Mismatch repair-mediated G2/M arrest by 6-thioguanine involves the ATR-Chk1 pathway

DNA mismatch repair (MMR) deficiency in human cancers is associated with resistance to a spectrum of clinically active chemotherapy drugs, including 6-thioguanine (6-TG). We and others have shown that 6-TG-induced DNA mismatches result in a prolonged G2/M cell cycle arrest followed by apoptosis in MMR(+) human cancer cells, although the signaling pathways are not clearly understood. In this study, we found that prolonged (up to 4 days) treatment with 6-TG (3microM) resulted in a progressive phosphorylation of Chk1 and Chk2 in MMR(+) HeLa cells, correlating temporally with a drug-induced G2/M arrest. Transfection of HeLa cells with small interfering RNA (siRNA) against the ataxia telangiectasia-related (ATR) kinase or against the Chk1 kinase destroyed the G2/M checkpoint and enhanced the apoptosis following 6-TG treatment. On the other hand, the induction of a G2/M population by 6-TG was similar in ATM(-/-) and ATM(+) human fibroblasts, suggesting that the ATM-Chk2 pathway does not play a major role in this 6-TG response. Our results indicate that 6-TG DNA mismatches activate the ATR-Chk1 pathway in the MMR(+) cells, resulting in a G2/M checkpoint response     

Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro

Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.     

Effects of corticosteroid-induced apoptosis on airway epithelial wound closure in vitro

Damage to the airway epithelium is common in asthma. Corticosteroids induce apoptosis in and suppress proliferation of airway epithelial cells in culture. Whether apoptosis contributes to impaired epithelial cell repair after injury is not known. We examined whether corticosteroids would impair epithelial cell migration in an in vitro model of wound closure. Wounds (approximately 0.5-1.3 mm2) were created in cultured 1HAEo- human airway epithelial cell monolayers, after which cells were treated with up to 10 microM dexamethasone or budesonide for 24 h. Cultured cells were pretreated for 24 or 48 h with dexamethasone to observe the effect of long-term exposure on wound closure. After 12 h, the remaining wound area in monolayers pretreated for 48 h with 10 microM dexamethasone was 43+/-18% vs. 10+/-8% for untreated control monolayers. The addition of either corticosteroid immediately after injury did not slow closure significantly. After 12 h the remaining wound area in monolayers treated with 10 microM budesonide was 39+/-4% vs. 43+/-3% for untreated control monolayers. The proportion of apoptotic epithelial cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling both at and away from the wound edge was higher in monolayers treated with budesonide compared with controls. However, wound closure in the apoptosis-resistant 1HAEo-.Bcl-2+ cell line was not different after dexamethasone treatment. We demonstrate that corticosteroid treatment before mechanical wounding impairs airway epithelial cell migration. The addition of corticosteroids after injury does not slow migration, despite their ability to induce apoptosis in these cells.     

Construction of a defective retrovirus containing the human hypoxanthine phosphoribosyltransferase cDNA and its expression in cultured cells and mouse bone marrow.

Defective ecotropic and amphotropic retroviral vectors containing the cDNA for human hypoxanthine phosphoribosyltransferase (HPRT) were developed for efficient gene transfer and high-level cellular expression of HPRT. Helper cell clones which produced a high viral titer were generated by a simplified method which minimizes cell culture. We used the pZIP-NeoSV(X) vector containing a human hprt cDNA. Viral titers (1 X 10(3) to 5 X 10(4)/ml) of defective SVX HPRT B, a vector containing both the hprt and neo genes, were increased 3- to 10-fold by cocultivation of the ecotropic psi 2 and amphotropic PA-12 helper cells. Higher viral titers (8 X 10(5) to 7.5 X 10(6] were obtained when nonproducer NIH 3T3 cells or psi 2 cells carrying a single copy of SVX HPRT B were either transfected or infected by Moloney leukemia virus. The SVX HPRT B defective virus partially corrected the HPRT deficiency (4 to 56% of normal) of cultured rodent and human Lesch-Nyhan cells. However, instability of HPRT expression was detected in several infected clones. In these unstable variants, both retention and loss of the SVX HPRT B sequences were observed. In the former category, cells which became HPRT- (6-thioguanine resistant [6TGr]) also became G418s, indicative of a cis-acting down regulation of expression. Both hypoxanthine-aminopterin-thymidine resistance (HATr) and G418r could be regained by counterselection in hypoxanthine-aminopterin-thymidine. In vitro mouse bone marrow experiments indicated low-level expression of the neo gene in in vitro CFU assays. Individual CFU were isolated and pooled, and the human hprt gene was shown to be expressed. These studies demonstrated the applicability of vectors like SVX HPRT B for high-titer production of defective retroviruses required for hematopoietic gene transfer and expression.     

The histone deacetylase inhibitor trichostatin A reduces nickel-induced gene silencing in yeast and mammalian cells

We have previously reported that nickel (Ni)-silenced expression of the URA3 gene in yeast (Saccharomyces cerevisiae) and gpt transgene in G12 Chinese hamster cells. In both cases, close proximity to a heterochromatic region was required for gene silencing. Yeast exposed to Ni exhibited reduced acetylation of the lysine residues in the N-terminal tail of histone H4. Ni-induced silencing of the gpt gene in mammalian cells involved hypermethylation of promoter region DNA. Yeast do not employ DNA methylation to silence gene expression. To determine if histone deacetylation participates in Ni-induced silencing of the URA3 and gpt genes, we exposed yeast and G12 hamster cells to the histone deacetylase inhibitor trichostatin A (TSA) prior to and concurrently with Ni. Treatment of yeast cells with 0.2-0.6mM NiCl(2) resulted in reduced expression of the URA3 gene as assessed by increased resistance to 1g/l 5-fluorotic acid (5-FOA). This effect was lessened when yeast were pre-treated with 50 microg TSA/ml. Similarly, treatment of G12 cells with 5 ng/ml TSA during and after exposure to 0.3 microg Ni(3)S(2)/cm(2) reduced silencing of the gpt gene as gauged by resistance to 10 microg/ml 6-thioguanine (6-TG). The ability of TSA alone and in combination with the DNA-demethylating agent (5-AzaC) to reactivate the gpt gene in Ni-silenced variants was also assessed. Although treatment with 100 ng/ml TSA for 48 h was partially effective in reactivating the gpt gene, treatment with 5 microM 5-AzaC was more efficacious. The greatest gpt gene reversion frequencies were observed following a sequential 5-AzaC/TSA treatment. Taken all together, our data from mammalian cells suggests that both DNA methylation and histone deacetylation participate in Ni-induced silencing of the gpt gene with DNA hypermethylation playing the more dominant role in maintaining the silenced state.     

用人DNA介导的基因转移修复中国仓鼠细胞的HPRT缺陷

 中国仓鼠卵巢细胞(CHO-K1)经N-甲基-N'-硝基-N-亚硝基胍(MNNG)诱变和6-巯基鸟嘌呤(6-TG)选择,得到稳定的次黄嘌呤磷酸核糖转移酶(HPRT)缺陷细胞株,酶活性仅为野生型的6.5％。用磷酸钙共沉淀法和电脉冲法向HPRT-细胞转移人宫颈癌细胞(HeLaS_3)基因组DNA,纠正了CHO细胞的HPRT缺陷。酶活性提高了6.9倍,达到野生型的45％。用Alu序列探针进行分子杂交,证实经过基因转移并连续传代15次以上的受体细胞中含人DNA序列。表明人的有关基因已稳定地整合到CHO细胞的染色体中。     

Biochemical and morphological modifications in dexamethasone-treated mouse bone marrow-derived mast cells

Addition of 1 microM dexamethasone (DM) to bone marrow-derived mast cells (BMMC) induced a time-dependent increase in cell histamine content. The latter reached a plateau of 2.5 micrograms/1 x 10(6) cells after 11 days in culture, compared with 100 ng/1 x 10(6) for untreated BMMC. Steroids, such as beta-estradiol, androsterone, and testosterone (1 microM), did not alter the histamine content of BMMC, whereas progesterone (1 microM) induced a moderate increase. Other glucocorticosteroids also enhanced histamine content, suggesting that the observed increase was specific for glucocorticosteroid. Treatment of BMMC with 1 microM DM for 14 days inhibited the Ag-induced, IgE-mediated release of histamine, beta-hexosaminidase, platelet-activating factor-acether, LTB4, and LTC4 by 65 +/- 3%, 66 +/- 1%, 93 +/- 3%, 66 +/- 2%, and 74 +/- 10%, respectively (mean +/- 1 SD, n = 3). In contrast with untreated cells which produce less than 2 ng/1 x 10(6) cells PGD2 after Ag challenge, DM-treated BMMC generated 16.8 +/- 0.3 ng/1 x 10(6) cells PGD2. Moreover, most of DM-treated BMMC became Alcian blue+/safranin+ and by ultrastructure, exhibited numerous cytoplasmic granules filled with abundant and uniform electron-dense matrix. The present results indicate that DM-treated BMMC exhibit biochemical and functional properties different from immature untreated cells, suggesting that a maturation-like process occurred in vitro during DM treatment.     

Thermal sensitivity and radiosensitization in Chinese hamster V79 cells exposed to 2-aminopurine or 6-thioguanine

Chinese hamster V79 cells were exposed to 10(-6) mol liter-1 2-aminopurine (2-AP) or 6-thioguanine (6-TG) for 18 or 40 h, and then tested for sensitivity to X rays, heat, or a combined treatment of heat and radiation. Cells exposed to 6-TG were sensitive to X rays, while those treated with 2-AP showed little or no sensitivity. At 42 degrees and 45 degrees C moderate sensitization resulted from 2-AP treatment, with greater sensitization resulting from treatment with 6-TG. Combined heat and X-ray treatment of cells exposed to 2-AP yielded sensitization similar to heat treatment alone, while cells exposed to 6-TG before receiving the combined treatment showed a degree of sensitization greater than that due to either treatment by itself but less than that of the two treatments added together. Uptake of the purine analogues into cellular DNA was measured by high-pressure liquid chromatography. At the 1% detection level, after either an 18- or a 40-h exposure of cells to 10(-6) mol liter-1 2-AP or 6-TG, no base substitution was found. Analysis of cell cycle distributions by flow cytometry revealed only very small changes following exposure of cells to the purine analogues.     

Thermosensitization by methylglyoxal bis(guanylhydrazone) in Chinese hamster cells

The modification of methylglyoxal bis(guanylhydrazone) (MGBG) by 42 degrees C hyperthermia-and/or radiation-induced cell killing was examined in Chinese hamster V-79 cells. At concentrations of more than 10 microM, cell survival decreased exponentially with increased MGBG exposure times. Cell lethality of MGBG (10 microM) was not specific for cell-cycle phases tested from G1/S through G2. When cells were treated with MGBG (10 microM) for 6 hr and then exposed to 42 degrees C hyperthermia with or without a 24-hr interval, cell survival decreased markedly compared with that for 42 degrees C alone. Cells became thermosensitive after MGBG treatment. Cells exposed to MGBG (10 microM) for 6 hr before or after X irradiation were slightly radiosensitive. When X irradiation was combined with MGBG and 42 degrees C hyperthermia, cells became more radiosensitive. From these results, it is suggested that MGBG may change the intracellular state to sensitize cells to the cytotoxic action(s) of hyperthermia.     

A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C

In order to create a novel in vitro test system for detection of large deletions and point mutations, we developed an immortalized cell line. A SV40 large T antigen expression unit was introduced into fibroblasts derived from gpt delta mouse lung tissue and a selected clone was established as the gpt delta L1 (GDL1) cell line. The novel GDL1 cells were examined for mutant frequencies (MFs) and for molecular characterization of mutations induced by mitomycin C (MMC). The GDL1 cells were treated with MMC at doses of 0.025, 0.05, and 0.1 microg/mL for 24h and mutations were detected by Spi- and 6-thioguanine (6-TG) selections. The MFs of the MMC-treated cells increased up to 3.4-fold with Spi- selection and 3.5-fold with 6-TG selection compared to MFs of untreated cells. In the Spi- mutants, the number of large (up to 76 kilo base pair (kbp)) deletion mutations increased. A majority of the large deletion mutations had 1-4 base pairs (bp) of microhomology in the deletion junctions. A number of the rearranged deletion mutations were accompanied with deletions and insertions of up to 1.1 kbp. In the gpt mutants obtained from 6-TG selection, single base substitutions of G:C to T:A, tandem base substitutions occurring at the 5'-GG-3' or 5'-CG-3' sequence, and deletion mutations larger than 2 bp were increased. We compared the spectrum of MMC-induced mutations observed in vitro to that of in vivo using gpt delta mice, which we reported previously. Although a slight difference was observed in MMC-induced mutation spectra between in vitro and in vivo, the mutations detected in vitro included all of the types of mutations observed in vivo. The present study demonstrates that the newly established GDL1 cell line is a useful tool to detect and analyze various mutations including large deletions in mammalian cells.     

Microcalorimetric evaluation of the effects of methotrexate and 6-thioguanine on sensitive T-lymphoma cells and on a methotrexate-resistant subline.

Isothermal microcalorimetry was used in order to continuously monitor and quantitatively assess the action of two antineoplastic drugs, methotrexate (MTX) and 6-thioguanine (6-TG), on a human T-lymphoma cell line, CCRF-CEM. The results with MTX were compared with data from experiments with a MTX-resistant subline, CEM/MTX. The slope of the power-time curve after drug injection relative to that obtained during unperturbed growth, was used to construct dose-response curves. The normal cell line was characterized by a D50 value (i.e., the dose producing half the maximal response) of 0.05 microM for MTX and 0.38 microM for 6-TG. For the MTX-resistant subline the D50 value was 8 microM of MTX. Comparisons of the continuous power-time curves showed the inhibitory effect of 6-TG to be faster than that of MTX.     

Investigation of coelectroporation as a method for introducing small mutations into embryonic stem cells.

We have investigated coelectroporation as a method for introducing minor genetic changes into specific genes in embryonic stem cells. A selectable marker (neo) and a targeting replacement vector designed to insert a 4-bp insertion into exon 3 of the mouse hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene were coelectroporated into embryonic stem cells and selected in G418 and 6-thioguanine (6-TG). HPRT-negative clones were obtained at a frequency of approximately 1 per 520 G418r clones. Southern analysis and the polymerase chain reaction were used to demonstrate that 3 of 36 of the 6-TG-resistant clones had the desired 4-bp insertion without any other disruption of the HPRT locus. Initial studies indicated that the other 33 6-TG-resistant clones probably resulted from the targeted integration of a concatemer containing both the targeting construct and the selectable neo gene.     

Effects of NaCN and ionomycin on neuronal viability and on the abundance of microtubule-associated proteins MAP1, MAP2, and tau in isolated chick cortical neurons

The effects of two toxins, sodium cyanide (NaCN) and ionomycin (IM), on neuronal viability and on the expression of the microtubule-associated proteins MAP1, MAP2, and tau were studied in isolated chick cortical neurons. Cytotoxic hypoxia due to NaCN treatment was performed to mimic acute neuronal damage, whereas long-term IM treatment was used as a model for chronic neuronal impairment. After 5 days in vitro, a cytotoxic lesion was induced either by addition of NaCN (0.01-10 mM) or IM (0.01-10 microM). The NaCN solution was aspirated after 30 min and cells were allowed to regenerate for 6 h, 24 h, 48 h, or 72 h; whereas the permanent IM lesions were left undisturbed during the same periods of time. Neuronal viability was assessed by MTT assay. The abundance of MAP1, MAP2, and tau was evaluated by immunoblotting and, for MAP2, by immunohistochemistry also. Results showed that NaCN and IM lesions dose-dependently decreased viability. Irreversible cell damage occurred after impairment with 10 mM NaCN and 1 microm or 10 microm IM, while neurons lesioned with lower concentrations regenerated partially or adapted to the toxic environment. However, the same level of viability as of untreated cells was never reached. Furthermore abundance of MAPs was changed after both lesions. But while after extended recovery from NaCN lesion protein expression was normalizing (MAP2) or at least still detectable (MAP1A, tau), the consequences of a permanent IM lesion were more severe, since neurons were not able to maintain or even restore their MAP expression. Immunohistochemical experiments for MAP2 revealed that, compared with controls, NaCN and, to a much higher extent, IM treatment resulted in a loss of immunoreactivity in neurites due to progressing cell death.     

Activation of a nonexpressed hypoxanthine phosphoribosyltransferase allele in mutant H23 HeLa cells by agents that inhibit DNA methylation.

HeLA H23 cells are a mutant female human tumor cell line harboring defective hypoxanthine phosphoribosyltransferase (HPRT; IMP-pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) as a result of a mutation that alters the isoelectric point of the enzyme (G. Milman, E. Lee, G. S. Changas, J. R. McLaughlin, and J. George, Jr., Proc. Natl. Acad. Sci. USA 73:4589-4592, 1976). As shown by Milman et al. and confirmed by us here, rare HAT+ revertants arise spontaneously at 1.9 X 10(-8) frequency and express both mutant and wild-type polypeptides. Thus, the H23 mutant also carries a silent wild-type HPRT allele that is activated in revertants. To test whether the silent allele was activated via hypomethylation of genomic DNA, H23 cells were treated with inhibitors of DNA methylation, and revertants were scored by HAT or azaserine selection. At an optimal dose of 5 microM 5-azacytidine, the reversion frequency was increased about 50-fold when assayed by HAT selection and over 1,000-fold when assayed by azaserine selection. HAT+ and azaserine revertants were heterozygous for HPRT, expressing both wild-type and mutant HPRT polypeptides. Like spontaneous revertants, they contained active HPRT enzyme and were genetically unstable, reverting at about 10(-4) frequency. Similar results were found after treatment with N-methyl-N'-nitro-N-nitrosoguanidine, a DNA-alkylating agent and potent inhibitor of mammalian DNA methylation. By contrast, the DNA-ethylating agent, ethyl methanesulfonate (EMS), did not increase the HAT+ reversion frequency; it did, however, increase the frequency by which H23 revertants heterozygous for HPRT reverted to 6-thioguanine resistance. Of nine EMS revertants, seven lacked HPRT activity and had a substantially reduced expression of the wild-type polypeptide. These observations support the hypothesis that DNA methylation plays an important role in human X-chromosome inactivation and that EMS can inactivate gene expression by promoting enzymatic methylation of genomic DNA as found previously for the prolactin gene in GH3 rat pituitary tumor cells (R. D. Ivarie and J. A. Morris, Proc. Natl. Acad. Sci. USA 79:2967-2970, 1982; R. D. Ivarie, J. A. Morris, and J. A. Martial, Mol. Cell. Biol. 2:179-189, 1982).     

The effects of inhibitors of DNA biosynthesis on the cytotoxicity of 6-thioguanine

The effects of several metabolic inhibitors of DNA synthesis on the antiproliferative activity of 6-thioguanine (6-TG) were examined using cultured L1210 leukemia cels. The presence of hydroxyurea (HU), 1-beta-D-arabinofuranosylcytosine (araC), or 5-fluorodeoxyuridine (FUdR) in cultures of L1210 leukemic cells during exposure of 6-TG did not increase the degree of inhibition of cellular replication produced by the 6-thiopurine, but instead partially protected cells against the delayed cytotoxicity of 6-TG, implying that DNA replication was essential for the expression of cytotoxicity by the purine antimetabolite. Consistent with these results was the finding that synchronized L1210 cells exposed to 6-TG were the most susceptible to the cytotoxic action of the 6-thiopurine during G1/S and S phase. However, G2 phase cells were also sensitive to 6-TG indicating that at least two metabolic lesions are responsible for the production of cytotoxicity. Alkaline sucrose gradient sedimentation of L1210 cells exposed to 6-TG revealed that the purine analog causes structural changes in DNA suggesting that these hitherto unreported lesions may be involved in the cytotoxicity caused by this agent.     

Leishmania donovani: role of CD2 on CD4+ T-cell function in Visceral Leishmaniasis

In this study, we investigated whether alteration in the CD2 mediated coordination of an immune response was associated with down regulation of CD4 associated Th1 cell response during Visceral Leishmaniasis (VL). Leishmania donovani (Ld) infection in VL patients markedly reduced expression of CD2 cell surface antigen on CD4+ cells. T-cells of VL patients were mostly in G0/G1 stage of the cell cycle (98.20%) with little or no activity of protein kinase C-alpha (PKC-alpha) isoform. However, pre-incubation with activating anti-CD2 monoclonal antibody (MAb) resulted in a corresponding increase up to 2.52-fold in T-cells of G2/M population supported by both activity and expression of PKC-alpha isoform. Furthermore, we observed that co-incubation of T-cell with anti-CD2 increased the lymphocyte-blast population in patients in whom the CD4 cells became more antigen responsive (CD4+ CD69+ cells). Consistent with these observations, it was shown that 59.3% of CD4 cells from patients responded to Ld by producing IFN-gamma. Even in the culture condition, when the T-cells from patients were depleted of APC, IFN-gamma production was noticed after CD2 activation. On the other hand, IL-4 production became low in the anti-CD2 antibody supplemented peripheral blood mononuclear cells (PBMNCs) culture. These findings imply that infection with L. donovani induces less CD2 on the surface of CD4+ T-cells, which once activated orchestrate the protective IFN-gamma dominant host defense mechanism via PKC-mediated signal transduction and cell cycle.     

Genotoxicity of 3'-azido-3'-deoxythymidine in the human lymphoblastoid cell line, TK6: relationships between DNA incorporation, mutant frequency, and spectrum of deletion mutations in HPRT.

Perinatal treatment with 3'-azido-3'-deoxythymidine (AZT) has been found to reduce the rate of maternal-infant transmission of HIV; however, AZT is genotoxic in mammalian cells in vitro and induces tumors in the offspring of mice treated in utero. The purpose of the present study was to investigate the relationships between incorporation of AZT into DNA, and the frequency and spectrum of mutations at the HPRT locus of the human lymphoblastoid cell line, TK6, following in vitro exposures to AZT. Cells were cultured in medium containing 0 or 300 microM AZT for 1, 3, or 6 day(s) (n = 5/group). The effects of exposure duration on incorporation of AZT into DNA and HPRT mutant frequency were determined using an AZT radioimmunoassay and a cell cloning assay, respectively. AZT accumulated in DNA in a supralinear manner, approaching a plateau at 6 days of treatment (101.9 +/- 14.7 molecules AZT/10(6) nucleotides). After 3 days of AZT exposure, HPRT mutant frequency was significantly increased (1.8-fold, p = 0.016) compared to background (mutant frequency = 3.78 x 10(-6)). Multiplex PCR amplification of genomic DNA was used to determine the frequency of exon deletions in HPRT mutant clones from untreated cells versus AZT-treated cells. Molecular analyses of AZT-induced mutations revealed a significant difference in the frequency of total gene deletions (44/120 vs. 18/114 in controls, p = 0.004 by the Mann-Whitney U-statistic). In fact, the Chi-square test of homogeneity demonstrate that the differences between the control and AZT-treatment groups is attributed mainly to this increase in total gene deletion mutations (p = 0.00001). These data indicate that the primary mechanism of AZT mutagenicity in human TK6 cells is through the production of large deletions which occur as a result of AZT incorporation into DNA and subsequent chain termination. The data imply that perinatal chemoprophylaxis with AZT may put children of HIV-infected women at potential risk for genetic damage.     

Differing responses of G2-related genes during differentiation of HL60 cells induced by TPA or DMSO

Differentiation leads to the cessation of cellular proliferation, but little is known about the molecular mechanisms of growth arrest. We compared the effect of two differentiation inducers, 12-o-tetradecanoyl 13-acetate (TPA) and dimethyl sulfoxide (DMSO) on both the cell-cycle and the modulation of G2-related genes in synchronized HL60 cells. TPA treatment of HL60 cells resulted in G1 arrest within 24 h. In contrast, the cell cycling of DMSO-treated cells was initially accelerated and they progressed to the second cycle before accumulating in the G1 phase. Expression of cyclin B, cdc25, wee1 and cdc2 was studied during cell cycle arrest by Northern blot hybridization. Expression of cyclin B, cdc25 and cdc2 fluctuated in association with cell cycle progression towards the G2/M phase, while wee1 expression remained constant in untreated cells. These four genes were highly expressed in TPA-treated cells for the first 12 h, but drastic down-regulation was seen at 18 h and expression became undetectable after 24 h. In contrast, no remarked changes of gene expression were seen in DMSO-treated cells. These findings suggest that cell cycle progression along with the initial process of differentiation in response to TPA differs from the response to DMSO and that the down-regulation of cdc2 expression by TPA-treated HL60 cells contributes to endorsement of G1 arrest.