The ubiquitin-proteasome system regulates p53-mediated transcription at p21waf1 promoter.

The ubiquitin (Ub)-proteasome system (UPS) promotes the proteasomal degradation of target proteins by decorating them with Ub labels. Emerging evidence indicates a role of UPS in regulating gene transcription. In this study, we provided evidence for the involvement of UPS in the transcriptional activation function of tumor suppressor p53. We showed that both ubiquitylation and proteasomal functions are required for efficient transcription mediated by p53. Disruption of transcription by actinomycin D, 5,6-dichloro-1-beta-D-ribofuranosyl-benzimadazole or alpha-amanitin leads to accumulation of cellular p53 protein. Proteasome inhibition by MG132 increases the occupancy of p53 protein at p53-responsive p21(waf1) promoter. In addition, the Sug-1 component of 19S proteasome physically interacts with p53 in vitro and in vivo. Moreover, in response to ultraviolet-induced DNA damage, both the 19S proteasomal components, Sug1 and S1, are recruited to p21(waf1) promoter region in a kinetic pattern similar to that of p53. These results suggested that UPS positively regulates p53-mediated transcription at p21(waf1) promoter.

Mdm2 mutant defective in binding p300 promotes ubiquitination but not degradation of p53: evidence for the role of p300 in integrating ubiquitination and proteolysis.

Turnover of the p53 tumor suppressor protein is mediated by Mdm2 through the ubiquitin proteolysis pathway. p300, a co-activator for p53, also participates in this process by complexing with Mdm2. We now report that the mutant Mdm2, defective in p53 binding, does not promote p53 ubiquitination and degradation in vivo or inhibit p53 transcriptional activation. By contrast, the mutant Mdm2, defective in p300 binding, still retains its activity to promote p53 ubiquitination and to inhibit p53 transcriptional activation but fails in promoting p53 degradation. We also show that both wild-type Mdm2 and the mutant Mdm2, defective in p300 binding, can promote the ubiquitination of cancer-derived p53 mutants, but only wild-type Mdm2 can cause their degradation. Furthermore, adenoviral oncoprotein, 12S.E.1A, but not its deletion mutant that lacks p300 binding, was shown to decrease in vivo ubiquitination of mutant p53. Taken together, these results provide genetic evidence that p300 plays a pivotal role in the regulation of Mdm2-mediated p53 turnover by integrating the cellular ubiquitination and proteolytic processes.

Human homologue of yeast Rad23 protein A interacts with p300/cyclic AMP-responsive element binding (CREB)-binding protein to down-regulate transcriptional activity of p53.

The tumor suppressor protein p53 regulates various cellular responses to DNA damage and plays a significant role in DNA repair. The nuclear p300/cyclic AMP-responsive element binding (CREB)-binding protein (CBP) proteins act as coactivators in supporting the transcription function of p53. We examined the role of the human homologue of yeast Rad23 protein A (hHR23A), one of the two human homologues of the Saccharomyces cerevisiae nucleotide excision repair gene product Rad23, in the p300/CBP-associated regulation of p53 activity. Overexpression of wild-type hHR23A inhibits the p53 transcriptional activity and results in a decreased steady-state protein level of cellular p53. The inhibitory effect of hHR23A can be overcome by the concomitant expression of p300, CBP, and p300 segments harboring C/H1 domain and neutralized by the coexpression of HIV accessory protein Vpr, which binds COOH terminus of hHR23A/B. Additionally, hHR23A was shown to interact in vitro and in vivo with p300 segments harboring C/H1 domain. These studies provide evidence for the involvement of hHR23A in the regulation of p53 activity through p300/CBP. Although the precise direct role of hHR23 proteins in regulation of p53 and DNA repair remains to be elucidated, our data suggest that the interaction between hHR23A and p300/CBP has important implications in cross-talk between the p53 pathway and DNA repair.

Modulation of Ki67, p53 and RARbeta expression in normal, premalignant and malignant human oral epithelial cells by chemopreventive agents.

BACKGROUND: Aberrant expression of Ki67, p53 and RARbeta are characteristic of many tumor types including those of the oral cavity. Chemopreventive agents may act by modulating their expression to more normal levels. MATERIALS AND METHODS: The effects of 21 chemopreventive agents on the expression of Ki67, p53 and RARbeta were determined using a human in vitro model of normal, premalignant and malignant oral epithelial cell lines. RESULTS: Ki67 and mutant p53 (mtp53) were overexpressed in both the premalignant and malignant cell lines, whereas expression of RARbeta was high in the normal, low in the premalignant and not detectable in the malignant cell lines. Most of the agents selectively inhibited the expression of Ki67 in the premalignant and malignant cell lines. Eight of the 21 agents increased, while four agents decreased, the levels of mtp53 protein in the premalignant cell line. In the malignant cell line, five of the agents increased, while ten agents decreased mtp53 protein levels. The agents increased RARbeta expression to near normal levels in the premalignant cell line. CONCLUSION: The data suggest that the suppression of Ki67 and mtp53 are good indicators of the effectiveness of agents in premalignant and malignant oral cells, whereas the enhancement of RARbeta is a measure of effectiveness in premalignant oral cells.

Modulation of transcriptional activity of p53 by ultraviolet radiation: linkage between p53 pathway and DNA repair through damage recognition.

The increase in the p53 activity in response to DNA damage is thought to be one of the important mechanisms by which p53 contributes to transcriptional activation of p21(wafl), mdm2, and other downstream regulatory genes. To investigate the p53 response to ultraviolet (UV) type of DNA damage, p53 protein level, its transcriptional activity and in vivo ubiquitination were compared in repair-proficient normal human fibroblasts (NHFs) and repair-deficient xeroderma pigmentosum (XP) group A and group C (XP-C) fibroblasts subsequent to irradiation with UV light. Accumulation of p53 protein level was observed with increasing UV doses in all the cell lines; however, discordance between p53 and p21(waf1) and mdm2 levels was observed in NHF and XP-A cells. Induction of p21(waf1) and mdm2 was inhibited by UV irradiation, requiring higher doses in NHF and lower doses in XP-A cells. However, inhibition of p21(waf1) and mdm2 induction was not observed in XP-C cells. Ubiquitin-p53 conjugates could be detected in irradiated or unirradiated NHF and XP-A cells but not in XP-C cells irradiated with 30 and 50 J/m(2) UV light. Using a p53 reporter assay, p53 transcriptional activities were found to be induced by 10 J/m(2) UV exposure and dramatically inhibited with increasing UV doses in NHF cells. Compared with repair-proficient NHF cells, UV inhibition of p53 transcriptional activity was relatively more sensitive in XP-A cells but resistant in XP-C cells. These results indicate that DNA damage by UV, in addition to inducing p53, acts as a trigger for inhibition of p53 transcriptional activity. Overall, recognition of DNA damage links both p53 induction and p53 degradation to DNA repair mechanisms.

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.

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.

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.

Expression of the O6-alkylguanine-DNA alkyltransferase gene is elevated in human breast tumor cells.

In tumors, resistance to chemotherapeutic drugs that alkylate the O6 position of guanine correlates with the levels of the DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT). The expression of AGT gene in human breast tumors was evaluated at the level of the single cell, to better understand the distribution of alkylation resistant cells within the tumor. Compared to normal breast ductal cells, the level of AGT expression in the breast tumor cells increased 2-fold. There was no significant association between AGT expression and tumor grade and metastatic malignancy. The up-regulation of AGT was not directly linked to the expression of cyclins D1 and D3, estrogen receptor, p53 and c-erbB-2, genes involved in cell cycle regulation and tumor growth. The elevated expression of AGT in human breast ductal carcinoma cells appeared to be a general characteristic of breast tumors, and suggests that prior treatment with analogs of O6-alkylguanine that inactivate AGT protein, should render the AGT expressing tumor cells sensitive to drugs that alkylate O6-guanine.

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.

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.

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.

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.

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.

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)

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.

Repair of DNA O-alkylation damage by various human organs.

Protein extracts from human adult liver, fetal liver, intestine, brain, kidney, lung and skin were tested against poly(dT)methylated X poly(dA), poly(dA)methylated X poly(dT) and methylated DNA. The suitability of various substrates was established in assays using E. coli extracts that removed O4-methylthymidine (O4-MedT), O2-MedT, and O6-methylguanine (O6-MeG). The human extracts efficiently removed O6-MeG and N3-methyladenine from methylated substrates. The adult liver exhibited low and fetal tissues negligible removal of O4-MedT. Only the liver showed limited removal of O2-MedT. The poor removal of the miscoding base O4-MedT by human organs could be an important factor in carcinogen induced mutagenesis, carcinogenesis and teratogenesis.

Repair of O6-methylguanine in human fetal brain and skin cells in culture.

The repair of O6-methylguanine was measured in cell cultures derived from human fetal brain and skin. Cells derived from nine different fetal specimens were treated in culture with N-methyl-N-nitrosourea (MNU). The brain cell cultures used were a mixture of glial and neuronal cell types, while the skin cell cultures were predominantly fibroblasts. The amount of O6-methylguanine initially induced and remaining in cellular DNA was quantitated as a function of time (0-4 h) by h.p.l.c. analysis of the acid hydrolyzates of in vitro alkylated cellular DNA. Very little (less than 10%) of the 7-methylguanine was lost from the DNA of both cell cultures 4 h post-treatment. Approximately 50% of the O6-methylguanine induced in cellular DNA by MNU was lost within 0.7 h in both the human fetal brain and skin cells in culture. Within 4 h, 80% of this methylated guanine was lost. The kinetics for the removal of O6-methylguanine in the brain and skin cells appeared biphasic. A rapid initial phase was followed by a gradual slower phase of repair. These studies indicate that cells derived from human fetal brain and skin exhibit the same degree of proficiency for the repair of the potential pro-carcinogenic O6-methylguanine lesion.