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PubMed1999

Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand.

Resistance to quinoline-containing compound has been associated with the Plasmodium falciparum multidrug resistance 1 (pfmdr1) gene. We analyzed wild P. falciparum isolates with high levels of chloroquine and mefloquine resistance for their macrorestriction maps of chromosome 5 and sequence of pfmdr1. Two types of chromosome 5 amplification were found. Eleven of 62 resistant isolates displayed Bgl 1 fragments larger than 100 kb. Twenty-nine isolates possessed multiple copies of the fragments. We failed to detect any amplification of this region on chromosome 5 in 22 mefloquine-resistant isolates, suggesting that other mechanisms can mediate the mefloquine-resistant phenotype. There was no direct association between pfmdr1 mutations and chloroquine sensitivity. Resistant lines could have Asn-86 and Tyr-184 or Phe-184, the predicted sequence of those chloroquine-sensitive isolates. No mutation at Asn-1042 and Asp-1246 was detected among these chloroquine-resistant isolates. Therefore, a few base substitutions in the pfmdr1 gene may not be sufficient to account for all chloroquine-resistant phenotypes.

Amodiaquine↗
PubMedDate not supplied

[Clinical importance and pathogenic mechanisms of insulin resistance in chronic renal insufficiency (part I): insulin resistance in patients with chronic renal insufficiency].

INTRODUCTION: This paper presents new investigations of abnormal insulin action in patients with chronic renal failure. Reduced tissue sensitivity to insulin action and the effects of the impairments on carbohydrate, protein and lipid metabolism have important pathophysiological implications in the genesis of the uremic syndrome. We analyzed confounding factors of reduced insulin sensitivity and potential sites of insulin resistance. INSULIN RESISTANCE IN HUMANS: Insulin resistance is associated with a number of metabolic and vascular abnormalities known as "syndrome X" or metabolic syndrome. Other features include obesity, particularly truncal distribution, glucose intolerance and non-insulin-dependent diabetes mellitus (NIDDM), hypertension, a specific dyslipidemia with raised triglyceride concentrations and a high low-density lipoprotein: high-density lipoprotein ratio, and hyperuricemia. These features, are all associated with accelerated atherogenesis and cardiovascular disease, the main cause of premature mortality. Some genetic (mutations affecting postreceptor signalling pathways) and environmental factors that could contribute to insulin resistance are discussed. INSULIN RESISTANCE IN CHRONIC RENAL FAILURE: The most prominent metabolic disturbance in uremic patients is insulin resistance due to a post-receptor defect. Insulin secretion is also impared, because the pancreatic beta-cell response to hyperglycemia is blunted. Insulin clearance by renal and extrarenal mechanisms is reduced. POSSIBLE SITES OF INSULIN RESISTANCE IN TERMINAL RENAL FAILURE: Increase in hepatic glucose production or impaired hepatic glucose uptake were overestimated. Impaired glucose uptake by peripheral tissues, primarily muscle and adipose tissue, has been extensively studied, and there is abundant evidence in patients with chronic renal failure. Decrease in renal glucose production would lead to a decrease in glucose appearance in circulation and decrease of insulin sensitivity. CONCLUSION: Cellular basis for insulin resistance in uremic patients is, however, unknown. It is now recognized that insulin-stimulated glucose transport in skeletal muscles and in other peripheral tissues is reduced. Although the majority of uremic patients are insulin resistant and about half of them are glucose intolerant, they are rarely diabetics. But, there are clinical implications of abnormal insulin metabolism in uremia. Cardiovascular complications are the most important consequences and significant cause of mortality in these patients.

Humans↗
PubMed2001

The antiviral resistance and replication of cidofovir-resistant adenovirus variants in the New Zealand White rabbit ocular model.

PURPOSE: To determine the antiviral resistance of three cidofovir (CDV)-resistant variants of adenovirus type 5 (Ad5) and their ability to replicate in the New Zealand White rabbit ocular model. METHODS: Rabbits were inoculated topically in both eyes with the CDV-resistant variants R1, R2, and R3, and the Ad5 parental strain. On day 1, rabbits from each virus inoculation were divided into two topical treatment groups: 0.5% CDV and PBS control. Treatment was administered twice daily in both eyes for 7 days. All eyes were cultured for virus on days 0, 1, 3, 4, 5, 7, 9, 11, and 14. Using viral outcome parameters, CDV resistance was determined for each virus by comparing each CDV-treated virus group to its respective PBS control, and altered pathogenesis was assessed by comparing viral replication in the PBS control groups of the Ad5 parent and the three resistant variants. RESULTS: Topical 0.5% CDV treatment demonstrated significant antiviral inhibitory activity in the Ad5 parental group (e.g., reduced total Ad5-positive cultures, reduced daily Ad5-positive cultures on days 5, 9, 11, and 14, and duration of ocular shedding), but had no effect on the three CDV-resistant variants. There were no significant differences in pathogenicity between the Ad5 parent and the CDV-resistant variants. CONCLUSIONS: The Ad5 variants R1, R2, and R3 were resistant to topical treatment with 0.5% cidofovir in the rabbit ocular model. However, the acquisition of CDV resistance did not alter the replication of the three Ad5 CDV variants on the rabbit eye.

Adenoviridae Infections↗
PubMed2002

Antibiotic resistance in uncomplicated urinary tract infection: problems with interpreting cumulative resistance rates from local community laboratories.

AIMS: To determine the resistance rates and patterns in bacteria causing uncomplicated urinary tract infections (UTIs) presenting to general practitioners (GPs) in Christchurch. METHODS: 82 randomly selected GPs in Christchurch participated in the study. Midstream urine (MSU) samples were prospectively collected for standard microbiological analysis on all women between the ages of 16 and 50 years presenting with symptoms of dysuria and frequency and who had positive dipstick testing to either (or both) nitrites or leucocytes. MSUs were submitted for bacterial colony counts and resistance testing of isolates present in adequate numbers. RESULTS: 374 specimens were collected. 299 filled the inclusion criteria, of which 94 fulfilled criteria for significant infection. Trimethoprim resistance was found in 8, (8.5%) (95%CI 2.8,14.2) overall with a resistance rate for Escberichia coli (E. coli) to trimethoprim of 11.5%. This compared with cumulative resistance rates from local community laboratories for E. coli to trimethoprim of 19%. For a woman in this age group presenting with symptoms of UTI we estimated that her probability of having a trimethoprim resistant organism was 2.7%. CONCLUSION: Trimethoprim remains a reasonable first line treatment for uncomplicated UTI in Christchurch. Actual resistance rates are significantly less than those derived from routine pooled laboratory specimens, and when used in an intention to treat calculation to inform empiric prescribing, become even less significant. While collection of these routine data is essential to provide early warning of emergent resistance, a truly representative rate should be determined to inform prescribing decisions if resistance appears to be increasing.

Adolescent↗
PubMed2002

Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance.

Previously, we have reported that a multidrug-resistant, mitoxantrone (MX)-selected cell line, MCF7/MX, is highly cross-resistant to the antifolate methotrexate (MTX), because of enhanced ATP-dependent drug efflux (E. L. Volk et al., Cancer Res., 60: 3514-3521, 2000). These cells overexpress the breast cancer resistance protein (BCRP), and resistance to MTX as well as to MX was reversible by the BCRP inhibitor, GF120918. These data indicated that BCRP causes the multidrug-resistance phenotype. To further examine the role of this transporter in MTX resistance, and in particular the role of amino acid 482, we analyzed a number of BCRP-overexpressing cell lines. MTX resistance correlated with BCRP expression in all of the cell lines expressing the wild-type transporter, which contains an Arg at position 482. In contrast, little or no cross-resistance was found in the MCF7/AdVp1000 and S1-M1-3.2 and S1-M1-80 cell lines, which contain acquired mutations at this position, R482T and R482G, respectively. Concomitantly, the greatest reduction in MTX accumulation was observed in the MCF7/MX cells (BCRP(Arg)) as compared with cells expressing the Thr and Gly BCRP variants. Furthermore, the reduction in drug accumulation was sensitive to BCRP inhibition by GF120918. In conclusion, we have demonstrated a novel role for BCRP as a mediator of MTX resistance and have provided further evidence for the importance of amino acid 482 in substrate specificity.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1992

Ouabain-resistant transfectants of the murine ouabain resistance gene contain mutations in the alpha-subunit of the Na,K-ATPase.

A 6.5-kilobase murine genomic DNA fragment isolated by Levenson et al. (Levenson, R., Racaniello, V., Albritton, L., and Housman, D. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1489-1493) (called the ouabain resistance gene) has been shown to produce ouabain resistance in primate cells. Preliminary sequence information has revealed no homology with the coding sequence of the Na,K-ATPase. We have introduced this murine sequence into monkey and murine cells in an attempt to characterize its mechanism of action. In our experiments, transfection of this DNA fragment is associated with the low frequency (1 in 8 x 10(5) cells) appearance of ouabain-resistant clones of CV1, COS, and NIH 3T3 cells, an event not seen in control transfections. Characterization of a new clone of ouabain-resistant CV1 cells (called OR8 cells) revealed a 5-fold increase in the IC50 for ouabain inhibition of rubidium uptake and a 10-fold increase in cell survival on ouabain. Although the murine sequence was detectable in Southern blots of ouabain-resistant cells soon after transfection, this exogenous DNA was rapidly lost despite continued exposure to ouabain. Furthermore, we were unable to detect message expression by this genomic sequence in any of the three cell types tested. Instead, we found that all three ouabain-resistant cell lines exhibited point mutations in a domain of the alpha-subunit that has been implicated in ouabain sensitivity (H1-H2). One of these mutations (Asp121-Asn121 in OR8 cells) has been previously reported to cause ouabain resistance (Price, E.M., Rice, D.A., and Lingrel, J.B. (1989) J. Biol. Chem. 264, 21902-21906). Other novel mutations in the H2 transmembrane domain were also detected. We postulate that the "ouabain resistance gene" is important in the early selection process on ouabain but that the permanent ouabain-resistant phenotype is due to a stable mutation in one allele of the alpha-subunit of the Na,K-ATPase.

3T3 Cells↗
PubMed2003

Genotypic resistance tests for the management of the patient failing highly active antiretroviral therapy: the resistance pattern in different biological compartments.

Single witness: HIV-1 drug resistance assays have been shown to be of value for guiding antiretroviral therapy (ART) decisions. Different tissues or body fluids in which HIV-1 can reside may contain viruses with distinct characteristics. HIV-1 variants with genotypic resistance markers are present in the male genital tract and evolve over time on incompletely suppressive ART. The magnitude of decline in the semen HIV-RNA level in with therapy is usually similar to the effect of treatment on the blood viral burden. Not all men on ART have complete suppression of HIV-1 replication in genital tract; thus, they may shed resistant HIV-1 strains. Failure of treatment to suppress HIV-RNA levels in the blood is common, resulting in the selection of resistant HIV-1 variants. Sexual transmission of resistant variants may have a negative impact on treatment outcome in newly infected individuals and on the spread of the diseases within a population. The use of rapid assessment of HIV resistance in plasma and semen may be useful in some situations, e.g. HIV transmitted infection by sexual contact from a viraemic patient. The recognition of resistant HIV isolates in the blood and semen of patients receiving ART leads to the question of the potential role of resistance testing. In many but not all infectious diseases, the choice of therapy is guided by sensitivity testing. Recent data suggest that testing the drug sensitivity of viral isolates from the blood can benefit patient management and response to therapy, albeit at considerable cost. However, since drug resistance may evolve independently in blood and semen, the public health benefits of such testing are unproven.

Antiretroviral Therapy, Highly Active↗
PubMed1992

Overcoming of vinblastine resistance by isoquinolinesulfonamide compounds in adriamycin-resistant leukemia cells.

We investigated the effects of seven isoquinoline derivatives in overcoming resistance to vinblastine in Adriamycin-resistant mouse leukemia P388/ADR cells and human myelogeneous leukemia K562/ADR cells. N-(2-Methylpiperazyl)-5-isoquinoline-sulfonamide (H-7), N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not reverse resistance to vinblastine in these resistant cells. N-[2-[N-[3-(4-Chlorophenyl)-2-propenyl]amino]ethyl]-5- isoquinolinesulfonamide (H-86) and N-[2-[N-[3-(4-chlorophenyl)-1-methyl-2-propenyl]- amino]ethyl]-5-isoquinolinesulfonamide (H-87) caused significant accumulation of intracellular vinblastine and marked reversal of the resistance to vinblastine in both resistant cell lines. Addition of a formyl group at the terminal amino group of H-86 (H-85) or addition of an aminoethyl group to the nitrogen atom at the sulfonamide group of H-86 (W-66) reduced those activities. The activity on vinblastine accumulation seems to correlated with the hydrophobicity of the compounds. The compounds that effectively reversed resistance to vinblastine inhibited [3H]vinblastine efflux and photoaffinity labeling of P-glycoprotein with a photosensitive analogue of vinblastine, N-(p-azido-(3-[125I]iodo)-salicyl)-N'-beta-aminoethylvindesine. Although these isoquinoline derivatives inhibited protein kinase A and protein kinase C with various potencies, these inhibitory activities did not correlate with the reversal of drug resistance. These results indicate that hydrophobic isoquinoline derivatives reverse multidrug resistance due to the suppression of drug binding to P-glycoprotein, without involvement of their activities on protein kinase A and protein kinase C.

Animals↗
PubMed2005

Re-emergence of multi-drug resistant Shigella dysenteriae with added resistance to ciprofloxacin in north India & their plasmid profiles.

BACKGROUND & OBJECTIVES: Multi drug resistant Shigellae pose a major threat in control of shigellosis with. Shigella dysenteriae being the most prevalent species at our centre before 1994. A gradual decrease in S. dysenteriae occurred over the years with a corresponding increase in S. flexneri which became the predominant serotype. From May to November 2003, an increase in number of patients admitted with clinical diagnosis of dysentery was noted in comparison to previous years, with a corresponding increase in the isolation of multi drug resistant S. dysenteriae. We report here the re-emergence of multi drug resistant S. dysenteriae at our tertiary care centre in north India after a gap of about 10 yr. Plasmid analysis of S. dysenteriae was also performed to study the origin and clonality of the isolates. METHODS: Stool samples were collected in Cary-Blair medium and processed by standard methods. Shigellae were confirmed by serotyping. Minimum inhibitory concentration was done by agar dilution method and E-test. Plasmid profiling of 18 isolates (16 S. dysenteriae 1 and 2 S. dysenteriae 2) was performed by modified alkali lysis method. Clinical details of patients were noted. RESULTS: A total of 64 patients with dysentery were admitted during the study period. Patients presented with unusually severe symptoms and six developed complications. Treatment failure with ciprofloxacin occurred in six patients who received cefotaxime and amikacin. There were 38 children below 5 yr of age. S. dysenteriae (18 isolates of S. dysenteriae 1 and 2 isolates of S. dysenteriae 2) were isolated from 20 of the 64 (31.2%) stool samples. S. dysenteriae re-emerged as the commonest isolate after a gap of nearly 10 yr. Fourteen of the 20 S. dysenteriae isolates were multi drug resistant; 12 were resistant to ciprofloxacin with MIC of 8-32 mug/ml. Plasmid profile analysis revealed that 6 of 11 ciprofloxacin resistant S. dysenteriae 1 had similar profiles. INTERPRETATION & CONCLUSION: Emergence of a clone of ciprofloxacin resistant S. dysenteriae 1 in north India is disturbing as treatment options in our geographic area are limited in view of already existing high drug resistance to nalidixic acid, co-trimoxazole and amoxycillin. A close monitoring of shifts in serogroup distribution and antibiotic resistance is required to guide clinicians for treatment of shigellosis.

Adolescent↗
PubMed2006

Assessment of resistance in multi drug resistant tuberculosis patients.

OBJECTIVE: To study MDR-TB isolates and to identify primary and secondary resistance at microbiology laboratory Aga Khan University, Karachi, Pakistan. METHODS: All samples positive for Mycobacterium tuberculosis (MTB) received during January - September 2004 were reviewed for drug resistance pattern as well as for history of previous antituberculous drugs exposure. RESULTS: Out of 216 Mycobacterium tuberculosis cultures, 138 (64%) showed resistance to one or more agents. Multi drug resistance (MDR) was observed in 102 (47%) isolates. Of 138 drug resistant isolates; primary resistance to any one or more agent was noted in 31(39%) and secondary (acquired) resistance in 107 (79%) isolates. On analysis of the 102 MDR-TB strains 8 (10%) showed primary resistance while 94 (69%) showed secondary resistance. CONCLUSION: In this group MDR-TB was mainly associated with previous anti-tuberculous treatment. However, primary MDR was also observed and reflects dissemination of MDR cases within the community.

Antitubercular Agents↗
PubMed1990

P-glycoprotein-independent mechanism of resistance to VP-16 in multidrug-resistant tumor cell lines: pharmacokinetic and photoaffinity labeling studies.

The interaction of etoposide (VP-16), Vinca alkaloids, and verapamil with the P-glycoprotein (P-gp) was studied in human breast (MCF-7) and Chinese hamster lung (DC3F) cell lines and the corresponding multidrug-resistant MCF-7/ADR and DC3F/ADX tumor cell lines, selected for resistance to Adriamycin and actinomycin D, respectively, and overexpressing P-gp. Verapamil (10 microM) markedly reversed resistance to vincristine (11-fold in DC3F/ADX and 125-fold in MCF-7/ADR; 1-hr exposure), but it had a very modest effect on resistance to VP-16 (3- to 4-fold; 1-hr exposure). Resistant cells accumulated 2- to 4-fold less VP-16 and vincristine than the parental cell lines. Verapamil (10 microM) significantly increased accumulation and retention of vincristine, but not of VP-16, in resistant cell lines. Photoaffinity labeling of resistant cell lines with radioactive analogs of verapamil [N(p-azido-3-125I-salicyl)-N'-beta-aminoethylverapamil (NASVP)] and vinblastine[N-(p-azido-3-125I-salicyl)-N'-beta-aminoethylvindesine (NASV)] showed distinctly labeled P-gp bands in both resistant cell lines, compared with wild-type cells. Excess nonradioactive vinblastine or verapamil effectively competed with the P-gp photolabeling by either NASVP or NASV, with IC50 levels of 0.6 and 10 microM, respectively. In contrast, nonradioactive VP-16 was 100- to 500-fold less potent than vinblastine in competing with P-gp photolabeling, suggesting that VP-16 has significantly lower affinity for P-gp than Vinca alkaloids have. Taken together, our data indicate that P-gp glycoprotein by itself may not be important in the transport/efflux of VP-16 and, thus, in the mechanism of resistance to VP-16 in these cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Trifluoperazine modulation of resistance to the topoisomerase II inhibitor etoposide in doxorubicin resistant L1210 murine leukemia cells.

Murine leukemia L1210 cells selected for progressive resistance to doxorubicin (DOX) display both the multidrug resistant (MDR) phenotype and reductions in drug induced topoisomerase II-mediated DNA cleavage in nuclear extracts (Ganapathi, R.; Grabowski, D.; Ford, J.; Heiss, C.; Kerrigan, D.; Pommier, Y., Cancer Commun. 1:217-224; 1989). The present study was performed to characterize the results of exposure of the sensitive (S) and progressively DOX-resistant (10-fold, R1, and 40-fold, R2) L1210 cells to the topoisomerase II inhibitor, etoposide, and to investigate the modulating effects of the calmodulin inhibitor, trifluoperazine (TFP). Immunoblotting experiments indicated no apparent decrease in the p170 or p180 isoforms of topoisomerase II in the resistant sublines versus parental sensitive cells. Cross-resistance to etoposide (VP-16) was similar to that of DOX (10- and 40-fold). A non-cytotoxic concentration of 5 microM TFP enhanced cell kill 1.5- fold in the sensitive and 3- to 5-fold in the progressively DOX-resistant cells. Accumulation of VP-16 was 30% to 50% lower in the resistant sublines versus similarly treated sensitive cells, and a marked enhancement of drug uptake in the presence of TFP was observed in the sensitive but not in the resistant cells exposed to equivalent extracellular levels of VP-16. Although equimolar concentrations of VP-16 produced fewer DNA single strand breaks (SSB) and DNA protein crosslinks (DPC) in the resistant versus sensitive cells, similar DNA damage was apparent when S and R1, but not R2, cells were treated at VP-16 concentrations that produced equivalent cell death.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[Combined effect of sulbactam/cefoperazone and other antibiotics against clinical isolates of multi-resistant strains. I. Effect of sulbactam against beta-lactams resistant strains and in vitro combined effect of sulbactam/cefoperazone with each of piperacillin, latamoxef, ceftazidime, fosfomycin and doxycycline].

We evaluated relationships between production of beta-lactamase and their resistances to beta-lactams, effect of sulbactam (SBT), a beta-lactamase inhibitor, against beta-lactam resistant strains, and combined effect of sulbactam/cefoperazone (SBT/CPZ) with other antibiotics against multi-resistant strains. Through these studies, we obtained the following results. 1. Most of the strains resistant to beta-lactams were beta-lactamase producers. 2. Relationships between the production of beta-lactamase and their resistances to beta-lactams indicate that their resistances generally were the highest in producers of both penicillinase (PCase) and cephalosporinase (CEPase), moderate in producers of either PCase or CEPase, and the lowest in beta-lactamase non-producers. Most of highly-resistant strains of MRSA appeared to be beta-lactamase non-producers though some exceptions were observed among methicillin-resistant Staphylococcus aureus (MRSA), Serratia marcescens and Pseudomonas aeruginosa. 3. SBT showed good effect against PCase producers, moderate effect against producers of both PCase and CEPase, little effect against CEPase producers, and no effect against beta-lactamase non-producers. 4. Results of combined effect of SBT/CPZ with other antibiotics indicated that good synergism was obtained by combining SBT/CPZ with fosfomycin (FOM) or piperacillin against multi-resistant strains of Proteus spp., Enterobacter cloacae, and S. marcescens, by combining SBT/CPZ with ceftazidime (CAZ) or FOM in methicillin-sensitive S. aureus and by combining SBT/CPZ with CAZ in P. aeruginosa. 5. Better synergism was obtained with the higher concentrations of antibiotics.

Anti-Bacterial Agents↗
PubMed1990

[Mechanism of beta-lactam-resistance in methicillin-resistant Staphylococcus aureus].

The role of penicillin-binding protein (PBP) 2' in the expression of beta-lactam-resistance was investigated using methicillin-resistant Staphylococcus aureus (MRSA) strains with different level of resistance. Both high- and moderate-level MRSA produced very similar PBP 2' with low affinities for beta-lactam antibiotics. Affinities of antibiotics for PBP 2' (I50, concentration which inhibits [14C] benzylpenicillin-binding by 50%) correlated well with their antibacterial activities (MIC) in a high-level MRSA, but did not in a moderate-level MRSA. High-level MRSA contained a larger amount of PBP 2' than moderate-level MRSA, and the amount of PBP 2' decreased by increasing the temperature of the culture; the extent of decrease was larger in a strain which was sensitive at 37 degrees C than a strain which exerted relatively high level resistance even at 40 degrees C. A cephamycin-resistant, methicillin-sensitive strain began to synthesize PBP 2' by adding cephamycin-type antibiotics to the medium and consequently acquired resistance to methicillin. Latent MRSA producing no PBP 2' generated clones which produced PBP 2' constitutively and were highly resistant to all beta-lactams. These results suggest that the presence of PBP 2' is critical for the expression of beta-lactam-resistance in MRSA and the degree of the resistance depends mainly on the amount of PBP 2' which differs from strain to strain and is influenced by environments such as temperature and the presence of inducer.

Anti-Bacterial Agents↗
PubMed1990

Tolerance to O6-methylguanine and 6-thioguanine cytotoxic effects: a cross-resistant phenotype in N-methylnitrosourea-resistant Chinese hamster ovary cells.

The biochemical and genetic characteristics of a clone of Chinese hamster ovary cells displaying resistance to N-methyl-N-nitrosourea (MNU) and 6-thioguanine (6-TG) were analyzed. The initial level of 7-methylguanine, 3-methyladenine, and O6-methylguanine formation and the repair rates for these alkylated bases were the same in the resistant and in the parental cell line, indicating that the resistance to alkylation damage is not due to differences in DNA alkylation. After exposure for 24 or 48 h to 6-TG (0.6 micrograms/ml) in culture medium, the resistant clone in contrast to them, was able to replicate the DNA containing the base analogue during the following 24 h. These data are in agreement with the hypothesis that resistant cells tolerate both O6-methylguanine and 6-TG present in DNA. The tolerance to MNU and 6-TG also included chromosomal damage induced by these two agents, and MNU-resistant cells incurred less sister chromatid exchanges after treatment with either MNU or 6-TG. 6-TG-resistant cells, selected by growth in 6-TG, exhibited cross-resistance to MNU but not to methyl methanesulfonate, confirming that a common pathway of tolerance is responsible for resistance to 6-TG and O6-methylguanine.

Alkylation↗
PubMed1989

Non-p-glycoprotein-mediated multidrug resistance in detransformed rat cells selected for resistance to methylglyoxal bis(guanylhydrazone).

Three independent variants (G2, G4, G5), resistant to methylglyoxal bis(guanylhydrazone), an anticancer drug, have been isolated by single step selection from an adenovirus-transformed rat brain cell line (1). These variants display selective cross-resistance to several natural product drugs of dissimilar structure and action. Multidrug resistance has recently been shown to be caused by overexpression of the membrane-associated p-glycoprotein, most often caused by amplification of the mdr gene. Several types of experiments were conducted to determine whether the observed drug resistance in our cell lines could be due to changes at the mdr locus. The following results were obtained: (a) the mdr locus was not amplified; (b) transcription of the mdr gene and p-glycoprotein synthesis were not increased; (c) multidrug resistance cell lines, which carry an amplified mdr locus, were not cross-resistant to methylglyoxal bis(guanylhydrazone); (d) verapamil did not reverse the resistance of G cells or mdr cells to methylglyoxal bis(guanylhydrazone), nor that of G cells to vincristine; and (e) methylglyoxal bis(guanylhydrazone) resistance was recessive and depended on a block to drug uptake, as opposed to mdr cells which are dominant and express increased drug efflux. The results obtained suggest that the drug resistance in the G2, G4, and G5 cells was atypical and may be due to a mechanism distinct from that mediated by the mdr locus.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Development of resistance to 1-beta-D-arabinofuranosylcytosine after high-dose treatment in childhood lymphoblastic leukemia: analysis of resistance mechanism in established cell lines.

Cell lines PER-163 and PER-164 are derived from a patient with acute lymphoblastic leukemia who developed resistance to 1-beta-D-arabinofuranosylcytosine (ara-C) after high-dose (HD) therapy. Both lines are highly resistant to ara-C and have maintained stable resistance for more than 18 mo. The resistance in PER-164 cells is the result of a selection process in vivo only, while PER-163 cells have in addition been exposed to ara-C in culture. Comparison with cell line PER-145, which is sensitive to ara-C and was established from the same patient before HDara-C therapy, revealed no differences with respect to surface markers, morphology, cytochemical stains, or requirements for growth in vitro. The leukemic origin of the three cell lines is indicated by the close similarities of all three cell lines to the patient's fresh cells. The analysis of the two resistant cell lines shows that resistance to ara-C is not due to lower ara-C transport capacity nor to cytokinetic reasons, since the percentage of cells in S-phase is similar in all three cell lines. In addition, the resistant cell lines do not show any increased cytidine deaminase activity. PER-164 cells show a markedly reduced deoxycytidine kinase activity, 4.8 nmol/h/mg of protein, compared to PER-145 cells with an enzyme activity of 21.48 nmol/h/mg of protein. In PER-163 cells, no deoxycytidine kinase activity could be detected. Furthermore, the two resistant cell lines show significantly different dCTP levels. The sensitive PER-145 cells generated 97.9 pmol of 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP)/10(7) cells during a 45-min incubation period in the presence of 10(-6) M ara-C. This contrasts with 0.16 and 12 pmol of ara-CTP/10(7) cells for PER-163 and PER-164 cells, respectively. These investigations suggest that cell phenotypes with distinct features can be generated after HDara-C treatment and that decreased deoxycytidine kinase activity appears to be one of the major mechanisms of resistance.

Arabinofuranosylcytosine Triphosphate↗
PubMed1988

Cross-resistance patterns in hydroxyurea-resistant leukemia L1210 cells.

Hydroxyurea is an inhibitor of ribonucleotide reductase and is specifically directed at the non-heme iron subunit (which contains the free radical) of this enzyme. Leukemia L1210 cells, grown in the presence of increasing concentrations of hydroxyurea, developed resistance to hydroxyurea. For hydroxyurea, the wild-type L1210 cells required a drug concentration of 85 microM to inhibit cell growth by 50%, and the hydroxyurea-resistant (HU-7-S7) cells required a concentration of approximately 2000 microM. The resistant L1210 cells were cross-resistant to 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole/Desferal. However, these HU-7-S7 cells remained sensitive to 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone and 1-isoquinolylmethylene-N-hydroxy-N'-amino-guanidine tosylate (inhibitors directed at the same subunit as hydroxyurea). The HU-7-S7 cells retained their sensitivity to deoxyadenosine/erythro-9-(2-hydroxy-3-nonyl)adenine and deoxyguanosine/8-amino-guanosine (inhibitors directed at the effector-binding subunit of ribonucleotide reductase). The L1210 cells developed for resistance to hydroxyurea were sensitive to the non-ribonucleotide reductase inhibitors, methotrexate and 1-beta-D-arabinofuranosylcytosine. Ribonucleotide reductase activity was elevated in the HU-7-S7 cells (CDP reductase, 5.5-fold increase; ADP reductase, 13.2-fold increase). The addition of exogenous effector-binding subunit caused much greater stimulation of reductase activities in the extracts from the resistant cells than from the wild-type cells. The reductase activity in cell-free extracts from the resistant cells was inhibited by hydroxyurea, 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole and dATP to the same extent as the activity from the wild-type L1210 cells. These data indicate that resistance to hydroxyurea in these L1210 cells is to some extent related to increased reductase activity. However, the specificity of resistance of these L1210 cells to inhibitors of ribonucleotide reductase depends on the nature of the inhibitor and the subunit at which the inhibitor is directed.

Adenine↗