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PubMed1995

Resistance-associated factors in human small-cell lung-carcinoma GLC4 sub-lines with increasing adriamycin resistance.

Previous studies have shown that the in vitro-selected adriamycin-resistant human small-cell lung-carcinoma cell line GLC4-ADR150 displays multidrug resistance as the result of 3-fold decreased DNA-topoisomerase II (topo II) activity and a 6-fold reduction in adriamycin accumulation. Not the MDR1 gene, but the MRP gene, was over-expressed in this cell line. The aim of our study was to establish which of these drug-resistance-associated factors are already involved in drug resistance occurring at early steps of selection with adriamycin. To address this question, changes in expression of topo II alpha/topo II beta, MRP and drug accumulation were measured along with adriamycin resistance (from 2- to 10- to 150-fold) and in a partial revertant cell line (10-fold resistant). Topo II alpha and II beta mRNA and protein levels were decreased in the resistant sub-lines, except in the 10-fold-resistant cell line. Cellular daunorubicin accumulation was decreased 1.2- to 5-fold with increasing resistance. MRP mRNA was over-expressed in all resistant sub-lines, with a marked increase in the 10-fold-resistant cells (level of expression as high as in the GLC4-ADR150 cells). Expression of an ATP-binding 190-kDa membrane protein and Western-blot analysis with anti-MRP anti-serum ASPKE, was in accordance with the expression of MRP mRNA in all cell lines. Expression of MRP mRNA and protein, however, was not proportional with the decrease in drug accumulation in all resistant sub-lines. This study also shows that drug accumulation, topo II and MRP expression were all changed at the earliest stage of resistance development of GLC4 cells upon adriamycin selection.

ATP-Binding Cassette Transporters↗
PubMed2001

Pyrethroid resistance and cross-resistance in the German cockroach, Blattella germanica (L).

A German cockroach (Blatella germanica (L)) strain, Apyr-R, was collected from Opelika, Alabama after control failures with pyrethroid insecticides. Levels of resistance to permethrin and deltamethrin in Apyr-R (97- and 480-fold, respectively, compared with a susceptible strain, ACY) were partially or mostly suppressed by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF), suggesting that P450 monooxygenases and hydrolases are involved in resistance to these two pyrethroids in Apyr-R. However, incomplete suppression of pyrethroid resistance with PBO and DEF implies that one or more additional mechanisms are involved in resistance. Injection, compared with topical application, resulted in 43- and 48-fold increases in toxicity of permethrin in ACY and Apyr-R, respectively. Similarly, injection increased the toxicity of deltamethrin 27-fold in ACY and 28-fold in Apyr-R. These data indicate that cuticular penetration is one of the obstacles for the effectiveness of pyrethroids against German cockroaches. However, injection did not change the levels of resistance to either permethrin or deltamethrin, suggesting that a decrease in the rate of cuticular penetration may not play an important role in pyrethroid resistance in Apyr-R. Apyr-R showed cross-resistance to imidacloprid, with a resistance ratio of 10. PBO treatment resulted in no significant change in the toxicity of imidacloprid, implying that P450 monooxygenase-mediated detoxication is not the mechanism responsible for cross-resistance. Apyr-R showed no cross-resistance to spinosad, although spinosad had relatively low toxicity to German cockroaches compared with other insecticides tested in this study. This result further confirmed that the mode of action of spinosad to insects is unique. Fipronil, a relatively new insecticide, was highly toxic to German cockroaches, and the multi-resistance mechanisms in Apyr-R did not confer significant cross-resistance to this compound. Thus, we propose that fipronil could be a valuable tool in integrated resistance management of German cockroaches.

Animals↗
PubMed2002

Cross-resistance to imidazolinone herbicides in chlorsulfuron-resistant Raphanus raphanistrum.

Raphanus raphanistrum L has evolved widespread resistance to sulfonylureas in the Western Australia (WA) wheat belt. With the introduction of imidazolinone-tolerant (IT) wheat (Tritcum aestivum L) and IT canola (Brassica napus L) in the WA wheat belt, it is important to understand the status of cross-resistance in this weed to sulfonylurea and imidazolinone (Imi) herbicides. A study was conducted to examine cross-resistance between chlorsulfuron and Imi herbicides (a mixture of imazapic and imazapyr) in 46 R raphanistrum populations collected from across the WA wheat belt. Plants were treated with herbicides and assessed for phytotoxicity under glasshouse conditions. Of the 46 R raphanistrum populations, 32 were resistant to chlorsulfuron and four were resistant to imazapic + imazapyr. Of the 70% chlorsulfuron-resistant populations, 13% showed cross-resistance to imazapic + imazapyr. However, the cross-resistant populations treated with imazapic + imazapyr showed a lower resistance level than the chlorsulfuron-treated populations. These results suggest that weed populations with such cross-resistance will not be controlled effectively by Imi herbicides. Although the resistance levels of the cross-resistant populations to Imi herbicides were low, the cross-resistance levels of R raphanistrum should be determined before growing IT crops, particularly IT canola.

Dose-Response Relationship, Drug↗
PubMed1987

Cross-resistance of drug-resistant murine leukemias to deoxyspergualin (NSC 356894) in vivo.

Deoxyspergualin, the 15-deoxy derivative of the antibiotic spergualin, is a novel guanidino analog structurally related to spermine. Deoxyspergualin has significant activity in selected experimental tumor models, and clinical trials have been initiated. Described here are in vivo evaluations of the therapeutic activity of deoxyspergualin against murine leukemia lines specifically resistant to eight clinically useful antitumor drugs. These were P388 lines resistant to doxorubicin, vincristine, L-phenylalanine mustard, cisplatin, ara-C, and methotrexate and L1210 lines resistant to 5-FU, L-phenylalanine mustard, and cyclophosphamide. Sensitivity to deoxyspergualin was evaluated in parallel comparisons of each resistant leukemia to the sensitive line from which it had been derived. All experiments were repeated at least once for confirmation of results. Responses were quantitated in terms of the change in tumor cell numbers from the beginning of treatment to the end of treatment as estimated from the median survival times of dying mice. The results indicated that P388 leukemia resistant to cisplatin (P388/DDPt) was cross-resistant to deoxyspergualin. No cross-resistance was observed in leukemias resistant to doxorubicin, vincristine, ara-C, methotrexate, or cyclophosphamide. L1210 resistant to 5-FU (L1210/5-FU) was collaterally sensitive to deoxyspergualin. Although cross-resistance was also observed in P388/L-PAM, L1210/L-PAM retained sensitivity to deoxyspergualin. Total glutathione concentrations in P388/L-PAM and L1210/L-PAM provided no apparent explanation for this unexpected result. It may be tentatively concluded that resistance to cisplatin, L-PAM, or other DNA alkylators or cross-linkers may increase the potential for cross-resistance to deoxyspergualin. This conclusion requires verification with additional alkylating agents, with drug-resistant human tumor cell lines, and with prospective clinical studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Resistance and cross-resistance of the IgM immunocytoma in the LOU/M Wsl rat for cisplatin, carboplatin, and iproplatin.

We investigated the antitumor activity of cis-diammine[1,1-cyclobutanedicarboxylato]platinum(II) (CBDCA, JM8) and cis-dichloro-trans-dihydroxybis(isopropylammine)platinum(IV) (CHIP, JM9) for the cis-DDP-sensitive and -resistant IgM immunocytoma in the LOU/M Wsl rat. The optimal dose for the antitumor effect of cis-diamminedichloroplatinum (cis-DDP) in this tumor model is 1 mg/kg body weight. In order to determine the dose range for antitumor activity of JM8 and JM9, tumor-bearing rats were treated i.p. (twice weekly) with 2, 4, 8, 16, or 32 mg/kg JM8 or with 2, 4, or 8 mg/kg JM9. The maximal antitumor activity of JM8 was found at a dose of 4-8 mg/kg and that of JM9, at 4 mg/kg. Doses of 16 or 32 mg/kg JM8 did not increase the antitumor activity. Recurrence of tumors was observed in JM8- and JM9-treated rats. It was demonstrated that these relapses during treatment with JM8 or JM9 involved tumor cell populations almost completely resistant against therapy with the respective drugs. The growth of cis-DDP-resistant tumors was not influenced by the analog JM9 (4 and 8 mg/kg). Only a high dose of JM8 (32 mg/kg) caused growth retardation of the cis-DDP-resistant IgM subline. The JM8-resistant tumor was resistant to treatment with cis-DDP (1 and 2 mg/kg). The JM9-resistant tumor was also resistant to this treatment (1 mg/kg); however, at a dose of 2 mg/kg cis-DDP, growth retardation of the tumor occurred. We conclude that cis-DDP, JM8, and JM9 induce resistance in the IgM immunocytoma tumor system; tumors resistant for cis-DDP were not sensitive to the treatment with JM8 or JM9. Although JM9 reacts in vitro distinctly differently with DNA than cis-DDP and JM8, no differences were found in the induction of Pt resistance. In this study tumor cells were readily made resistant, which allows us to study in more detail the induction of (cross-) resistance by cis-DDP, JM8, and JM9.

Animals↗
PubMed2006

Novel mechanisms of platinum drug resistance identified in cells selected for resistance to JM118 the active metabolite of satraplatin.

PURPOSE: The goal of this study was to identify molecular determinants of sensitivity and resistance to JM118, the active metabolite of satraplatin, an orally bioavailable cisplatin analog that has activity in prostate cancer. EXPERIMENTAL DESIGN: Human ovarian carcinoma 2008/JM118 cells were derived from parental 2008 cells by repeated exposure to JM118; the revertant 2008/JM118/REV subline was isolated from the 2008/JM118 cells by growth in the absence of drug. Drug sensitivity was determined by clonogenic assay and Pt levels were measured by ICP-MS. RESULTS: Eight sequential rounds of selection yielded the 2008/JM118 subline that was 4.9-fold resistant to JM118 and cross-resistant at varying levels to satraplatin, cisplatin, carboplatin, and oxaliplatin. Cross-resistance to the other Pt drugs was lost as resistance to JM118 waned. The same parental 2008 cells selected for resistance to cisplatin were partially cross-resistant to JM118. The 2008/JM118 cells accumulated significantly more Pt than the 2008 cells when exposed to low concentrations of either JM118 or cisplatin indicating a detoxification process that involves intracellular sequestration. In contrast, 2008 cells selected for cisplatin resistance accumulated less cisplatin and less JM118 reflecting a mechanism involving reduced accumulation. The 2008 and 2008/JM118 cells did not differ in their uptake or efflux of 64Cu, expression of Cu efflux transporters ATP7A or ATP7B or their glutathione content. The 2008/JM118 cells exhibited 3.0-7.7-fold hypersensitivity to docetaxel, paclitaxel and doxorubicin. Expression profiling identified 4 genes that were significantly up-regulated and 19 that were down-regulated in the 2008/JM118 cells at a false discovery rate of 1 gene. CONCLUSIONS: While the cellular defense mechanisms that protect cells against JM118 also mediate resistance to the other Pt drugs, these mechanisms are quite different from those commonly found in cells selected for resistance to cisplatin. JM118-resistant cells accumulate more rather than less Pt and rely on an intracellular detoxification mechanism different from that involved in cisplatin resistance. This is consistent with clinical evidence suggesting that satraplatin has activity in diseases in which cisplatin does not. In this model, JM118 resistance is associated with substantial collateral hypersensitivity to docetaxel, paclitaxel, and doxorubicin.

Antineoplastic Agents↗
PubMed1996

Increased DT-diaphorase expression and cross-resistance to mitomycin C in a series of cisplatin-resistant human ovarian cancer cell lines.

In a series of ovarian carcinoma cell lines selected in vitro for resistance to cisplatin by continuous exposure to increasing drug concentrations, the level of resistance is proportional to the expression of gamma-glutamylcysteine synthetase (gamma-GCS). To determine if other detoxicating genes are coordinately expressed, we measured the activity of DT-diaphorase and cytochrome P450 reductase. The specific activity of DT-diaphorase, but not that of cytochrome P450 reductase, increased with increasing resistance to cisplatin. Steady-state mRNA levels for DT-diaphorase correlated with enzyme activity and hence with cisplatin resistance. Since the activity of DT-diaphorase has been associated with sensitivity to quinones, we studied the cytotoxicity of mitomycin C under oxic conditions. Unexpectedly, resistance to mitomycin C increased proportionally with that to cisplatin (r = 0.997). Pretreatment with buthionine sulfoximine, which inhibits glutathione (GSH) synthesis, failed to sensitize either the sensitive or the resistant lines to mitomycin C. Thus, the basis for collateral resistance to mitomycin C in the cisplatin-resistant lines under oxic conditions is unrelated to overproduction of GSH. Under hypoxia, the toxicity of mitomycin C to the most sensitive (A2780) cell line was unchanged. However, the most resistant (C200) line was 2-fold more resistant to mitomycin C under hypoxic conditions. The coordinate overexpression of DT-diaphorase and gamma-GCS in the resistant cell lines is thus associated with hypoxic cell resistance, and supports the involvement of shared mechanisms of gene regulation in the observed resistant phenotype.

Antineoplastic Agents↗
PubMed1996

Cross-resistance to cis-diamminedichloroplatinum(II) of a multidrug-resistant lymphoma cell line associated with decreased drug accumulation and enhanced DNA repair.

HOB1/VCR, a multidrug-resistant subline of the immunoblastic B lymphoma cell line, was established by sequential selection in increasing concentrations of vincristine. The expression of the human mdr l gene, as analyzed by reverse transcription and polymerase-chain reaction (RT-PCR), revealed a 10-15-fold overexpression in this resistant cell line. A complete inhibition of vincristine resistance by verapamil was observed in the vincristine-resistant HOB1/VCR cells, which suggests that acquired resistance may be mainly due to P-glycoprotein. HOB1/VCR cells also developed a 67-fold cross-resistance to the anticancer drug cis-diamminedichloroplatinum (cisplatin). DNA repair of the resistant and the parental cell lines was investigated by in situ detection with a cisplatin-DNA adduct-specific antibody and by measurement of repair-associated host cell reactivation of damaged plasmid DNA. HOB1/VCR cells exhibited a 2-fold decrease in the level of cisplatin-DNA adducts, compared to the parental cells. The DNA repair rate following peak accumulation of cisplatin-DNA adducts (which took approximately 4 h) was also enhanced in the resistant cells. This was supported by the measurement of the cisplatin level remaining in cells by atomic absorption spectrophotometry, which showed a 2.7-fold reduction in the resistant cells. In addition, the acquired resistance and enhanced DNA repair in HOB1/VCR cells were partially reversed by nontoxic aphidicolin, a DNA polymerase-alpha and DNA repair inhibitor. Inhibition of the intracellular level of glutathione by DL-buthionine-[S,R]-sulfoximine demonstrated that cell viability was inhibited 4-fold more in the resistant cells than in the parental cells. The results suggest that the reduced formation of cisplatin-DNA adducts and the increased glutathione content of the multidrug-resistant cells play a major role in phenotypic cross-resistance to cisplatin.

Antineoplastic Agents↗
PubMed1980

Resistance to methyl mercaptopurine riboside in cultured hamster cells. Preliminary characterization of resistant cells and conditions affecting their selection in quantitative mutation studies.

Cells resistant to high concentrations of methyl mercaptopurine riboside (MMPR), an anlogue of adenosine, were found at a frequency of about 6 x 10(-6) per viable cell in untreated cultures of V79 Chinese hamster cells. Resistant cells were selected less efficiently if purines were added to the MMPR-medium, but high cell densities had little effect upon selection. 6 independently-isolated spontaneous MMPR-resistant sublines were characterized by their resistance to the toxicity of different purines, rate of purine excretion, incorporation of radioactive adenosine, electrophoresis of cell extracts, and expression of resistance in hybrids to an MMPR-sensitive line. 5 of these sublines showed recessive expression of MMPR-resistance in hybrids and had characteristics consistent with loss of adenosine kinase activity, while the remaining subline was much less resistant to MMPR and showed semi-dominant expression of resistance without loss of adenosine kinase activity. Cells with high resistance to MMPR were not found in a "tetraploid" (hybrid) V79 line or in freshly-isolated human cell cultures, but occurred at a comparable frequency to V79 in another commonly-used aneuploid hamster line, CHO-K1. The frequency of MMPR-resistant cells in V79 cultures was increased to a similar extent by treatment with gamma-rays or with ethyl methanesulphonate, providing a suitable post-treatment interval was allowed for the expression of resistance. A genetic interpretation of these data is given in which it is proposed that resistance most usually arises through mutation of an autosomally-linked gene of which one copy has been inactivated or lost in V79 and in CHO-K1 cells. In comparison to published data on the selection of "mutants" resistant to 6-thioguanine, it is argued that MMPR could be as useful a selective agent as thioguanine and may select a different range of types of mutagenic event.

Adenosine Kinase↗
PubMed1992

Characterisation of melarsen-resistant Trypanosoma brucei brucei with respect to cross-resistance to other drugs and trypanothione metabolism.

An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.

Animals↗
PubMed1997

Reflections on the development of resistance during therapy for advanced breast cancer. Implications of high levels of activity of docetaxel in anthracycline-resistant breast cancer patients.

Anthracyclines play a central role in the treatment of breast cancer. They are perhaps the most active single agents available for the treatment of this disease. For patients with primary breast cancer for whom chemotherapy would be appropriate, anthracyclines are often incorporated in adjuvant regimens. For patients with endocrine-therapy-resistant metastatic disease, anthracyclines are nearly always included in the first or second-line therapeutic regimes. Unfortunately, despite the fact that anthracyclines and anthracycline-containing regimens can achieve impressive objective response rates, this impressive activity has not translated into a great improvement in disease-free survival for patients with early stage breast cancer or overall survival for patients with metastatic disease. The addition of anthracyclines to adjuvant therapy regimens in general only modestly improves their efficacy and the use of anthracyclines in metastatic disease does not cure these patients. The clinical utility of anthracyclines would be greatly improved if we could predict which patients would be anthracycline resistant, interfere with the development or expression of anthracycline resistance and predict which anticancer agents would be non-cross-resistant with anthracyclines. Some progress is being made in all these areas. Preclinical studies have identified several intracellular processes that are perturbed by anthracyclines, or that may modulate anthracycline sensitivity of cells. These processes include topoisomerase II activity, drug and toxin transmembrane pumps, intracellular detoxification systems (such as that related to gluatathione), stress-related proteins and apoptotic mechanisms. Although measurement of the components of these systems has not yet shown clinical utility in breast cancer, some preclinical work and exploratory studies with small numbers of patients suggest that we may in the future be able to predict which patients will respond or be resistant to anthracyclines. An important avenue of work is the identification of anticancer agents that are non-cross-resistant with anthracyclines in breast cancer patients. These agents would be particularly valuable in patients with metastatic disease who had progressed while on anthracyclines. In general, such patients have a very poor prognosis with a median survival of less than 1 year. Also of importance in non-cross-resistant agents is that they might be used in combination with anthracyclines in regimens with very high response rates. Recently completed work suggests that taxoids might be such agents. This finding opens up exciting possibilities in the treatment of metastatic disease and, even more importantly, in the adjuvant arena. The identification of agents that clinically are non-cross-resistant with anthracyclines depends on the careful interpretation of clinical trial data. Unfortunately, a number of definitions of anthracycline resistance have been used in the medical literature. These range from very weak definitions, which include many patients with only prior anthracycline exposure (for example, in an adjuvant regimen), to more biologically and clinically appropriate definitions, such as documented progression while receiving an anthracycline. This distinction is very important because it is clear that many patients who have relapsed after an anthracycline-based adjuvant therapy will respond to anthracyclines for metastatic disease and are, therefore, not truly anthracycline resistant. In this regard, the recently completed trials with docetaxel in patients with rigorously defined anthracycline resistance are particularly provocative. These trials show that docetaxel maintains much of its excellent first-line levels of efficacy in patients with anthracycline-resistant breast cancer. Agents with this type of maintenance of efficacy in anthracycline-resistant tumours may find immediate utility in this clinical scenario as single agents but, more importantly, have gr

Antibiotics, Antineoplastic↗
PubMedDate not supplied

Mechanisms of antibiotic resistance and their dissemination of resistance genes in the hospital environment.

The dissemination of resistance determinants among bacterial populations depends on ecological and epidemiological properties as well as additional factors: 1) the mechanism of resistance or its specificity toward a certain drug, and 2) the genetic basis in relation to the mobility of the genetic material and its survival in bacteria. From two resistance mechanisms directed toward old-fashioned drugs, namely sulfonamides (Su) and streptomycin (Sm), we can deduce that a resistance mechanism is encoded by a special sort of genetic material. Thus the linked SmSu resistance mediated by a sulfonamide-resistant dihydropteroatsynthetase II and the aminoglycoside phosphotransferase APH-(3") is always located on very small pBP1-like plasmids. Such plasmids survive without selective pressure of drugs in Enterobacteriaceae in the bowel flora of humans and animals. Both resistance determinants can be mediated by a transposon which codes for the production of a dihydropteroatsynthetase I in connection with an aminoglycoside adenylyltransferase AAD-(3"). These two mechanisms are genetically linked as well. The basic structure is a transposon designated Tn2411, which belongs to a whole family of transposons, all including the basic structure; however, their genetic exchange and substitution leads to structures coding for many different enzymatic characters: ANT-(2") (Gentamicin resistance), CAT (Chloramphenicol resistance), AAC-(6') (resistance to all modern aminoglycosides), TEM-1, OXA-1, OXA-2, or PSE (beta-lactam resistance). Resistance to the modern beta-lactamase-stable antibiotics is mediated by mutation in the regulatory genes of chromosomally-determined beta-lactamases. A spread of these resistance mechanisms can be avoided as long as the responsible genes are not located on sufficient structures like small plasmids or efficient transposons.

Anti-Bacterial Agents↗
PubMed1994

Characterisation of a navelbine-resistant bladder carcinoma cell line cross-resistant to taxoids.

A bladder carcinoma cell line (J82) was selected for resistance to the new vinca alkaloid navelbine. The resistance factor of the resistant subline (J82-NVB) to navelbine was 17. P-glycoprotein was not detected in the membrane of J82-NVB cells. The lack of cross-resistance to multidrug-resistant (MDR) drugs such as doxorubicin, epipodophyllotoxins and colchicine, the absence of increase in navelbine efflux and the fact that a reduced accumulation of the drug cannot account for the resistance level confirmed that the phenotype of resistance of J82-NVB cells is not a classical MDR phenotype. Moreover, verapamil did not reverse the resistance of J82-NVB cells. The cells were cross-resistant to vinca alkaloids and taxoids which share the same target protein: tubulin. Analysis of microtubules using immunofluorescence showed that disassembly of the microtubular network occurred for the same concentration of navelbine in sensitive and resistant cells. However, after treatment with a concentration of navelbine inducing depolymerisation in both sensitive and resistant cells, reassembly of the microtubular network was observed only in resistant cells. This study suggests that the mechanism of resistance of J82-NVB cells involves recovery from the inhibition of microtubule dynamics induced by drug treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Mechanism by which phosphonoformic acid resistance mutations restore 3'-azido-3'-deoxythymidine (AZT) sensitivity to AZT-resistant HIV-1 reverse transcriptase.

The development of phosphonoformic acid (PFA) resistance against a background of 3'-azido-3'-deoxythymidine (AZT) resistance in human immunodeficiency virus type 1 (HIV-1) restores viral sensitivity to AZT. High level AZT resistance requires multiple mutations (D67N/K70R/T215F/K219Q). In order to characterize the mechanism of PFA resistance-mediated resensitization to AZT, the A114S mutation associated with PFA resistance was introduced into the reverse transcriptase (RT) of both wild type and drug-resistant virus. We previously showed that pyrophosphorolytic removal of chain-terminating AZT is the primary mechanism of the AZT resistance phenotype (Arion, D., Kaushik, N., McCormick, S., Borkow, G., and Parniak, M. A. (1998) Biochemistry 37, 15908-15917). Introduction of A114S into the AZT resistance background significantly diminishes both the enhanced pyrophosphorolytic activity and the DNA synthesis processivity associated with the AZT-resistant RT. The A114S mutation also alters the nucleotide-dependent phosphorolysis activity associated with AZT resistance. The presence of the A114S mutation therefore severely impairs the mutant enzyme's ability to excise chain-terminating AZT. The decrease in phosphorolytic activity of RT conferred by the PFA resistance A114S mutation resensitizes AZT-resistant HIV-1 to AZT by allowing the latter to again function as a chain terminator of viral DNA synthesis. These data further underscore the importance of phosphorolytic removal of chain-terminating AZT as the primary mechanism of HIV-1 AZT resistance.

Anti-HIV Agents↗
PubMed2001

Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines.

Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Transposition of gentamicin resistance to staphylococcal plasmids encoding resistance to cationic agents.

Plasmid pWG115 isolated from a methicillin-resistant Staphylococcus aureus encodes resistance to cationic surface-active agents and trimethoprim. It has a molecular weight of ca 14.6 megadaltons and can be transferred to other strains of staphylococci in mixed-culture transfer with propamidine isethionate as a selective agent. Gentamicin resistance in Australian methicillin-resistant Staph. aureus isolates can be either chromosomal or plasmid-borne. The most common gentamicin resistance plasmid is 18.0 megadaltons and also encodes resistance to trimethoprim and cationic surface-active agents. This suggested that pWG115 was related to gentamicin resistance plasmids and that it may provide a target for the postulated gentamicin resistance transposon. This paper demonstrates that the chromosomal gentamicin resistance determinant from WG523 can transpose into pWG115 to generate an 18.0 megadalton plasmid, phenotypically indistinguishable from the naturally occurring gentamicin resistance plasmids such as pWG53. EcoR1 restriction enzyme analysis demonstrated that gentamicin resistance can transpose into at least two sites on pWG115. One of these sites generates EcoR1 restriction fragments identical to pWG53. The 5.2 kilobase pair (3.4 megadalton) element involved confers low-level resistance to gentamicin, cross resistance to tobramycin and kanamycin, and has been designated Tn3851.

Benzamidines↗
PubMed2000

Macrolide resistance and erythromycin resistance determinants among Belgian Streptococcus pyogenes and Streptococcus pneumoniae isolates.

Resistance of streptococci to macrolide antibiotics is caused by target-site modification or drug efflux. The phenotypic expression of target-site modification can be inducible or constitutive. The prevalence of the three phenotypes among Belgian erythromycin-resistant Group A streptococci (GAS) and Streptococcus pneumoniae isolates was surveyed, their MICs for seven antibiotics were determined and the clonality of the isolates was explored. Of the 2014 GAS isolates tested 131(6.5%) were erythromycin resistant (MIC > 1 mg/L): 110 (84.0%) showed the M-resistance phenotype whereas the remaining 21 strains (16.0%) were constitutively resistant. No inducibly resistant strains were detected. Of 100 S. pneumoniae isolates, 33 were erythromycin resistant (MIC > 1 mg/L). In contrast to the GAS isolates, only 9.1% of the 33 erythromycin-resistant S. pneumoniae isolates showed the M-resistance phenotype. The presence of mefA/E and ermB genes in the M-resistant and constitutively and inducibly resistant strains, respectively, was confirmed by PCR analysis. Genomic analysis based on pulsed-field gel electrophoresis (PFGE) using the restriction enzyme SfiI, revealed 54 different PFGE patterns among the 131 erythromycin-resistant GAS isolates, of which an M6 clone represented 16.0% of the strains; all other clones, exhibiting different M-types, represented <7% of the strains. The S. pneumoniae isolates also appeared to be polyclonally based, as determined by arbitrarily primed PCR. The macrolides miocamycin and rovamycin, the lincosamide clindamycin and the ketolide HMR 3647 showed excellent activity against the M-resistant GAS and S. pneumoniae strains.

Anti-Bacterial Agents↗
PubMed2005

Molecular mechanisms of resistance in multidrug-resistant serovars of Salmonella enterica isolated from foods in Germany.

OBJECTIVES: The objectives of this study were to determine antimicrobial susceptibility and to characterize the molecular mechanisms of multidrug resistance among German food-borne Salmonella isolates of different serovars. METHODS: A total of 319 epidemiologically independent multidrug-resistant isolates from German foodstuffs comprising 25 different serovars were tested for their antimicrobial susceptibility by broth microdilution. The presence of antimicrobial resistance genes, integrons of classes 1 and 2 and their integrated resistance gene cassettes as well as the Salmonella genomic island 1 (SGI1) was investigated by PCR and DNA sequencing. Localization of integrons and relevant resistance genes was done by Southern hybridization. Sequence analysis revealed mutations in the quinolone resistance-determining region of the gyrA gene. RESULTS: The most prevalent resistances found in the multidrug-resistant serovars of Salmonella enterica from foods were to streptomycin (94%), sulfamethoxazole (92%), tetracycline (81%), ampicillin (73%), spectinomycin (72%), chloramphenicol (48%) and trimethoprim (27%). Twenty-four resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, sulphonamides, tetracycline, and trimethoprim) were identified in the food isolates, many of them integrated as gene cassettes in class 1 and class 2 integrons. Class 1 integrons were detected in 65% of the multidrug-resistant Salmonella isolates comprising 16 different serovars, while class 2 integrons were found in 10% of the isolates belonging to two serovars only. The results demonstrate a clear predominance of both SGI1-borne resistance genes and class 1 integrons in Salmonella serovar Typhimurium DT104 and of class 2 integrons in Salmonella serovar Paratyphi B (d-tartrate positive). Nalidixic acid resistance found in 15% of the isolates was associated with single mutations in the gyrA gene. CONCLUSIONS: This study confirms the role of foods of animal and other origin as a reservoir of multidrug-resistant Salmonella and underlines the need for continuing surveillance of food-borne zoonotic bacterial pathogens along the food chain.

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