmission of tuberculosis, especially multidrug resistant forms, in the former Soviet Union . Int J Oncol, 1998 May, 12(5), 1143 - 9 Multidrug resistance modulation in rhabdomyosarcoma and neuroblastoma cell lines; Cowie FJ et al.; Four rhabdomyosarcoma and three neuroblastoma cell lines were characterised for the presence of P-glycoprotein and MDR-1 expression using immunohistochemistry, northern analysis, RT-PCR and in situ mRNA hybridisation . None of the rhabdomyosarcoma lines were unequivocally positive in contrast to all three neuroblastoma lines . Chemosensitivity to cytotoxic agents was determined using the MTT assay and chemosensitisation by cyclosporin and verapamil was evaluated . In a single rhabdomyosarcoma line (HX 170) there was sensitisation to etoposide using verapamil but not to other drugs or using cyclosporin A . In contrast, in all three neuroblastoma lines both cyclosporin and verapamil sensitised to vincristine and doxorubicin . No evidence of sensitisation to etoposide was apparent . The sensitisation was most marked for vincristine, using either modulator and therefore the influence of modulator scheduling was evaluated with this drug in the neuroblastoma line SK N BE . Prolonged pre-exposure to modulator did not appear necessary and maximum sensitisation was apparent where either cyclosporin or verapamil was added 1-3 h prior to and post vincristine . Continuity of exposure was important and even a break of 30 min appeared to reduce sensitisation . These data confirm the potential for chemosensitisation in MDR-1 positive neuroblastoma cell lines and provide some basis for rational schedule design in clinical practice . Because of the probability that vincristine resistance is predominantly related to MDR-1 and less multifactorial than for other drugs such as doxorubicin or etoposide, this agent should be considered for inclusion in any clinical evaluation of MDR reversal strategies. Int J Oncol, 1998 May, 12(5), 1137 - 42 P-glycoprotein-mediated multidrug resistance is modulated by pretreatment with chemosensitizers in HCT-8 carcinoma cells in vitro; Haberl I et al.; The effects of pretreatment with the multidrug resistance (MDR) modulators verapamil (VPM), tamoxifen (TMX), cyclosporin A (CsA), and SDZ PSC833 (PSC) on drug sensitivity of the P-glycoprotein (Pgp) expressing human ileocecal carcinoma cell line HCT-8 is described . Following pretreatment of 2, 16 and 48 h with the individual modulators, rhodamine 123 efflux (RHO), transepithelial vinblastine transport (VIN) across treated HCT-8 monolayers, and chemosensitivity to doxorubicin (DOX) were determined and compared to Pgp protein expression and phosphorylation . After 2 h, VPM, TMX, CsA and PSC inhibited RHO efflux and VIN transport and increased the chemosensitivity of HCT-8 to DOX significantly . Prolonged exposure failed to further increase inhibition of Pgp-mediated transport, but in contrast maximized phosphorylation of Pgp (16 h) and Pgp protein expression (48 h), respectively. Leuk Lymphoma, 1998 Feb, 28(5-6), 469 - 82 Prognostic value of P-glycoprotein and leukocyte differentiation antigens in chronic myeloid leukemia; Stavrovskaya A et al.; P-glycoprotein (Pgp) mediated multidrug resistance is often the cause of therapy failure in some tumors . Pgp expression was shown to have prognostic value in several hematological malignancies, especially in acute myeloblastic leukemia (AML) and acute lymphoblastic leukemia (ALL) . In chronic myeloid leukemia (CML) Pgp is expressed by peripheral blood (PB) cells more often in the terminal disease stages (20-50% of patients have Pgp+ phenotype) . Sequential studies show that Pgp+ cells often disappear from the PB during the course of therapy . Nevertheless Pgp expression has some prognostic value in blast crisis (BC) predicting shorter BC, while CD13 has the same predictive value in BC . 10% of patients formed a distinct group with large numbers of Pgp+CD34+ blasts in the PB and also had shorter BC . Cases with inactive Pgp were found in chronic and accelerated phases of CML but not in BC. Int J Tuberc Lung Dis, 1998 May, 2(5), 390 - 6 Use of DNA fingerprinting for primary surveillance of nosocomial tuberculosis in a large urban hospital: detection of outbreaks in homeless people and migrant workers; Lemaitre N et al.; SETTING: A large urban teaching hospital in the southeast of Paris . OBJECTIVE: Primary surveillance of nosocomial transmission of tuberculosis (TB) by systematic restriction fragment length polymorphism analysis (RFLP) of isolates (n = 161) recovered from smear-positive pulmonary TB patients identified from 1 March 1993 to 28 February 1994, and from all TB patients (with any form of tuberculous infection) identified from 1 March 1994 to 30 April 1995 . RESULTS: Systematic RFLP analysis revealed 12 clusters of patients (n = 40) infected by strains of Mycobacterium tuberculosis showing matching RFLP patterns . None of the isolates were multidrug-resistant . Compared with non-clustered patients, clustered patients were more likely to be homeless (55% vs 19%, P < or = 0.001), or Africans living in hostels for migrant workers (20% vs 6%, P = 0.01), and had fewer previous admissions to hospital (12% vs 28%, P = 0.05) . Further epidemiological investigations showed that the clustered TB cases actually resulted not from nosocomial transmission, but from transmission in the community, very likely in homeless shelters and hostels for migrant workers . CONCLUSION: No nosocomial transmission of TB was identified among the patients included during the study period . Systematic RFLP analysis using hospital-based sampling can detect the spread of TB in specific environments in the community where transmission is occurring. Oncol Res, 1998, 10(1), 29 - 41 Characterization of 2-chloro-N10-substituted phenoxazines for reversing multidrug resistance in cancer cells; Thimmaiah KN et al.; Twenty-one 2-chloro-N10-substituted phenoxazines have been synthesized and characterized as potential modulators of multidrug resistance (MDR) . Many of the compounds, at a concentration of 100 microM, enhanced accumulation of vinblastine (VLB) in drug-resistant KB8-5 cells to a greater extent than the same concentration of verapamil (VRP) . However, the effects on VLB accumulation were specific, because these derivatives had little activity in the parental drug-sensitive line KB3-1 . The compounds slowed the efflux of VLB from KB8-5 cells, suggesting that the chlorophenoxazines, like VRP, can inhibit P-glycoprotein (P-gp)-mediated efflux of VLB from this cell line . Two of the chlorophenoxazine derivatives, and also VRP, were able to stimulate the vanadate-sensitive ATPase activity attributable to P-gp in membranes isolated from MDR1 baculovirus-infected Sf9 cells . This result suggests that these modulators exert their effect by directly interacting with P-gp . Apart from the parent unsubstituted molecule, 2-chlorophenoxazine, there was a good correlation between log10P and the ability of the compounds to enhance VLB accumulation in KB8-5 . This suggests that lipophilicity of a modulator is important, but is not the sole determinant of potency . Within this series of compounds, the optimal structural features for MDR modulation include a hydrophobic phenoxazine ring with a -Cl atom in the C-2 position and a tertiary amine group four carbons from the tricyclic ring . Many of the agents at the IC10 concentration completely reversed the 37-fold VLB resistance in KB8-5 cells . The most active agents in KB8-5 were able to partially reverse VLB resistance in an MDR colon carcinoma cell line GC3/c1 and completely reversed the 86-fold VLB resistance in the MDR1-overexpressing breast carcinoma cell line BC19/3 . These same agents could only partially sensitize BC19/3 cells to taxol and doxorubicin, suggesting that the chlorophenoxazine derivatives show some specificity for modulating VLB resistance. Orv Hetil, 1998 May 10, 139(19), 1139 - 45 {Tuberculosis at the threshold of the 21st century}; Hutas I; The author summarizes the most important results of the present history of tuberculosis obtained both in Hungary and abroad . He deals with the state of the art of the tuberculosis epidemics all over the world and in Hungary, presenting also the potential genetic risks . He also discusses the risk groups with special regard to the extremely large regional differences, searching for the underlying causes, as well as to that of the relationships between the epidemic and the social factors . He reviews the up-to-date methods of the diagnostics of Mycobacterium tuberculosis . He evaluates the significance of mass screening in the early diagnosis of tuberculosis and lung cancer in Hungary . He gives an overview of the current strategy of treatment, of the principals of directly observed therapy short course (DOTS) and the international results obtained . In the Hungarian surveillance programme, these principles are adapted under domestic conditions . Based on the data from Hungary and abroad, he presents the risk of development of multidrug-resistant strains and the mechanism of their development, presuming that genetic methods will also play a role in the future management of tuberculosis. Nucleic Acids Res, 1998 Jun 15, 26(12), 3001 - 5 Binding of the modified daunorubicin WP401 adjacent to a T-G base pair induces the reverse Watson-Crick conformation: crystal structures of the WP401-TGGCCG and WP401-CGG{br5C}CG complexes; Dutta R et al.; 2'-Bromo-4'-epi-daunorubicin (alpha-manno configuration, denoted WP401) is a new anthracycline drug that exhibits promising activity toward multidrug-resistant cancer cells . We carried out X-ray diffraction analyses of the complexes formed in the presence of formaldehyde between WP401 and two DNA hexamers, TGGCCG and CGG{br5C}CG . The two complexes crystallized in different crystal lattices with respective crystal data of space group P4322, a = b = 37.20 A, c = 70.53 A and space group P43212, a = b = 37.23 A, c = 61 . 96 A . These new crystal forms are different from the P41212 form of other daunorubicin/doxorubicin complexes studied previously . The refined crystal structures at approximately 2.0 A resolution revealed that the entire 2:1 drug-DNA complex is in the asymmetrical unit . Two WP401 drug molecules bind to the duplex, with the aglycones intercalated between the CpG or TpG steps and their modified daunosamines in the minor groove . As observed earlier, in the presence of formaldehyde, WP401 more readily forms a covalent adduct with (C/T)GG*:CCG than with (C/T)GC:G*CG (G* is the crosslink site), the opposite of what is seen for daunorubicin and doxorubicin . Surprisingly, the two T-G mismatched base pairs in the WP401-TGGCCG complex adopt the reverse Watson-Crick conformation, instead of the wobble conformation . The unusual T-G reverse Watson-Crick conformation may be required in order to maintain favorable stacking interactions between the base pair and the aglycone of WP401 . Our results show that chemical modifications like bromo or iodo substitution on anthracycline drugs have significant effects on their DNA binding properties. Int J Cancer, 1998 May 29, 76(5), 729 - 37 Drug binding to P-glycoprotein is inhibited in normal tissues following SDZ-PSC 833 treatment; Jette L et al.; Rats were treated with daily injections of SDZ-PSC 833 (PSC) to study the interaction of this potent modulator of multidrug resistance (MDR) with P-glycoprotein (P-gp) expressed in normal tissues . After 2 days of treatment, the level of P-gp expression, detected by Western blot analysis, was not modified in renal brush border membranes (BBMs) and brain capillaries . However, the amount of P-gp detected with the photoaffinity probe {125I}-arylazidoprazosin (IAAP) was decreased in both tissues, suggesting that the drug binding properties of P-gp were altered by PSC treatment . This effect was further characterized by treating rats with PSC for 10 days . Following these treatments, the amount of immunodetected P-gp was increased in renal BBMs and brain capillaries . However, no increase in P-gp expression was observed in photolabeling experiments, suggesting that induced P-gp was not functional . In vitro experiments performed with renal BBMs showed that the inhibition of P-gp photolabeling by cyclosporin A (CsA), verapamil and vinblastine could be reversed by performing washing steps to remove these drugs before incubating the samples with IAAP . However, the inhibition mediated by PSC was less reversible since photolabeling of P-gp remained inhibited following the washing steps . Pre-incubation of intact CHRC5 cells with PSC, CsA and verapamil also inhibited P-gp photolabeling and increased rhodamine 123 accumulation . For PSC pre-treated samples, these effects were not completely reversed following washing, but were abolished for CsA and Ver pre-treated samples . Our results suggest that PSC could block P-gp function by a different mechanism from that of CsA and verapamil, involving modification of the drug binding sites. Int J Cancer, 1998 May 29, 76(5), 702 - 8 Induction of broad drug resistance in small cell lung cancer cells and its reversal by paclitaxel; Su GM et al.; The H82 "variant" and the H69 "classic" small cell lung cancer (SCLC) cell lines were treated with low levels of epirubicin (69 and 14 nM) which caused little cell death but produced the H82/E8 and H69/E8 extended-multidrug resistant sublines . Both were resistant to drugs associated with multidrug resistance (MDR), and to chlorambucil (9.5- and 5.6-fold, respectively) and cisplatin (2.3- and 8.5-fold, respectively) . There was increased expression of the multidrug resistance-associated protein (MRP1) in the H82/E8 subline while P-glycoprotein expression was not detected in any cells or sublines . Treatment of the H82 cells for 1 hr with 69 nM epirubicin increased MRP1-mRNA expression within 4 hr and this was associated with an increase in the resistance to epirubicin, chlorambucil, cisplatin and paclitaxel . Further, a 1 hr treatment with non-cytotoxic doses of chlorambucil (2.5 microM), cisplatin (1.3 microM) or paclitaxel (13 nM), drugs not normally associated with MRP1-mediated MDR, also increased MRP1-mRNA expression in the H82 cells with paclitaxel causing the highest increase (4.5-fold) . For chlorambucil treatment, this increased MRPI-mRNA expression was accompanied by increased drug resistance while paclitaxel treatment had no effect on drug resistance in the H82 cells . For the drug resistant H82/E8 subline, these drug treatments had no effect on the MRP1-mRNA expression and little effect on increasing the subline drug resistance . However, pretreatment with paclitaxel sensitised the H82/E8 subline to chlorambucil and cisplatin returning the subline to the sensitivity of the H82 cell line . We conclude that treatment with low levels of MDR and non-MDR drugs can induce extended-multidrug resistance in SCLC cells, a process that probably involves the co-ordinate upregulation of MRP1 and other resistance mechanisms . The results also suggest paclitaxel may have a role as a response modifier in the treatment of refractory SCLC. Free Radic Biol Med, 1998 Apr, 24(6), 913 - 23 Antiproliferative effect of the piperidine nitroxide TEMPOL on neoplastic and nonneoplastic mammalian cell lines; Gariboldi MB et al.; The stable nitroxide 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL) is widely used as a probe in biophysical studies and as an antioxidant in several experimental models . The potential cytotoxic effects of TEMPOL were tested on a panel of human and rodent cell lines, and the nitroxide proved to be significantly more effective in inhibiting the growth of neoplastic than nonneoplastic cell lines after a 96-h exposure . More detailed studies on MCF-7/WT cells indicate that at least 24 h are necessary for TEMPOL to induce irreversible cell damage, which seems to be related to the reactivity of the nitroxyl group . This observation, together with the antagonistic effect of N-acetylcysteine, suggests an involvement of free radical-mediated processes . Cell cycle studies indicate a biphasic effect of TEMPOL, with a short-term accumulation of the cells in the G1 phase and a later increase in G2/M phase; the pattern of DNA fragmentation observed in TEMPOL-treated cells points to an apoptotic mode of cell death . In conclusion, our data suggest that, while the possible cytotoxic effects of TEMPOL should not be overlooked when using this compound as a biophysical probe or antioxidant, these same properties could be exploited as a novel approach to cancer chemotherapy, especially in tumor cells exhibiting unfavorable characteristics, such as a multidrug-resistant phenotype or loss of hormone receptors. Clin Cancer Res, 1998 May, 4(5), 1305 - 14 Bryostatin 1 down-regulates mdr1 and potentiates vincristine cytotoxicity in diffuse large cell lymphoma xenografts; Al-Katib AM et al.; The down-regulation of multidrug resistance (mdr1) gene expression as detected by competitive reverse transcription-PCR and the antitumor activity of bryostatin 1 (Bryo1) are investigated in a newly established cell line from a patient with relapsed diffuse large cell lymphoma (DLCL) . The cell line (WSU-DLCL2) grows in liquid culture and forms s.c . tumors in mice with severe combined immune deficiency . WSU-DLCL2 is a mature B-cell line (IgG lambda) that is negative for EBV nuclear antigen, expresses the multidrug resistance phenotype, and has t(14;18)(q32;q21) plus other chromosomal aberrations . Exposure of the WSU-DLCL2 cells to Bryo1 in culture reversed the multidrug resistance phenotype within 24 h . A functional assay revealed a 4-fold increase in {3H}vincristine accumulation in Bryo1-treated cells compared with control . Vincristine (VCR), doxorubicin, Bryo1, and 1-beta-D-arabinofuranosylcytosine showed no clinically significant activity when given alone to WSU-DLCL2-bearing severe combined immune deficiency mice . However, when given 24 h before each cytotoxic agent, Bryo1 substantially increased the antitumor activity of VCR but not 1-beta-D-arabinofuranosylcytosine . There was a statistically significant (P < 0.001) decrease in the expression of P-glycoprotein in WSU-DLCL2 tumors taken from Bryo1-treated animals compared with untreated controls . In vivo, a competitive reverse transcription-PCR assay revealed decreased mdr1 RNA expression 24 h after Bryo1 treatment . These findings taken together indicate that Bryo1-induced down-regulation of mdr1 might be one mechanism by which Bryo1 potentiates VCR activity . The sequential use of both agents resulted in clinically significant antitumor activity in this lymphoma model. Receptors Channels, 1997, 5(3-4), 175 - 83 Drug accumulation in the presence of the multidrug resistance pump: dissociation between verapamil accumulation and the action of P-glycoprotein; Ayesh S et al.; We studied the interaction between the multidrug transporter, P-glycoprotein, and two compounds that interact with it: vinblastine, a classical substrate of the pump, and verapamil, a classical reverser . Steady-state levels of accumulation of these two drugs were determined in a multidrug resistant P388 leukemia cell line, P388/ADR . The time course of accumulation of these drugs, and the effect of energy starvation and the presence of chloroquine on the level of their steady-state accumulation were quite disparate . Vinblastine inhibited the accumulation of verapamil whereas it enhanced the accumulation of daunomycin, another classic substrate of P-glycoprotein . Verapamil did not compete with the intracellular binding sites of vinblastine . In all these aspects, vinblastine behaved as a typical substrate of P-glycoprotein but verapamil did not . Our data suggest that verapamil is a reverser of P-glycoprotein but that its intracellular accumulation is not affected by this membrane-bound transporter. Neoplasma, 1997, 44(6), 366 - 9 Human multidrug-resistant (MRP,p190) myeloid leukemia HL-60/ADR cells in vitro: resistance to the mevalonate pathway inhibitor lovastatin; Hunakova L et al.; Mevalonate pathway inhibitor lovastatin inhibited proliferation of human multidrug-resistant promyelocytic leukemia HL-60/ADR cells in vitro, with MRP-gene coded p190 mediated drug resistance, to a markedly lesser extent than that of the parental drug sensitive HL-60 cells and also that of the other human multidrug resistant (MDR-1, P-glycoprotein) myeloid leukemia cell line HL-60/VCR . The sensitivity of the examined human leukemia cell lines to the cytostatic activity of lovastatin correlated approximately with the potential of lovastatin to induce the characteristic cell cycle alteration (i.e . the accumulation of lovastatin-treated cells in the G0/G1 phase of the cell cycle) . The P-glycoprotein positive HL-60/VCR cells and the parental drug sensitive HL-60 cells were more sensitive to this cell cycle alteration than the HL-60/ADR multidrug resistant leukemia cells with MRP drug resistance . Lovastatin (72 hours, 20 micromol) induced apoptosis and cell necrosis in HL-60 cells, apoptosis but not cell necrosis in HL-60/VCR cells and neither apoptosis nor necrosis in HL-60/ADR cells. Blood, 1998 Jun 1, 91(11), 4106 - 17 Rhodamine-123 staining in hematopoietic stem cells of young mice indicates mitochondrial activation rather than dye efflux; Kim M et al.; Low-intensity fluorescence of rhodamine-123 (Rh-123) discriminates a quiescent hematopoietic stem cell (HSC) population in mouse bone marrow, which provides stable, long-term hematopoiesis after transplantation . Rh-123 labels mitochondria with increasing intensity proportional to cellular activation, however the intensity of staining also correlates with the multidrug resistance (MDR) phenotype, as Rh-123 is a substrate for P-glycoprotein (P-gp) . To address the mechanisms of long-term repopulating HSC discrimination by Rh-123, mouse bone marrow stem and progenitor cells were isolated based on surface antigen expression and subsequently separated into subsets using various fluorescent probes sensitive to mitochondrial characteristics and/or MDR function . We determined the cell cycle status of the separated populations and tested for HSC function using transplantation assays . Based on blocking studies using MDR modulators, we observed little efflux of Rh-123 from HSC obtained from young (3- to 4-week-old) mice, but significant efflux from HSC derived from older animals . A fluorescent MDR substrate (Bodipy-verapamil, BodVer) and Rh-123 both segregated quiescent cells into a dim-staining population, however Rh-123-based separations resulted in better enrichment of HSC function . Similar experiments using two other fluorescent probes with specificity for either mitochondrial mass or membrane potential indicated that mitochondrial activation is more important than either mitochondrial mass or MDR function in defining HSC in young mice . This conclusion was supported by morphologic studies of cell subsets separated by Rh-123 staining. Eur J Nucl Med, 1998 Apr, 25(4), 401 - 9 Application of SPET using technetium-99m sestamibi in brain tumours and comparison with expression of the MDR-1 gene: is it possible to predict the response to chemotherapy in patients with gliomas by means of 99mTc-sestamibi SPET? Yokogami K, Kawano H, Moriyama T, Uehara H, Sameshima T, Oku T, Goya T, Wakisaka S, Nagamachi S, Jinnouchi S, Tamura S. Technetium-99m sestamibi (MIBI) is thought to be passively taken up by metabolically active tumour cells and effluxed from them by P-glycoprotein (Pgp) . This 170-kDa membrane-bound protein, encoded by the MDR-1 gene, acts as an energy-dependent efflux pump for several antineoplastic agents, resulting in multidrug resistance . For this reason, it is of interest whether the tumour's response to chemotherapy can be predicted by MIBI single-photon emission tomography (SPET) . In this study, MIBI SPET was compared with thallium-201 (Tl) SPET using magnetic resonance imaging as a guide in 16 patients with untreated brain tumours {ten glioblastomas (GBs), two anaplastic astrocytomas (AAs), two low-grade gliomas (LGASs) and two metastatic brain tumours) and in four patients who had received treatment for with brain tumours (two GBs, two AAs) . In addition, we investigated the expression of the MDR-1 gene and its product Pgp in the same patients, and compared the results with MIBI SPET findings . MIBI, as well as Tl, was highly accumulated and retained in the enhanced region of malignant gliomas . In addition, MIBI SPET yielded sharp and well-contrasted images, and the margin of the tumour was more clearly defined than with Tl SPET due to a good signal-to-noise ratio . Follow-up MIBI SPET in patients who had received therapy showed marked uptake in a patient with malignant transformation, who deteriorated clinically . Patients with no uptake on MIBI SPET showed no sign of recurrence . Semiquantitative analysis of untreated patients showed a relationship between the early uptake index (UI, ratio of average count/pixel in the lesion to that in the contralateral area on early images) and the degree of malignancy (early UI = 1.08+/-0.06 in LGASs, 4.10+/-0.84 in AAs, 5.71+/-3.47 in GBs, and 7.52+/-1.52 in metastatic brain tumours) . The retention index (RI, ratio of delayed to early UI) of MIBI was significantly lower than that of Tl in metastatic brain tumours (P<0.05), but not in malignant gliomas . Histological and biological investigation of gliomas showed that the MDR-1 gene and its product Pgp were expressed only in normal endothelial cells and not in tumour cells or proliferating endothelial cells; Pgp tended to decrease as the degree of malignancy rose . Hence, the presence of Pgp and the grade of malignancy were inversely related in gliomas . By contrast, immunohistochemical study showed strong accumulation of Pgp in metastatic brain tumour cells . These histopathological findings and MIBI SPET findings are compatible with experimental data; MIBI was washed out by Pgp . The main cause of chemoresistance is probably not an increasing drug efflux by Pgp in gliomas . Thus, MIBI SPET is useful for detecting the active lesions, but may not be useful for predicting the response to chemotherapy in gliomas. J Biol Chem, 1998 Apr 24, 273(17), 10733 - 40 Multidrug resistance protein . Identification of regions required for active transport of leukotriene C4; Gao M et al.; Multidrug resistance protein (MRP) is a broad specificity, primary active transporter of organic anion conjugates that confers a multidrug resistance phenotype when transfected into drug-sensitive cells . The protein was the first example of a subgroup of the ATP-binding cassette superfamily whose members have three membrane-spanning domains (MSDs) and two nucleotide binding domains . The role(s) of the third MSD of MRP and its related transporters is not known . To begin to address this question, we examined the ability of various MRP fragments, expressed individually and in combination, to transport the MRP substrate, leukotriene C4 (LTC4) . We found that elimination of the entire NH2-terminal MSD or just the first putative transmembrane helix, or substitution of the MSD with the comparable region of the functionally and structurally related transporter, the canalicular multispecific organic anion transporter (cMOAT/MRP2), had little effect on protein accumulation in the membrane . However, all three modifications decreased LTC4 transport activity by at least 90% . Transport activity could be reconstituted by co-expression of the NH2-terminal MSD with a fragment corresponding to the remainder of the MRP molecule, but this required both the region encoding the transmembrane helices of the NH2-terminal MSD and the cytoplasmic region linking it to the next MSD . In contrast, a major part of the cytoplasmic region linking the NH2-proximal nucleotide binding domain of the protein to the COOH-proximal MSD was not required for active transport of LTC4. Ann Oncol, 1998 Jan, 9(1), 85 - 93 No evidence of significant activity of the multidrug resistance gene product in primary human breast cancer; Hegewisch-Becker S et al.; BACKGROUND: The discovery of the multidrug resistance (MDR1) gene product P-glycoprotein (P-gp) has been widely seen as an important milestone in our understanding of the mechanisms underlying the clinical phenomenon of the emergence of resistant cells . MDR1 expression has been shown for numerous solid tumors and for virtually all hematologic malignancies . Nevertheless, results regarding MDR1/P-gp expression in human breast cancer have been controversial and the results of clinical trials on modulation of P-gp activity have not been encouraging . PATIENTS AND METHODS: MDR1/P-gp expression and the function of the P-gp pump were investigated in 61 tumor samples from patients with primary breast cancers by multiparameter analysis using MDR1-RT-PCR, immunohistochemistry with two MAbs (UIC2 and MRK16) and the rhodamine 123 (Rh123) efflux assay . The cellular composition of the tumor cell suspension was analyzed by using specific MAbs against the P-gp expressing lymphocyte subsets CD4, CD8 and CD56, as well as against the HER-2/neu gene product, which was used to identify breast carcinoma cells . RESULTS: UIC2 and MRK16 revealed a staining positivity in 72% and 75% of samples, respectively . A positive MDR1-RT-PCR signal was detected in 62% of the samples . Nevertheless, no correlation between immunohistochemistry and RT-PCR could be established . Furthermore, there was no correlation between HER-2/neu expression and MDR1-RT-PCR or P-gp immunohistochemical assays . A contamination by CD8+ and CD4+ lymphocytes was established in 100% and 84% of tumor cell suspensions, respectively . As assessed by the Rh123 efflux assay CD8+ and the CD4+ lymphocytes exhibited marked P-glycoprotein activity, whereas such activity was not detectable in a single instance for the breast carcinoma cells . In MDR1-RT-PCR positive samples, contamination by CD8 lymphocytes averaged 4.3%, while the contamination of CDS cells in the MDR1 mRNA-negative samples was only 2.4% (P = 0.007) . This signal vanished after elimination of the lymphocyte subpopulations by T-cell rosetting . CONCLUSIONS: In primary breast cancer detection of MDR1 gene expression by means of RT-PCR or immunohistochemical assays is not indicative for the MDR phenotype, since there is no evidence of significant activity of the P-gp pump. Am J Respir Crit Care Med, 1998 May, 157(5 Pt 1), 1609 - 15 Preliminary results of collapse therapy with plombage for pulmonary disease caused by multidrug-resistant mycobacteria; Jouveshomme S et al.; Seven patients underwent collapse therapy with polystyrene sphere plombage for pulmonary disease caused by multidrug-resistant mycobacteria . Four patients were infected with multidrug-resistant strains of Mycobacterium tuberculosis, two with Mycobacterium xenopi, one with Mycobacterium avium . All patients were heavily pretreated before surgery, had extensive, bilateral cavitary disease and were considered unsuitable for resection because of extensive disease or functional respiratory impairment . Six patients had active disease at time of surgery . Collapse therapy with insertion of six to 18 spheres resulted in long-standing bacteriological conversion in six patients . Collapse therapy was unilateral in six and bilateral in one . No immediate postoperative complication or death was observed . Hospital stay was short (mean 12 d) . Collapse therapy is a conservative alternative therapy in patients with pulmonary disease caused by multidrug-resistant mycobacteria at high risk of treatment failure considered unsuitable for pulmonary resection. Genes Dev, 1998 May 15, 12(10), 1453 - 63 Regulation of the fission yeast transcription factor Pap1 by oxidative stress: requirement for the nuclear export factor Crm1 (Exportin) and the stress-activated MAP kinase Sty1/Spc1; Toone WM et al.; The fission yeast Sty1 stress-activated MAP kinase is crucial for the cellular response to a variety of stress conditions . Accordingly, sty1- cells are defective in their response to nutrient limitation, lose viability in stationary phase, and are hypersensitive to osmotic stress, oxidative stress, and UV treatment . Some of these phenotypes are caused by Sty1-dependent regulation of the Atf1 transcription factor, which controls both meiosis-specific and osmotic stress-responsive genes . However, in this report we demonstrate that the cellular response to oxidative stress and to treatment with a variety of cytotoxic agents is the result of Sty1 regulation of the Pap1 transcription factor, a bZip protein with structural and DNA binding similarities to the mammalian c-Jun protein . We show that both Sty1 and Pap1 are required for the expression of a number of genes involved in the oxidative stress response and for the expression of two genes, hba2+/bfr1+ and pmd1+, which encode energy-dependent transport proteins involved in multidrug resistance . Furthermore, we demonstrate that Pap1 is regulated by stress-dependent changes in subcellular localization . On imposition of oxidative stress, the Pap1 protein relocalizes from the cytoplasm to the nucleus in a process that is dependent on the Sty1 kinase . This relocalization is the result of regulated protein export, rather than import, and involves the Crm1 (exportin) nuclear export factor and the dcd1+/pim1+ gene that encodes an Ran nucleotide exchange factor. J Pharm Pharmacol, 1998 Mar, 50(3), 343 - 9 Effect of the number of samples on Bayesian and non-linear least-squares individualization: a study of cyclosporin treatment of haematological patients with multidrug resistance; Wu G et al.; We have studied whether the prediction of drug concentrations improves as the number of samples used for individualization is increased, and whether the Bayesian method of individualization is superior to the non-linear least-squares method . Data were obtained from ten adult haematological patients with multidrug resistance who were treated with cyclosporin . The predictions of blood-cyclosporin concentrations were made using the Abbott PKS program . The number of samples used for individualization was increased from 1 to 30 for the Bayesian method and from 4 to 30 for the non-linear least-squares method . Linear regression, percentage prediction error, and absolute and relative predictive performance were used to evaluate the predictions . The results show that the Bayesian method affords greater precision than the non-linear least-squares method, but that the non-linear least-squares method is more accurate and results in less bias . Whereas for linear regression predictions improve as the number of samples is increased, other evaluations show improvement in the range from 5 to 11 samples; linear regression, percentage prediction errors and prediction bias support the opinion that the Bayesian method progressively becomes the non-linear least-squares method as the number of samples used for individualization is increased, but the accuracy and precision of prediction do not support this opinion . The study supports the statement that Bayes' law requires parameters from an infinite population, otherwise the advantage of the Bayesian method might be marginal. Am J Trop Med Hyg, 1998 May, 58(5), 630 - 7 Characterization of Plasmodium falciparum isolated from the Amazon region of Brazil: evidence for quinine resistance; Zalis MG et al.; The prevalence and severity of drug-resistant malaria is emerging rapidly in the Amazon basin of Brazil . In support of clinical trials using the new antimalarial drug combination of atovaquone and proguanil, we performed in vitro drug sensitivities, molecular characterization of parasite populations using the circumsporozoite protein, merozoite surface antigen-1 (MSA-1), and MSA-2 markers, and an analysis of the Plasmodium falciparum multidrug resistance (pfmdr1) gene sequence and copy number in 26 isolates of P . falciparum obtained in a gold-mining endemic area in Peixoto de Azevedo, Mato Grosso State . All 26 isolates were found to be resistant to chloroquine (50% inhibitory concentration {IC50} = 100-620 nM) and sensitive to mefloquine (IC50 < 23 nM) and halofantrine (IC50 < 6 nM) . The isolates also show reduced susceptibility to quinine (IC50 = 48-280 nM) . Sequence analysis of the pfmdr1 gene revealed Asn, Phe, Cys, Asp, and Tyr in positions 86, 184, 1034, 1042, and 1246, respectively . These point mutations were similar to that previously described in other Brazilian isolates . Southern blot analysis revealed no amplification of the pfmdr1 gene . These results suggest that three different mechanisms for drug resistance exist for chloroquine, mefloquine, and quinine. J Med Invest, 1998 Feb, 44(3-4), 185 - 91 The role of cyclosporin A on antibody-dependent monocyte-mediated cytotoxicity against human multidrug-resistant cancer cells; Yano S et al.; A P-glycoprotein (P-gp) inhibitor, cyclosporin A (CsA) was found to enhance the susceptibility of multidrug resistant (MDR) cancer cells to anti-P-gp antibody-dependent cellular cytolysis (ADCC) by monocytes, but the exact mechanism is unknown . In this study, we examined whether CsA enhanced the susceptibility of MDR cells through its inhibitory effect of P-gp function by using anti-ganglioside GM2 (GM2) monoclonal antibody (Ab), KM966, instead of anti-P-gp Ab, MRK16 . Monocyte-ADCC induced by both KM966 and MRK16 against P-gp positive human MDR ovarian cancer cells was significantly augmented by addition of CsA . KM966, but not MRK16, induced monocyte-ADCC against P-gp negative human ovarian cancer cells and CsA enhanced this ADCC activity, indicating that suppressive effect of P-gp function by CsA was not essential to the enhancement of ADCC . Moreover, pretreatment of tumor cells with CsA augmented their susceptibility to monocyte-ADCC irrespective of P-gp expression . Interestingly, KM966 or MRK16 induced monocyte-ADCC against various human lung cancer cells expressing either GM2 or P-gp, but CsA did not affect these ADCC . These findings suggest that CsA may enhance the susceptibility to the monocyte-ADCC of ovarian cancer cells, but not of lung cancer cells, irrespective of its suppressive effect of P-gp function. Exp Cell Res, 1998 May 1, 240(2), 312 - 20 An active efflux system for heavy metals in cisplatin-resistant human KB carcinoma cells; Chen ZS et al.; The mechanism for cisplatin resistance in cisplatin-resistant KCP-4 cells was studied . Although multidrug resistance-associated protein (MRP) was not detected in KCP-4 cells, the cells were more resistant to heavy metals than multidrug-resistant C-A120 cells that overexpressed MRP . KCP-4 cells expressed metallothionein, but it was scarcely involved in cisplatin resistance in these cells . KCP-4 cells did not express canalicular multispecific organic anion transporter (cMOAT) . The glutathione (GSH) level was 4.7-fold higher in KCP-4 cells than in KB-3-1 cells . When the GSH level in KCP-4 cells was decreased by treating the cells with buthionine sulfoximine and nitrofurantoin, the accumulation of and sensitivity to cisplatin in the cells were increased . C-A120 cells were only 3.0-fold more resistant to cisplatin than KB-3-1 cells and this resistance was not affected by the increased glutathione level . The accumulation of platinum in C-A120 and KCP-4 cells was 68.5 and 20.4% of that in KB-3-1 cells, respectively, while the intracellular levels of antimony potassium tartrate in C-A120 and KCP-4 cells were 13.2 and 9.9% of that in KB-3-1 cells, respectively . The ATP-dependent efflux of antimony was enhanced in both C-A120 and KCP-4 cells . These results, taken together, suggest an efflux pump for heavy metals different from MRP and cMOAT is involved in cisplatin resistance in KCP-4 cells. Zhonghua Jie He He Hu Xi Za Zhi, 1996 Dec, 19(6), 338 - 41 {Application of PCR-SSCP technique in detection of rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis}; He X et al.; OBJECTIVE: To evaluate rpoB gene mutation in rifampin-resistant Mycobacterium tuberculosis and its relationship with rifampin resistance . METHODS: Forty clinical isolates of Mycobacterium tuberculosis were analyzed by PCR-SSCP technique, with H37Rv reference strains as control group . RESULTS: The sensitivity of amplication products of 411bp and 258bp were found to be 5 pg/microliters, 500 organisms per milliliter and 1 pg/microliter, 500 organisms per milliliter respectively . rpoB gene belongs to genus specificity . Characteristics of SSCP graph of 258bp fragment: Ten sensitive strains were the same as H37 Rv . Thirty strains of rifampin-resistant or multidrug resistance, including rifampin, were different from H37Rv except for three strains . Positive rate was 90%, while specificity 100% . CONCLUSIONS: Results showed that PCR-SSCP technique could detect rPOB gene mutation, which might associate with rifampin resistance and be helpful to rapid detection and research of rifampin-resistant Mycobacterium tuberculosis. Blood, 1998 May 1, 91(9), 3163 - 71 Inhibition of P-glycoprotein and recovery of drug sensitivity of human acute leukemic blast cells by multidrug resistance gene (mdr1) antisense oligonucleotides; Motomura S et al.; To overcome the problem of multidrug resistance, we investigated the effectiveness of phosphrothioate antisense oligonucleotides (MDR1-AS) in suppressing multidrug resistance gene (mdr1) expression in drug-resistant acute myelogenous leukemia (AML) blast cells and the K562 adriamycin-resistant cell line K562/ADM . The percentage of cells with the mdr1 gene product P-glycoprotein (P-gp) was decreased from 100% to 26% by 20 micromol/L MDR1-AS in the K562/ADM cells, and from 48.1% to 10.2% by 2.5 micromol/L MDR1-AS in the AML blast cells . Western blot analysis also showed a decrease in the amount of P-gp in the MDR1-AS-treated K562/ADM cells . This effect was specific to MDR1-AS, and not observed with sense or random control oligonucleotides . The expression of mdr1 mRNA in K562/ADM and AML blast cells treated with MDR1-AS was decreased compared with the random control . Intracellular rhodamine retention and {3H}daunorubicin also increased after antisense treatment . Chemosensitivity to daunorubicin increased in MDR1-AS-treated blast cells up to 5.9-fold in the K562/ADM cells and 3.0- to 6.4-fold in the AML blast cells . The expression of mdr1 mRNA derived from colony cells decreased in the MDR1-AS-treated groups . No inhibitory effect of the oligonucleotides on normal bone marrow progenitors was observed . These findings suggest that MDR1-AS is useful to overcome multidrug resistance in the treatment of leukemia. Biochim Biophys Acta, 1998 May 8, 1380(3), 329 - 35 Cytotoxicity and DNA binding characteristics of dextran-conjugated doxorubicins; Yang M et al.; The antitumor antibiotic doxorubicin was conjugated with polymeric dextrans of various molecular weights and the cytotoxicity of the conjugates against human carcinoma KB-3-1 cells and its multidrug-resistant subclone KB-V-1 cells was measured by tetrazolium salt MTT assay . The conjugates were much less toxic to the KB-3-1 cells than the free doxorubicin but exhibited similar toxicity to the KB-V-1 cells . The conjugate-DNA interactions were monitored in real-time using an optical biosensor based on evanescent wave detection to obtain the association (ka) and dissociation (kd) rate constants as well as the equilibrium binding constants (KA) of the bindings . Both ka and kd values for the conjugates are more than three magnitudes smaller than those for free doxorubicin, while the KA values of the conjugate-DNA complexes are only about 10 times smaller than that of the free doxorubicin-DNA complex . The results indicate that the cytotoxicity and the DNA-binding kinetics of doxorubicin may be modified with dextran conjugation . The KA values obtained from the biosensor measurements were in close agreement with those determined in solution by fluorescent titration method, verifying the utility of the label-free biosensing measurements as an efficient method for studying ligand-DNA interactions . Rinsho Byori, 1998 Apr, 46(4), 380 - 90 {Quantitative analysis of multidrug resistance phenotype in hematological malignancies}; Kobayashi H et al.; Cells with multidrug resistance (MDR) phenotype express P-glycoprotein (P-gp) on the cell membrane, which functions as a drug-efflux pump . To quantify the expression of the gene encoding P-gp (multidrug resistance 1; MDR1) and assess P-gp function, we developed competitive reverse transcription-polymerase chain reaction (RT-PCR) assay using heterologous competitor RNA and flow cytometric analysis using rhodamine 123 (Rh123; an artificial substrate for P-gp), respectively . First, we adjusted the assays by analyzing leukemia sublines showing various levels of MDR1 expression . The MDR1 expression in leukemia sublines quantified by competitive RT-PCR assay showed linearity over a wide range, and the results were parallel with those of MDR1 expression measured by Northern blot analysis, the P-gp antigen expression measured by flow cytometric analysis using MRK16, P-gp function analysis by Rh123 dye-efflux assay, and MDR phenotype . Then, we applied these assays to leukemic cells from patients with hematological malignancies . All 69 samples from 64 patients were successfully assayed, and the range of MDR1 expression was from 1.6 to 100 amol/microgram RNA . Since subpopulations of normal lymphocytes show a low degree of P-gp function, strict gating of leukemia cells was mandatory for dye-efflux assay of clinical samples . MDR1 expression in normal lymphocytes was below 8 amol/microgram RNA . By comparison to MDR1 expression quantified by competitive RT-PCR assay with P-gp function assessed by Rh123 dye-efflux assay in gated leukemic cells, more than 8 amol/microgram RNA was regarded as positive MDR1 expression in the leukemic cells themselves . These data suggest that these assays are suitable for evaluating P-gp expression and function in clinical samples when the proper cut-off value is used and may provide insights into the contribution of P-gp to drug resistance in hematological malignancies. Chin Med J (Engl), 1997 Mar, 110(3), 167 - 72 Establishment of doxorubicin-resistant human bladder cancer cell line (BUI-87/ADMR) and its mechanism of multidrug resistance; Guo H et al.; OBJECTIVE: To establish a doxorubicin-resistant human bladder cancer cell line, BIU-87/ADMR, and to study its biological characteristics and mechanism of drug resistance . METHODS: A human bladder cancer cell line resistant to doxorubicin, BIU-87/ADMR, has been established in vitro by exposing BIU-87 parent cells to progressively increasing concentrations of the drug over a period of 8 months . The cell line has been characterized in terms of growth kinetics, morphology, cross-resistance to other anticancerous agents, pharmacokinetics of daunorubicin and expression of P-glycoprotein (P-gp) which is closely related to the MDR phenotype . RESULTS: The BIU-87/ADMR cell line was 6.3 times more resistant to doxorubicin than the parent BIU-87 . It exhibited cross-resistance to doxorubicin derivatives (epirubicin, daunorubicin), vincristine and etoposide, but not to cisplatin and mitomycin C . Compared to the parent cells, the resistant cells have a slower growth rate and lower confluent density . Unlike the parent BIU-87, about 75% of the BIU-87/ADMR cells showed a positive reaction with monoclonal antibody against P-gp, JSB-1 . Intracellular drug accumulation studies with fluorescence spectrometry indicated that the resistance exhibited by the BIU-87/ ADMR line was mainly caused by an increased active efflux . CONCLUSIONS: The results suggest that MDR is an important phenomenon in bladder cancer and that more than one pathway of MDR may be present in human bladder cancer cell lines . BIU-87/ADMR may be a useful model for the development of new chemotherapeutic strategies in overcoming drug-resistance in the treatment of bladder cancer. Haematologica, 1998 Apr, 83(4), 290 - 7 P-glycoprotein (PGP), and not lung resistance-related protein (LRP), is a negative prognostic factor in secondary leukemias; Damiani D et al.; BACKGROUND AND OBJECTIVE: In cell lines, there is an ongoing debate about the role of the lung resistance-related protein (LRP) whereas the role played by P-glycoprotein (PGP) in determining a multidrug resistance is well known . The aim of this study was to evaluate the frequency and the role of a PGP and an LRP overexpression in affecting the intracellular daunorubicin accumulation (IDA) and in predicting the therapy outcome on a subset of overt secondary acute non lymphocytic leukemias (ANLL) . An adjunctive point was to evaluate the efficacy of the reversal agent SDZ PSC 833 (PSC) in counteracting impaired IDA . DESIGN AND METHODS: By flow cytometry, PGP and LRP expression and the IDA were evaluated on 54 overt secondary ANLL PGP and LRP overexpressions were respectively defined by an MRK-16 mean fluorescence index (MFI) > or = 6 (PGP+) and by an LRP-56 MFI > or = 5 i.e . by MRK-16 and LRP-56 MFIs higher than the one observed in normal leukocytes . The blasts' IDA was studied after a two-hour incubation in 1000 ng/mL daunorubicin in the presence or in the absence of the MDR reversal agent SDZ PSC 833 (PSC) 1.6 mumol . RESULTS: A PGP overexpression was detected in 40/54 (74%) cases while an LRP overexpression was observed on 33/54 (61%) cases . No differences were found in terms of PGP and LRP expressions between ANLL developing after chemo/radiotherapy (therapy-related ANLL) or evolving from a myelodysplastic syndrome (MDS-related ANLL) . Compared to the PGP-, the PGP+ cases showed a significantly lower mean IDA (DNR NMFI 196 +/- 46 vs . 267 +/- 53, p < 0.001) . The co-incubation of DNR with the PSC significantly increased only the mean IDA of the PGP+ cases, that grew from a DNR NMFI of 196 +/- 46 to a DNR NMFI of 284 +/- 67 (p < 0.0001) . With respect to normal leukocytes, even the PGP- cases had an impaired IDA suggesting that other mechanisms, including an LRP overexpression, could affect the IDA . A strongly negative correlation was observed between PGP overexpression and therapy outcome, in fact, 8/10 (80%) PGP- but only 2/27 (7%) PGP+ patients obtained complete remission (p = 0.0002) . Moreover, 7/33 (21%) cases showing an impaired IDA (NMFI < 280) but 4/4 (100%) with NMFI > 280 had complete remission (p = 0.006) . No correlation was found between therapy response and LRP or CD34 expression . INTERPRETATION AND CONCLUSIONS: This data suggests that an important role in determining therapy outcome is played by PGP in secondary leukemias . Even if the LRP is frequently overexpressed in secondary leukemias and is likely to contribute to the reduction of the intracellular drug accumulation, the role played by LRP in determining the therapy-outcome has still to be cleared. Zhonghua Nei Ke Za Zhi, 1996 Sep, 35(9), 591 - 4 {Detection of multidrug resistance in patients with leukemia by using flow cytometry and RNA in situ hybridization}; Li S et al.; Multidrug resistance (MDR) in leukemia and the reversal of MDR by cyclosporin A (CsA) in vitro have been studied through intracellular accumulation of daunorubicin (DNR) in leukemic myeloblasts . The study was carried out with real-time flow cytometry and relative expression levels of MDR, which is estimated by RNA in situ hybridization in 26 patients suffering from leukemia . The change of intracellular DNR accumulation in vitro after adding CsA was also analyzed . The results showed that intracellular DNR accumulation in newly diagnosed and treated patients (MDR1 negative) with remission increased significantly than that in refractory and relapsing patients (P < 0.01) . Good reversal effect was obtained in ref