Drug Resistance Assays Research Articles

-mediated expression in

The b-Zip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the KNQ1 gene, encoding a multidrug transporter of the major facilitator superfamily in Kluyveromyces lactis and containing two potential Yap1p response elements in its promoter, is a putative transcriptional target of KlYap1p, the structural and functional homologue of ScYap1p. In this work, we provide evidence that KlYAP1 controls the expression of the KNQ1 gene. Using a P(KNQ1)-gusA fusion construct we showed that the expression of KNQ1 is induced upon cell treatment with the oxidizing agents H2O2 and menadione and that this induction is mediated by KlYap1p. These results were confirmed by Northern-blot analysis showing that the expression of KNQ1 is responsive to hydrogen peroxide and dependent on the presence of KlYap1p. The role of KlYAP1 in the control of KNQ1 expression was further demonstrated by EMSA experiments and drug resistance assays. These results clearly demonstrate the involvement of the KlYap1p transcription factor in the control of KNQ1 gene expression.

The Change of Intracellular pH is Involved in the Cisplatin-Resistance of Human Lung Adenocarcinoma A549/DDP Cells

We had reported that the intracellular pH (pHi) of human lung adenocarcinoma A549 cells, which is sensitive to cisplatin, was more acidic than that of cisplatin-resistant A549/DDP cells. The correlation between the change of the pHi and cisplatin-resistance of A549/DDP cells was further studied by altering pHi in consequence of the change of CO2 concentration of the incubator. The pHi alterations of the cells were monitored by using the fluorescence probe of BCECF-AM. The results indicated that the pHi was more alkaline at lower CO2 concentration (2% CO2 in the incubator) and more acidic at higher CO2 concentration (8% CO2 in the incubator) for both A549 and A549/DDP cells compared with those of both A549 and A549/DDP cells cultured at 5% CO2 as the normal condition. Accumulation of bodipy-cisplatin, a fluorescence probe used for drug resistance assays, in A549 cells incubated at 2%, 5%, and 8% CO2 was increased 8.4%, 17.4%, and 23.5% compared to A549/DDP cells, respectively. Intracellular sequestration and distribution of bodipy-cisplatin imaged by laser scanning confocal microscopy indicated that bodipy-cisplatin was more encapsulated in acidic compartments of A549/DDP cells as shown with acridine orange, a dye that specifically labels acidic organelles in the cells. These results can be further confirmed in liposome systems with different pH gradients. It is proposed from the above results that the change of pHi in especially more acidic compartments in A549/DDP cells involves their cisplatin resistance.

The Change of Intracellular pH is Involved in the Cisplatin-Resistance of Human Lung Adenocarcinoma A549/DDP Cells

We had reported that the intracellular pH (pHi) of human lung adenocarcinoma A549 cells, which is sensitive to cisplatin, was more acidic than that of cisplatin-resistant A549/DDP cells. The correlation between the change of the pHi and cisplatin-resistance of A549 /DDP cells was further studied by altering pHi in consequence of the change of CO2 concentration of the incubator. The pHi alterations of the cells were monitored by using the fluorescence probe of BCECF-AM. The results indicated that the pHi was more alkaline at lower CO2 concentration (2% CO2 in the incubator) and more acidic at higher CO2 concentration (8% CO2 in the incubator) for both A549 and A549/DDP cells compared with those of both A549 and A549/DDP cells cultured at 5% CO2 as the normal condition. Accumulation of bodipy-cisplatin, a fluorescence probe used for drug resistance assays, in A549 cells incubated at 2%, 5%, and 8% CO2 was increased 8.4%, 17.4%, and 23.5% compared to A549/DDP cells, respectively. Intracellular sequestration and distribution of bodipy-cisplatin imaged by laser scanning confocal microscopy indicated that bodipy-cisplatin was more encapsulated in acidic compartments of A549/DDP cells as shown with acridine orange, a dye that specifically labels acidic organelles in the cells. These results can be further confirmed in liposome systems with different pH gradients. It is proposed from the above results that the change of pHi in especially more acidic compartments in A549/DDP cells involves their cisplatin resistance.

Optophoresis Accurately Predicts Response to Chemotherapy in CLL.

In vitro drug resistance assays have shown efficacy in predicting response to chemotherapeutic drugs in a number of solid tumors. In chronic lymphocytic leukemia (CLL) many assays are limited by the difficulty involved in culturing these cells ex vivo. We have developed a novel, optical-based methodology that is sensitive to broad cellular physical characteristics, such as morphology, size, refractive index, density and surface properties. This measurement, known as Optophoresis, quantifies cell motion induced by exposure to a moving optical gradient, generated from a near-infrared laser beam (Forster AH, et al. Anal Biochem 2004, 327(1):14–22 and Wang MM, et al. Appl Opt 2003, 42(28):5765–73). In Optophoresis small numbers of cells are analyzed intact, in their native state. No labels are required for quantification of functional responses, but cell subpopulations may be identified using fluorescent tags. We have used this assay to predict response to chemotherapeutic agents in patients with CLL.Methods: We performed Optophoresis with six drugs (fludarabine, chlorambucil, vincristine, cyclophosphamide, cladribine and prednisolone) on 74 CLL patient samples. 68 of these were classic B-CLL; there was one hairy cell variant, two were T-CLL, two evolved to PLL and one evolved to lymphoma. 21 of the samples were from patients for whom clinical data on response to chemotherapy was available for 33 drug treatments. Response to therapy was defined as a decrease in RAI stage.Results: 21 patients aged 58 to 93 years; 71% Rai stage III or IV were included in this analysis. Six patients were evaluated prospectively, with the assay performed before chemotherapy administration, and 15 patients were evaluated retrospectively with the assay performed after a course of treatment. Optophoresis accurately predicted response to chemotherapy in 88% of the 33 treatments evaluated. In the four instances for which the Optophoresis results did not match the retrospective clinical outcome; the patient was historically sensitive to treatment and Optophoresis results indicated current resistance to the drug. Subsequent resistance to drug therapy is known to occur in a significant percentage of treated CLL patients.Conclusion: Optophoresis of CLL cells accurately predicts response to chemotherapy in CLL. Further studies using results of Optophoresis to guide CLL treatment are warranted.

In vitro synthesis of enzymatically active HIV-1 protease for rapid phenotypic resistance profiling

Given the expanding antiretroviral therapy, inexpensive and fast HIV drug resistance assays are urgently needed. In this view, we have developed a novel phenotypic resistance test for HIV-1 protease inhibitors (PIs) based on recombinant expression of patient-derived HIV PR in Escherichia coli and subsequent enzymatic testing in a fluorescent readout. To facilitate and expedite the test procedure, we have introduced coupled in vitro transcription/translation using a commercially available technology called RTS for producing enzymatically active HIV-1 protease (PR). We expressed one wild type PR and one highly resistant mutant starting from molecular clones as well as three patient-derived PRs. The amplified PR gene was either ligated into an expression vector or directly used as a template for the in vitro transcription/translation reaction. Enzymatic susceptibility data derived from in vitro expressed PRs were correlated to the respective results from E. coli expression and genotypic evaluation. All tested enzymes were obtained in sufficient quantities for complete resistance profiling to five PIs. The PRs required no purification prior to the enzymatic assay. Inhibition constants and enzymatic resistance factors compared well to corresponding data from PRs expressed in parallel in E. coli. Enzymatic resistance was in good agreement with the respective PR genotype. The presented in vitro transcription/translation system represents a novel approach for HIV PR expression starting from molecular clones or patient samples. Coupled with the enzyme-kinetic PR assay recently developed in our group it allows to sensitively quantify resistance to PIs. The test system is significantly less laborious and faster than currently available phenotypic drug resistance assays.

Detection of mutations associated with isoniazid and rifampin resistance in Mycobacterium tuberculosis isolates from Samara Region, Russian Federation.

High incidence rates of isoniazid-, rifampin-, and multiple-drug-resistant tuberculosis have been reported in countries of the former Soviet Union (FSU). Genotypic (unlike phenotypic) drug resistance assays do not require viable cultures but require accurate knowledge of both the target gene and the mutations associated with resistance. For these assays to be clinically useful, they must be able to detect the range of mutations seen in isolates from the population of tuberculosis patients to which they are applied. Two novel macroarrays were applied to detect mutations associated with rifampin (rpoB) and isoniazid (katG and inhA) resistance. In a sample of 233 isolates from patients in Samara, central Russia, 46.5% of isolates possessed mutations in both the rpoB and the katG (or inhA) genes. Combined results from the macroarrays demonstrated concordance in 95.4 and 90.4% of phenotypically defined rifampin- and isoniazid-resistant isolates, respectively. The contribution of different mutations to resistance was comparable to that reported previously for non-FSU countries, with 90% of rifampin-resistant isolates and 93% of isoniazid resistant isolates due to rpoB531 and katG315 mutations, respectively. The percentage of phenotypically resistant rifampin isolates with no mutations in the rpoB codons 509 to 536 was 4.2%, which was similar to previous reports. Novel macroarrays offer a rapid, accurate, and relatively cheap system for the identification of rifampin-, isoniazid-, and multiple-drug-resistant Mycobacterium tuberculosis isolates.

Performance of drug-resistance genotypic assays among HIV-1 infected patients with predominantly CRF02_AG strains of HIV-1 in Abidjan, Côte d’Ivoire

We evaluated the performance of three genotypic assays to detect resistant mutations among HIV-1 infected patients with known antiretroviral drug resistance profile in Abidjan, Cote d'Ivoire, most of whom had the circulating recombinant form (CRF02_A/G) of HIV-1. The 56 patients analyzed in this study were enrolled in a pilot program to make available antiretroviral therapy (ART) to HIV-infected patients in Abidjan through the UNAIDS Drug Access Initiative (DAI). These patients had failed ART, as demonstrated by rebound in RNA viral load. Their plasma samples had been previously analyzed for ART genotypic drug-resistance by VircoGEN (Mechelen, Belgium) and were known to have primary and secondary resistance mutations, and also had phenotypic drug-resistance by a recombinant virus assay technology (Mechelen, Belgium). The two assays we evaluated were: VircoGEN, TruGene HIV-1, and ViroSEQ HIV-1 assays. For the reverse transcriptase gene, all 27 samples that had the T215Y/F mutation were detected by VircoGEN , ViroSEQ, and TrueGene. All 19 (100%) samples that had the K70R/E mutation detected by VircoGEN were detected by ViroSEQ, and 18 (94.7%) by TrueGene. All ten samples with the M184V mutation, three with the K65R, two with the G190A mutation, one with the K103N mutation, and one with the V75T mutation were detected similarly by all three assays. For the protease gene, all three assays detected the I84V (n = 1), M46I (n = 1), and L90M (n = 1) mutations. These results suggest that any of these assays should be considered for monitoring the occurrence of drug resistance among HIV-infected patients receiving antiretroviral therapy in West Africa.

An in vitro short time-high dose drug exposure assay for predicting 5FU-resistance of colorectal cancer

The goal of this study was to develop a simple and rapid in vitro drug resistance assay to ascertain the effectiveness of 5-fluorouracil (5-FU) for the individual therapy of colorectal cancer. Colorectal cancer cells were isolated from tumor specimens and, after 4 h exposure to high doses of 5-FU cell viability was measured with an ATP assay. The average IC 50 concentration for 5-FU was calculated as 4000 μg/ml from 35 patients’ tumors. The tumor cells were defined as extreme drug resistance with a survival rate 1 standard deviation (SD) over IC 50, low drug resistance (LDR) with a survival rate 1 SD below IC 50, and intermediate drug resistance (IDR) with survival rate between these two. The drug resistant assay for 102 patients’ cancer cells showed that the proportion of patients with LDR to 5-fluorouracil was 19%. The in vitro drug resistance of the cancer cells was not correlated with cancer stages or by patient sex or age. However, most mucinous and poor differentiated cancer cells showed extreme or IDR. The in vitro ATP assay values for 25 Duke's D patients receiving postoperative 5-FU chemotherapy were comparable with clinical postchemotherapy responses. The sensitivity and specificity of the assay were 100 and 95%, respectively. This short time-high dose drug exposure assay may serve as an aid to improve 5-FU treatment for individual chemotherapy.

404. Cloning and Expression of Canine O6-Methylguanine–DNA Methyltransferase Gene in Ex Vivo Cultured Cells

The human O6-methylguanine-DNA-methyltransferase (MGMT) gene and its mutants have been used previously for in vivo selection of transduced hematopoietic stem cells with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) alone or in combination with O6-benzylguanine (BG). To allow similar in vivo selection in dogs, without the risk of inducing of an immune response, we have cloned the canine MGMT drug resistance gene (GenBank Accession No. AY423553). Comparison of canine and human MGMT coding regions indicated that there was about 62% amino acid identity and 78% similarity between the two MGMTs. The canine MGMT was also longer by 9 amino acids. Proline at position 140 and the surrounding amino acids of the human MGMT were highly conserved in the canine sequence. To determine if mutation of the proline residue at position 144 to lysine (P144K) in the canine MGMT would provide a similar advantage for selection with BG and BCNU of transduced cells as the human P140K mutant, we created the corresponding P144K mutant of canine MGMT. Bicistronic Moloney murine leukemia virus and human immunodeficiency type 1 vectors were used to express separately canine and human MGMTs in cultured canine CTAC cells. Drug-resistance assays demonstrated that the wild-type and the P144K mutant canine MGMTs provided resistance to the BCNU or BG together with BCNU selection, respectively that was comparable to the human MGMT counterparts.

Oligo-3-hydroxybutyrates as potential carriers for drug delivery

In the present paper we describe the synthesis and toxicity studies of well-defined tailor made oligo-[ R, S]-3-hydroxybutyrates (OHBs). The results indicate potential applicability of these nano-polymers as drug delivery carriers. Several OHBs of number average molecular weight ( M n) ranging from 800 to 2400 have been synthesized and tested on transformed hamster V79 fibroblasts and murine melanoma B16(F10) cells using the 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) based drug resistance and clonogenic survival assays. We show that 96-h incubation of cells with 1–9 μg/ml of OHBs did not affect cell viability. Incubation of OHBs with rat hepatoma FTO-2B cells stably transfected with chloramphenicol acetyltransferase (CAT) gene ligated to heat-inducible hsp70i gene promoter demonstrated that OHBs did not induce cellular stress response. Finally, we demonstrate that doxorubicin conjugated with OHB is effectively taken up by murine melanoma B16(F10) cells in vitro and localizes in the cytoplasm. These data show for the first time that tailor-made biodegradable and biocompatible oligomers of 3-hydroxybutyric acid can be taken into consideration as effective, non-toxic vectors for delivery of drugs in a conjugated form.

Determination of functional viral titer by drug-resistance colony assay, expression of green fluorescent protein, and beta-galactoside staining.

With this method, antibiotic selection of the infected cells gives rise to a countable number of colonies after 7–10 d (, , , ). Other reporter genes are based on green fluorescent protein (GFP; ref. ) or the Escherichia coli lacZ gene, which encodes for β-galactosidase for X-gal staining (,). GFP-expressing cells can be easily visualized under an ultraviolet microscope. The main advantage is that GFP expression can be detected in living cells (). Retroviral and lentiviral transfer vectors can be engineered with an internal phosphoglycerate kinase (pgk) promoter driving the expression of a bacterial gene that confers resistance to a certain antibiotic (, ). Usually, the bacterial genes used are based on neomycin phosphotransferase, hygromycin B phosphotransferase, or puromycin N-acetyltransferase, which render mammalian cells resistant to G418, hygromycin B, and puromycin, respectively (). In addition, either the internal pgk or SV40 early promoter can drive the expression of lacZ gene (,) or GFP (). Functional titer does not provide a consistent measurement of virion concentration because it depends upon the transduction efficiency of the cell line being used to determine the titer. Therefore, several investigators use preferentially direct quantification for determining virus particle concentration. There are several bibliographic references for the counting of selected colonies (,).

In vitro chemoresistance and biomarker profiles are unique for histologic subtypes of epithelial ovarian cancer

Objectives. To determine whether there is a relationship between histologic subtype of epithelial ovarian cancer and chemoresistance, we evaluated ovarian carcinomas of six histologic subtypes and correlated histology with in vitro drug response. Biomarker profiles (p53, Her-2 neu, and EGFR) were also evaluated to determine if their expression patterns were associated with histology. Methods. In vitro drug response profiles for different histologic subsets of epithelial ovarian carcinomas exposed to standard relevant chemotherapy agents were determined in the Extreme Drug Resistance assay (EDR). Immunohistochemistry techniques were employed to determine biomarker expression. Results. Of 5195 referred serial cases of epithelial ovarian cancer, there were 2660 papillary serous, 303 endometrioid, 142 mucinous, 102 clear cell, 952 undifferentiated carcinomas, and 42 tumors of low malignant potential. For the samples as a whole, the incidences of extreme drug resistance to the tested chemotherapeutic agents were cisplatin 10%, carboplatin 16%, cyclophosphamide 16%, doxorubicin 40%, gemcitabine 21%, paclitaxel 22%, and topotecan 13%. When compared to papillary serous tumors, mucinous tumors were more frequently resistant to cisplatin (10% vs. 18%) but less frequently resistant to topotecan (13% vs. 5%) and doxorubicin (42% vs. 16%). Endometrioid tumors were less resistant to cisplatin (10% vs. 6%) and doxorubicin (42% vs. 20%). Clear cell and undifferentiated tumors had the lowest rates of EDR to paclitaxel (13% and 18%) and cyclophosphamide (7% and 11%), while borderline tumors showed high rates of EDR to these agents (52% and 63%, respectively). With respect to biomarker profiles, mP53 was detected in 46%, Her-2 neu in 16%, and EGFR in 30% of the cases evaluated. As compared to all other subtypes, clear cell carcinomas had significantly higher Her-2 neu expression (19%). Relative to papillary serous carcinomas, borderline tumors exhibited significantly lower rates of mP53 expression (60% vs.17%). Conclusions. We found significant differences in the frequencies of extreme drug resistance to chemotherapeutic agents and biomarker expression among histologic subtypes of epithelial ovarian cancer. The data collected in this investigation may provide a guide for stratification of patients entering clinical trials based on histology and biomarker expression.

Survival outcomes in patients with recurrent ovarian cancer who were treated with chemoresistance assay–guided chemotherapy

Objective The purpose of this study was to determine the outcome of patients with recurrent ovarian carcinoma after extreme drug resistance assay-directed therapy. Study design Fifty women who were treated with chemotherapy based on extreme drug resistance assay guidance were compared with 50 well-balanced control subjects who were treated empirically. Results In the platinum-sensitive group, patients with extreme drug resistance–directed therapy had an overall response rate of 65% compared with 35% in the patients who were treated empirically ( P = .02). The overall and progression-free median survival were 38 and 15 months in the extreme drug resistance assay group compared with 21 and 7 months in the control group, respectively ( P = .005, overall; P = .0002, progression free). In the platinum-resistant group, there was no improved outcome in the patients who underwent assay-guided therapy. In multivariate analysis, platinum-sensitive disease, extreme drug resistance–guided therapy and early stage of disease were independent predictors for improved survival. Conclusion In this retrospective analysis, our results indicate an improved outcome in patients with recurrent ovarian carcinoma who have platinum sensitive disease and who underwent extreme drug resistance–directed chemotherapy. Randomized, prospective, controlled trials are needed.

Stable, stoichiometric delivery of diverse protein functions

As contemporary “genomics” steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.

“All-in-One Assay”, a direct phenotypic anti-human immunodeficiency virus type 1 drug resistance assay for three-drug combination therapies that takes into consideration in vivo drug concentrations

A novel phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay is described. Three drugs at concentrations equivalent to those determined in in vivo pharmacokinetics, were mixed in a well, serially diluted by 10-folds, and added to incubations of clinical HIV-1 isolates and CCR-5 expressing HeLa/CD4+ cells which was previously reported as the MAGIC-5 cells [Antimicrob. Agents Chemother. 45 (2001) 495] to determine the 95% inhibitory dilution (ID 95) of the combination regimens. The ID 95 of efavirenz (EFV)-containing regimens was ten-times lower than that of nevirapine (NVP)-containing regimens against HIV-1 isolated from antiviral therapy naive patients. However, the difference was not apparent by the conventional fold resistance measurement based on the 50% inhibitory concentration. Furthermore, the synergistic effects of drug combinations against clinical HIV-1 isolates can be evaluated by our assay. The ID 95s of EFV- and nelfinavir (NFV)- containing regimens against HIV-1 from naive patients were less than 0.01 whereas those against resistant viruses were over 0.05, although the clinical cut-off values are to be determined in larger clinical studies. Our assay, designated “All-in-One Assay”, that can examine resistance to three drugs simultaneously under consideration of in vivo drug concentrations described above might be useful in practice.

Performance of drug resistance assays in testing HIV-1 non-B subtypes

Antiretroviral susceptibility analyses were performed in plasma samples collected from 32 HIV-1 non-B-infected individuals, most of whom had received antiretroviral drugs. Reverse transcriptase (RT) and protease gene sequences were obtained, and 15 anti-HIV drugs were tested in a recombinant virus phenotypic assay. Phenotypic results were obtained in 25 (78.1%) samples, while genotypic data were recorded in 19 (59.4%). In seven samples (21.9%), neither genotypic nor phenotypic results were obtained. Ten of 13 samples with plasma HIV RNA below 2000 copies/mL did not yield genotypic results. Resistance assays work accurately when testing HIV-1 non-B subtypes. However, as for subtype B variants, a low viral load is the most important factor limiting the application of these tests.

Low rate of HIV-1 codon 215 revertants in antiretroviral-experienced patients.

Novel codon T215C/D/N/S mutations have been described in antiretroviral-naive patients infected with zidovudine-resistant HIV-1-carrying 215F/Y. Viruses with codon 215-revertant genotypes are sensitive to zidovudine and stavudine, but rapidly develop resistance under exposure to these drugs. In pre-treated HIV-infected patients who discontinue zidovudine or stavudine there is no evidence of reversion to these unusual genotypes, because of an overgrowth of pre-existing wild-type viruses. Therefore, these novel codon 215 mutations almost always reflect a past primary infection with zidovudine-resistant viruses.

Actinomycin D induces high-level resistance to thymidine analogs in replication of human immunodeficiency virus type 1 by interfering with host cell thymidine kinase expression.

Actinomycin D (ActD) is a transcription inhibitor and has been used in the treatment of certain forms of cancer. ActD has been reported to be a potential inhibitor of human immunodeficiency virus type 1 (HIV-1) replication due to its ability to inhibit reverse transcription. In contrast to what was expected, low concentrations of ActD (1 to 10 nM) upregulated HIV-1 replication 8- to 10-fold in MT-2 cells and had no effect on HIV-2 replication or on HIV-1 replication in MT-4, Jurkat, or peripheral blood mononuclear cells. The upregulation of HIV-1 replication was associated with an increase in HIV-1 transcription and a decrease in CD4 and CXCR4 expression. To further evaluate the effects of ActD on emergence of drug resistance in HIV-1 replication, a series of drug resistance assays were performed. Of interest, treatment of MT-2 cells with ActD also led to a high level of resistance to thymidine analogs (>1,000-fold increase in resistance to zidovudine and >250-fold to stavudine) but not to other nucleoside reverse transcriptases (RT), nonnucleoside RT, or protease inhibitors. This resistance appeared to be due to a suppression of host cell thymidine kinase-1 (TK-1) expression. These results indicate that ActD leads to a novel form of thymidine analog resistance by suppressing host cell TK-1 expression. These results suggest that administration of combination drugs to HIV-1-infected patients may induce resistance to antiretroviral compounds via a modification of cellular factors.

Isolation and molecular characterization of a nelfinavir (NFV)-resistant human immunodeficiency virus type 1 that exhibits NFV-dependent enhancement of replication.

During the use of a phenotypic anti-human immunodeficiency virus type 1 (HIV-1) drug resistance assay in a large set of clinical virus isolates, we found a unique variant (CL-4) that exhibited a high level of nelfinavir (NFV) resistance and rather enhanced replication under subinhibitory concentrations of NFV (0.001 to 0.1 micro M). Comparison of gag-pol sequences of the CL-4 variant and its predecessor virus isolates showed a stepwise accumulation of a total of 19 amino acid substitutions in protease (PR) and Gag p17 during 32-month NFV-containing antiretroviral therapy, while other Gag regions including the cleavage sites of the p55 precursor remained highly conserved. To understand the relationship between the genetic and phenotypic changes in CL-4, we constructed chimeric viruses using pNL4-3, replacing the PR, p24PR, or p17PR gene segment of CL-4 or its predecessor. A series of tissue culture infections with the chimeras in the absence or presence of increasing concentrations of NFV demonstrated that only the p17PR segment of CL-4 could confer the NFV-dependent replication enhancement phenotype on NL4-3. Our data suggest a novel adaptation mechanism of HIV-1 to NFV, in which coevolution of Gag and PR genes generates a variant that replicates more efficiently in the cellular environment in the presence of NFV than without the drug.

Relationship between DNA repair capacity and resistance to genotoxins in four human cell lines

We have developed fast, reliable and simple fluorescent method to assess and compare repair capacity of cells. To this end plasmid pEGFP containing the gene for the enhanced green fluorescent protein was damaged in vitro by genotoxic agents and introduced into cells by transfection. The repair capacity of the cells was determined from the number of fluorescent cells counted with a fluorescent microscope 24 h after transfection. The ability of four human tumor cell lines—HEK293, HeLa, Namalwa and K562 to repair DNA lesions inflicted by cis-diamminedichloroplatinum(II), UV light, 8-methoxypsoralen and 4′,5′-8-trimethylpsoralen were determined and compared to the survival rates of the cells after treatment with the same genotoxic agents. In most but not all cases, there was a good correlation between repair capacity and cell survival. This finding indicates that the DNA repair capacity could be used as a biomarker in risk assessment and/or drug resistance assays.