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S M D'Ambrosio

Publications and source records attributed to S M D'Ambrosio.

At least 19 recordsLinked to original sources
PubMed2001

Mechanisms of nitric oxide-induced cytotoxicity in normal human hepatocytes.

Chronic exposure of hepatocytes to reactive nitrogen species (RNS) following liver injury and inflammation leads not only to functional and morphological alterations in the liver but also to degenerative liver diseases and hepatocellular carcinoma. Previously, we showed that S-nitroso-N-acetylpenicillamine-amine (SNAP), which generates nitric oxide, and 3-morpholinosydnonimine (Sin-1), which generates equal molar concentrations of superoxide and nitric oxide resulting in peroxynitrite production, exhibited different levels of cytotoxicity to normal human hepatocytes in culture. The aim of the present study was to elucidate some of the molecular and cellular pathways leading to hepatocyte cell death induced by RNS. Following treatment of the hepatocytes with SNAP or Sin-1, gene-specific DNA damage was measured in mtDNA and a hprt gene fragment using a quantitative Southern blot analysis. Both agents induced dose-dependent increases in DNA damage that was alkaline labile, but not sensitive to both formamidopyrimidine-DNA glycosylase (fpg) and endonuclease III, which recognize 8-oxoguanine, thymine glycol, and other oxidized pyrimidines. DNA damage was two- to fivefold greater in mtDNA than in the hprt gene fragment. There was a persistent and marked increase in DNA damage posttreatment that appeared to arise from the disruption of electron transport in the mitochondria, generating reactive species that saturated the repair system. DNA damage induced by Sin-1 and SNAP led to cell-cycle arrest in the S-phase, growth inhibition, and apoptosis. The data support the hypothesis that the functional and morphological changes observed in liver following chronic exposure to RNS are, in part, the result of persistent mitochondrial and nuclear DNA damage.

Adult↗
PubMed2001

Protection of human keratinocyte mtDNA by low-level nitric oxide.

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

Base Sequence↗
PubMed1998

Enhanced expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase gene in human breast tumor cells.

The expression of the 8-oxo-7,8-dihydrodeoxyguanosine triphosphatase (8-oxo-dGTPase) gene in human breast tumors was evaluated at the level of the single cell to better understand how breast tumor cells regulate expression in response to oxidative stress. Compared to normal breast ductal cells, the level of 8-oxo-dGTPase expression in the breast tumor cells increased from non-detectable levels in normal breast to expression in 30-85% of the tumor cells in individual tumors. There was no significant association between 8-oxo-dGTPase expression and tumor grade and metastatic malignancy. The upregulation of 8-oxo-dGTPase was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53, Ki-67 and c-erbB-2, which are genes involved in cell cycle regulation and tumor growth. The elevated expression of 8-oxo-dGTPase in human breast ductal carcinoma cells appears to be a general characteristic of breast tumors and may provide the tumor cell with a cellular defense mechanism to prevent the incorporation of 8-hydroxy-deoxyguanosine during DNA replication.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1997

Sensitivity of human hepatocytes in culture to reactive nitrogen intermediates.

The cytotoxic effects of 3-morpholinosydnonimine (Sin-1) and S-nitroso-N-acetylpenicillamine-amine (SNAP) on replicatively active human hepatocyte cells in culture was determined as a function of oxidant type. Both Sin-1 which yields nitric oxide and peroxynitrite following the generation of superoxide anion plus nitric oxide, and SNAP which generates nitric oxide, induced dose dependent decreases in the colony forming capabilities of the human hepatocytes. Sin-1 was much more cytotoxic (LD50 = 400 microM) than SNAP (LD50 = 1250 microM). Comparatively, both compounds were much less cytotoxic than H2O2 (LD50 = 96 microM). Sin-1 induced 4-fold higher levels of cellular nitrite than that generated by the chemical in cell free medium. Nitrotyrosine, a marker of peroxynitrite formation in cells, was immunohistochemically detected in hepatocytes treated with both Sin-1 and SNAP. The formation of 3-nitrotyrosine by hepatocytes incubated with SNAP, suggests that hepatocytes generate intracellular superoxide which reacts with the exogenous nitric oxide derived from SNAP to produce intracellular peroxynitrite, resulting in the SNAP cytotoxicity. The enhanced levels of Sin-1 cytotoxicity on the hepatocytes is suggested to be due both to the chemical generation of peroxynitrite and superoxide anion by Sin-1. These data indicate that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication, and that peroxynitirite may play a significant role in the pathogenesis of liver disease.

Cells, Cultured↗
PubMed1997

Mechanism of enhancement of esophageal tumorigenesis by 6-phenylhexyl isothiocyanate.

6-Phenylhexyl isothiocyanate (PHITC) enhances esophageal tumorigenesis induced by the carcinogen N-nitrosomethylbenzylamine (NMBA) in rats while its shorter chain analog, phenethyl isothiocyanate (PEITC), inhibits NMBA-induced esophageal tumorigenesis. A significant increase in O6-methylguanine levels in esophageal DNA at 72 h after NMBA administration to rats pretreated with PHITC suggested that PHITC might enhance NMBA metabolic activation or inhibit DNA repair. To test this hypothesis, groups of 20 rats were administered PEITC or PHITC at concentrations of 0, 1.0, or 2.5 mmol/kg in modified AIN-76A diet for 2 weeks. The esophagi were removed from rats, stripped, split, and maintained in HEPES buffered saline (HBS) for assays of NMBA metabolism (n = 5 per group) or were snap frozen for DNA repair assays (n = 15 per group). The principal metabolites of NMBA produced by esophageal explants were: two unidentified peaks, benzyl alcohol (at 4 h only), and benzoic acid. Esophageal explants from PEITC-treated animals showed a significantly decreased ability to metabolize NMBA as expected. PHITC-treated animals showed a slight inhibition in the formation of most NMBA-related metabolites, rather than an overall increase in NMBA activation. This inhibition was less than that observed with PEITC. No inhibitory effects were observed on O6-alkylguanine transferase (AGT) activity in the esophagi of rats treated with 1.0 micromol/g or 2.5 micromol/g PHITC. Thus, effects of PHITC on esophageal metabolism and DNA repair do not account for the enhancement of NMBA tumorigenicity by PHITC.

Animals↗
PubMed1997

Molecular characterization of mutations in the hprt gene of normal human skin keratinocytes treated with N-ethyl-N-nitrosourea: influence of O6-alkylguanine alkyltransferase.

O6-Alkylguanine-DNA alkyltransferase (AGT) is responsible for repairing the O6-alkylguanine lesion in DNA. There is wide variation in the levels of AGT between organ and cell types, which appears to correlate with cell and tissue type sensitivity to the mutagenic and carcinogenic effects of alkylating agents. In order to investigate the role of AGT in modulating the frequency and types of mutations induced in one type of normal human parenchymal cells, we examined the types and frequency of mutations in the hypoxanthine (guanine) phosphoribosyltransferase (hprt gene in 116 mutants derived from two N-ethyl-N-nitrosourea (ENU)-treated normal human skin keratinocyte cell lines. O6-Benzylguanine (O6-BZ; 5 microM x 2 hours) was used to specifically inhibit AGT activity before ENU treatment (0 to 5 mM x 1 hour). O6-BZ increased both the cytotoxic and mutagenic effects of ENU by 1.8- and 3- to 5-fold, respectively. In both treatment groups, most of the mutations were base substitutions (72%). The proportion of GC to AT transitions in the O6-BZ group (14/31) was twice that in the group treated with ENU alone, consistent with the loss of AGT activity in these cells. There was no strand specificity of GC to AT and AT to GC transitions in both groups. Base transversions accounted for 28% of total base substitutions. A lower than expected proportion of AT to TA transversions were observed in both cell lines, which decreased in the O6-BZ pretreated group. A strand bias was observed for GC to TA and AT to TA transversions. Most of the G to A and G to T base substitutions had one or more purines flanking 3' to the mutated deoxyguanosines. There were more deletion mutants with the deletion of exon 1, 4, 6, and 8 in the BZ group than in the control group. These data, characterizing the mutational spectra of ENU in normal human keratinocytes treated in vitro, indicate that GC to AT and AT to GC transition mutations predominate in these cells depleted or not depleted of AGT.

Cell Line↗
PubMed1997

Expression of molecular biomarkers in primary breast tumors implanted into a surrogate host: increased levels of cyclins correlate with tumor progression.

BACKGROUND: The overexpression or amplification of tumor suppressor and proto-oncogenes are important factors in the progression of breast cancer. Recent attention has focused on the cyclin genes, whose involvement in signal transduction pathways regulate cell cycle progression. The amplification of the cyclins D1 and D3 genes usually leads to loss of normal growth control and is thought to play an important growth regulatory role in tumor development and progression. In this report, we investigate the association of altered cyclin expression with other prognostic indicators (histological grade, lymph node status, estrogen receptor, p53, and c-erbB-2) in the progression of human breast cancer. MATERIALS AND METHODS: Surgical tumor specimens were obtained from 16 breast tubular ductal, and invasive ductal carcinomas and grafted onto gnotobiotic nude (nu/nu) mice. The expression diversity and distribution of the localization of the protein products of the c-erbB-2, cyclins D1 and D3, p53, and estrogen receptor were characterized immunohistochemically and the results in the original tumor (T0) were compared with those in the tumors that developed in nude mice (T1) xenografts. RESULTS: The T0 tumors exhibited a diversity of cellular morphology in the tumor matrix and diversity in expression of these proteins. These specific changes were also preserved in the T1 tumors. Whereas 67% of the T1 tumors exhibited high numbers of estrogen receptorpositive nuclei, only 50% of these tumors grew when grafted onto nude mice. The histological grade (14/15 were G2 to G3) and metastatic malignancy in the lymph nodes (10/15) did not appear to be related to tumor growth in the nude mouse. There was no relationship between those tumors which exhibited high percentages of c-erbB-2- and p53-positive cells and growth in nude mice. A strong association (p < 0.001) was observed between the overexpression of cyclin D1 transcripts in the T0 tumors and the continued growth of the T1 tumors in nude mice. In the T1 tumors, both cyclins D1 and D3, estrogen receptor, and p53 were observed in 49% to 86% of the cells of the T1 tumors examined; the number of cells expressing c-erbB-2 protein varied widely in these tumors. CONCLUSIONS: The results indicate that the tumor matrix exhibits a diversity in the level of phenotypic expression of genes involved in cellular growth of breast tumors in both the T0 or T1 host environment. Changes in cyclin activity appear to correlate with the vigorous level of breast tumor growth and progression.

Adult↗
PubMed1995

Differential expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human breast and skin tissue: in situ mapping of cell type-specific expression.

O6-Alkylguanine is a major toxic, mutagenic, and carcinogenic lesion in cellular DNA that is repaired by O6-alkylguanine-DNA alkyltransferase (ATase). The expression of this gene is directly related to the cellular sensitivity of alkylating agents, with levels of this protein varying widely among human organs, tumors, and cell types. To better understand specific cell-type responses to repairing O6-alkylguanine lesions in DNA, we used colorimetric in situ hybridization, with an ATase-specific antisense oligomer probe, to map the cellular distribution of ATase mRNA in tissue sections of normal adult human breast and neonatal foreskin tissues. This is the first report of mapping ATase gene expression directly in normal human breast and skin tissues. Paraffin-embedded tissue sections were hybridized with a digoxigenin-labeled, 39-base antisense ATase oligomer. Hybridization of the probe to cells expressing the ATase gene was visualized after immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. After color development, we simultaneously identified tissue architecture and cell types and measured the expression of the ATase gene. There was no hybridization-specific color when sections were mock hybridized, hybridized with a sense probe, or treated with RNase. In the breast tissue, 93% of the cells in the loosely connective tissue and 84% of the myoepithelial cells expressed high levels of ATase mRNA. Most of the luminal ductal epithelial cells (61%) were devoid of stain, indicating undetectable levels of ATase mRNA. In skin dermis, 93% of the fibroblasts appeared to express high levels of ATase mRNA. Within the epidermis, approximately 64% of the basal and 65% of the granular epithelial cells expressed ATase mRNA. Expression was undetectable in the epithelial cells of the suprabasal layer of the epidermis. There was very little interindividual variation (< 17%) in the distribution of expression of ATase within the same cell types of different individuals. These data illustrate the differential potential of individual cell types within the organ matrix to repair O6-alkylguanine damage in cellular DNA. This data may provide insights into the understanding of cell type-specific responses to carcinogens.

Breast↗
PubMed1995

Cell type-specific expression of human 8-oxo-7,8-dihydroguanosine triphosphatase in normal breast and skin tissues in vivo.

8-Oxo-7,8-dihydroguanosine triphosphate (8-oxo-dGTP) is formed from the oxidation of GTP in the nucleotide pools of cells during normal cellular metabolism and from exogenous sources. 8-Oxo-dGTP is a potent mutagenic substrate for DNA synthesis causing transversion mutations. In human cells this oxidized base is hydrolyzed to 8-oxo-7,8-dihydroguanosine monophosphate by 8-oxo-7,8-dihydroguanosine triphosphatase (8-oxo-dGTPase) to prevent the misincorporation of 8-oxo-dGTP into cellular DNA. In order to better understand specific human tissue and cell type responses to oxidative stress, we used colorimetric in situ hybridization, with an 8-oxo-dGTPase-specific antisense oligomer probe, to map, for the first time, the cellular distribution of 8-oxo-dGTPase mRNA in tissue sections of normal neonatal foreskin and adult human breast tissues. Paraffin embedded tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to 8-oxo-dGTPase cDNA. Hybridization of the probe to cells expressing the 8-oxo-dGTPase gene was visualized following immunodetection with an alkaline phosphatase-conjugated anti-digoxigenin antibody. Following color development, we were able to simultaneously identify tissue architecture and cell types with expression of the 8-oxo-dGTPase gene. There was no hybridization-specific color when sections were 'mock' hybridized, hybridized with a sense probe or treated with RNase. In skin dermis, fibroblasts express high levels of 8-oxo-dGTPAse mRNA. Within the epidermis, a gradient of expression was observed, from high to moderate levels in the replicating basal epithelial cells to undetectable in the non-mitotic suprabasal and granular epithelial cells. In the breast tissue, fibroblasts in the loosely connective tissue and myoepithelial cells expressed high levels of 8-oxo-dGTPase mRNA, while expression in the luminal epithelial cells was not detectable. Our data suggest that expression of 8-oxo-dGTP is heterogenous between cell types within an organ and may help to explain cell type-specific responses to oxidative stress, especially in replicating and potentially replicating cells with low levels of this protective protein.

Adult↗
PubMed1995

Identification and isolation of human epithelial cell colonies that express specific gene products.

A new and simple method for the identification and isolation of human cell clones expressing specific gene products is presented. This technique is analogous to the colony blotting techniques described for prokaryotes. In this technique, human epithelial cells are grown in tissue-culture dishes, and cells from the colonies on the dish are transferred in situ to a high-density cationized quaternary amine-charged nylon membrane. The membrane can then be processed multiple times, using antibody and/or nucleic acid probes, for the identification of those cells producing the desired protein, mRNA or DNA sequence. Once identified, the desired cell colony is isolated for expansion, direct DNA sequencing and/or cell function. We demonstrate the potential of the technique by identifying and isolating colonies of replicating normal human liver hepatocytes producing albumin and keratin.

Albumins↗
PubMed1994

Evaluation of the genotoxicity data on caffeine.

The potential health effects of caffeine have been investigated for over two decades in a variety of model systems including limited human populations. Thus, it is probably one of the most extensively studied natural occurring dietary chemicals. One area which has received a great deal of attention is the potential genotoxic property of caffeine. To better understand whether caffeine itself or in combination with other agents exhibits genotoxic effects, hundreds of research studies published over the past 5 years have been reviewed. These studies have utilized a number of animal, prokaryotic, eukaryotic, and mammalian cell culture model systems. They have investigated the effects of caffeine alone or in combination with other physical and chemical agents on many aspects of cell division, chromosome stability, toxicity, and mutagenicity. A number of effects have been observed. However, they usually appear after very high doses (> 1 mM) of caffeine in combination with genotoxins, and are usually specific to certain cell types and/or cellular parameters. Humans, on the other hand, consume much less caffeine in the diet, with peak serum levels in the micromolar range 10- and 1000-fold higher compared to levels in animal and cell culture models. Thus, it is difficult to implicate caffeine, even at the highest levels of dietary consumption, as a genotoxin to humans.

Animals↗
PubMed1993

Cell type-specific expression of the O6-alkylguanine-DNA alkyltransferase gene in normal human liver tissues as revealed by in situ hybridization.

The O6-alkylguanine-DNA alkyltransferase (ATase) is known to overcome the effects of promutagenic, precarcinogenic O6-alkylguanine induced in DNA by exposure to environmental, chemotherapeutic and dietary alkylating agents. Within an organ, the cell type-specific responses to these agents may be attributed, in part, to varying expression of critical DNA repair genes, like ATase. In order to determine the cell-specific expression of the human ATase gene, in situ hybridization was used to map the cellular distribution of ATase mRNA in tissue sections of normal human fetal and adult livers. Tissue sections were hybridized with a digoxigenin-labeled 39 base oligomer, antisense to ATase cDNA. Following immunodetection, using an alkaline phosphatase-conjugated anti-digoxigenin antibody, the ATase-specific mRNA levels were visualized in parallel with liver cell type identification. The specificity of the antisense probe and hybridization to human ATase mRNA was demonstrated by: (i) staining of Mer+ and not Mer- cells by the antisense probe; (ii) faint staining of liver sections when the antisense probe was not used during hybridization; (iii) no hybridization of liver sections by the sense probe; (iv) no staining of sections preincubated with RNase before hybridization; and (v) the retention of cell type-specific staining patterns in tissue sections incubated with DNase prior to hybridization with the antisense probe. The staining patterns appeared similar in adjacent sections of tissues obtained from the same liver and in sections obtained from either adult or fetal livers of different individuals. The expression of the ATase mRNA, as noted by stain intensity, appeared highest in all of the bile ductal cells. There was a heterogenous expression in hepatocytes, which varied from moderate to high stain. Staining in Kupffer cells also appeared to be high. Sinusoidal cells, endothelial cells of the hepatic artery and cells of the connective tissue showed weak hybridization, indicating low levels of ATase mRNA. These data explain, in part, the basis for a differential response of various cell types within the liver to the mutagenic and carcinogenic effects of alkylating agents.

Base Sequence↗
PubMed1992

In situ hybridization of human kidney tissue reveals cell-type-specific expression of the O6-methylguanine-DNA methyltransferase gene.

The cell-type-specific expression of human O6-methylguanine-DNA methyltransferase (O6-MT) was determined in paraffin-embedded sections of human kidney. A 39 base oligomer complementary to O6-MT cDNA was labeled with digoxigenin and visually detected in situ using an alkaline phosphatase-conjugated anti-digoxigenin antibody. This allowed direct determination of O6-MT-specific mRNA levels, while simultaneously identifying the structures and cell types in the kidney section. Expression of O6-MT was high in distal tubular and glomerular epithelial cells and low in the cells of the Bowman's capsule, collecting and proximal tubular cells. Hybridization of the oligomer was specific to RNA, since RNase and not DNase eliminated the signal. Expression was uniform in all the cell types, except the glomerular cells exhibited varying levels of high intensity. Cell-specific expression was constant between tissue sections from the same and different kidney tissues. These data may help explain the differential response of various cell types to alkylating agents.

DNA Repair↗
PubMed1991

An enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of the tumor marker 1-methylinosine in human urine.

A highly sensitive enzyme-linked immunoassay (ELISA) was developed to detect and quantify the tumor marker, 1-methylinosine (m1I), in human urine. The rabbit antisera was highly specific for m1I with negligible or no inhibition by other nucleosides excreted into urine. Using the competitive ELISA, nanogram amounts of m1I were easily measured directly in urine. The assay agreed with our previous hplc analysis of m1I in urine for identifying those individuals with chronic myelogenous leukemia. Thus, this assay should greatly facilitate the quantitation of m1I as a tumor marker.

Antibody Specificity↗
PubMed1990

Repair of O4-alkylthymine damage in human cells.

The capacity of a cell to repair damage is the first step in preventing the deleterious consequences of DNA structural alterations induced by the exposure to mutagenic carcinogens. Mammalian cells, having complex genetic organization, have evolved sophisticated mechanisms for the maintenance of integrity of their genome and normal cell function (Bohr and Hanawalt, 1988; Sancar and Sancar, 1988; Pienta et al., 1989). However, many DNA repair processes in mammalian cells are similar to those in prokaryotic cells. For example, the unique damage reversal mechanism by transferase, specific for the repair of O6-alkylGua, results in the restoration of intact guanine base in both bacteria and mammalian cells (Olsson and Lindahl, 1980; D'Ambrosio and Wani, 1989). The proteins involved, however, are different and vary in their specificities. Mammalian transferase specific for O6-alkylGua, more closely resembles the bacterial ogt gene product (Potter et al., 1987; Rebeck et al., 1988). The main O6-alkylGua specific transferase activity in E. coli resides in the product of the ada gene (Demple et al., 1985; Nakabeppu et al., 1985). This protein possesses multiple activities including a specificity for the transfer of alkyl group from O4-alkylthy in DNA. Such a transferase activity specific for O4-alkylThy has not been detected in mammalian cells either as an individual activity or part of a multi-activity protein (Brent et al., 1988). Nevertheless, there is tangible evidence for the active removal of O4-alkylthy in mammalian cells, particularly human cells. The nature, level, and mode of the O4-alkylThy repair activity has not been fully established. Whether the repair occurs by the well known or some novel mechanism(s) has yet to be determined. Recently Boyle et al., (1987) has provided genetic evidence for an alternate mode of repair of O6-alkylGua in mammalian cells. The human cells, that lack O6-MT activity were able to repair O6-nButylGua in cellular DNA. Additional experiments, with mammalian V79 cell lines, indicated a differential specificity for various alkyl groups. It has been suggested that in these cells the repair occurs by an excision process, which is known to recognize the distortions of the DNA duplex rather than the adduct itself (Sancar and Sancar, 1988). Support for the excision mechanism is also provided by in vitro experiments, showing repair of O6-MeGua by purified E. coli ABC excinuclease enzyme (Voigt et al., 1989). It is quite likely that the repair of O4-alkylThy in mammalian cells occurs by a similar process.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkylating Agents↗
PubMed1990

A human DNA repair activity specific for O4-ethylthymine: identification and partial characterization.

An O4-ethylthymine-specific antibody and immunoslot blot assay were used to identify an enzymatic activity in human tissue and cell extracts, specific for removing O4-ethylthymine from ethylated DNA in vitro. The assay allowed the quantitation of activity in the range of 2 fmol O4-ethylthymine removed per mg cell-free protein extract. The specific activities in human brain, kidney and liver tissue extracts ranged from 5.2 to 26.0 with mean levels of 12.8, 9.9 and 12.3 fmol/mg protein respectively. The activity in extracts prepared from cultured kidney and liver epithelial cells ranged from 8.4 to 16.0 fmol/mg protein, exhibiting mean levels of 10.3 and 13.5 fmol/mg protein respectively. The similar levels of repairing O4-ethylthymine in human liver, kidney and brain contrast the organ-specific activity of the O6-alkylguanine DNA-methyltransferase. The repair activity for O4-ethylthymine was not inhibited by preincubation of the extracts with either O4-methylthymine or O6-methylguanine, indicating that the loss of O4-ethylthymine was not mediated by an alkyltransferase. However, there was no detectable activity in extracts prepared from the GM006 mer- cell line lacking O6-alkylguanine DNA-methyltransferase. These data suggest that the activity for repairing O4-ethylthymine may be due to a protein distinct from the O6-alkylguanine DNA-methyltransferase. The low, but significant, level of repair activity specific for O4-ethylthymine identified in human tissue and cell extracts is consistent with the slow, but active, repair of this adduct in vivo.

Brain↗
PubMed1989

Absence of DNA damage-mediated induction of human methyltransferase specific for precarcinogenic O6-methylguanine.

The ability of cultured normal human fetal liver and kidney epithelial cells to repair the premutagenic and precarcinogenic O6-methylguanine (O6-MeGua) DNA adduct was determined by directly monitoring its loss in cellular DNA and quantitating the number of O6-MeGua-DNA-methyltransferase (O6-MT) molecules per cell. Following treatment of the epithelial cells with the direct acting carcinogen N-methyl-N-nitrosourea (MNU), the loss of the O6-MeGua adduct was biphasic, exhibiting a half-life of 2.0 and 1.5 h in the liver and kidney cells, respectively. The activity of O6-MT in the liver and kidney epithelial cells in culture was 0.19 pmol/mg protein or 18,500 molecules/cell. The activity of O6-MT was maintained throughout the life of the cultures, i.e., 20 subpassages or 50 cumulative population doublings for the liver and kidney. In order to ascertain whether human fetal epithelial cells exhibit an induction of O6-MT, the cell cultures were treated with single and multiple conditioning doses of N-methyl-N-nitro-N-nitroso-guanidine (MNNG) or gamma-irradiated and assayed for the amount of O6-MT. A 1 h exposure of cells to 2, 4, and 8 microM MNNG resulted in an 80-100% decrease of the initial O6-MT activity which was restored to the constitutive levels within 48 and 72 h post-treatment. Rat hepatoma cells, used as a positive control, increased their levels of O6-MT to 2.8-fold the constitutive levels following treatment with MNNG. Treatment of the human liver and kidney epithelial cells with chronic low doses of MNNG exhibited O6-MT levels identical to untreated cells. The O6-MT activity in epithelial cells remained unaffected upon pre-irradiation with 1.2 or 2.5 Gy of gamma-irradiation.

Animals↗