The Zn-finger domain of MdmX suppresses cancer progression by promoting genome stability in p53-mutant cells.

The MDMX (MDM4) oncogene is amplified or overexpressed in a significant percentage of human tumors. MDMX is thought to function as an oncoprotein by binding p53 tumor suppressor protein to inhibit p53-mediated transcription, and by complexing with MDM2 oncoprotein to promote MDM2-mediated degradation of p53. However, down-regulation or loss of functional MDMX has also been observed in a variety of human tumors that are mutated for p53, often correlating with more aggressive cancers and a worse patient prognosis. We have previously reported that endogenous levels of MdmX can suppress proliferation and promote pseudo-bipolar mitosis in primary and tumor cells derived from p53-deficient mice, and that MdmX-p53 double deficient mice succumb to spontaneously formed tumors more rapidly than p53-deficient mice. These results suggest that the MdmX oncoprotein may act as a tumor-suppressor in cancers with compromised p53 function. By using orthotopic transplantation and lung colonization assays in mice we now establish a p53-independent anti-oncogenic role for MdmX in tumor progression. We also demonstrate that the roles of MdmX in genome stability and in proliferation are two distinct functions encoded by the separate MdmX protein domains. The central Zn-finger domain suppresses multipolar mitosis and chromosome loss, whereas the carboxy-terminal RING domain suppresses proliferation of p53-deficient cells. Furthermore, we determine that it is the maintenance of genome stability that underlies MdmX role in suppression of tumorigenesis in hyperploid p53 mutant tumors. Our results offer a rationale for the increased metastatic potential of p53 mutant human cancers with aberrant MdmX function and provide a caveat for the application of anti-MdmX treatment of tumors with compromised p53 activity.

Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay.

DNA damage is thought to be the initial event that causes sulfur mustard (SM) toxicity, while the ability of cells to repair this damage is thought to provide a degree of natural protection. To investigate the repair process, we have damaged plasmids containing the firefly luciferase gene with either SM or its monofunctional analog, 2-chloroethyl ethyl sulfide (CEES). Damaged plasmids were transfected into wild-type and nucleotide excision repair (NER) deficient Chinese hamster ovary cells; these cells were also transfected with a second reporter plasmid containing RENILLA: luciferase as an internal control on the efficiency of transfection. Transfected cells were incubated at 37 degrees C for 27 h and then both firefly and RENILLA: luciferase intensities were measured on the same samples with the dual luciferase reporter assay. Bioluminescence in lysates from cells transfected with damaged plasmid, expressed as a percentage of the bioluminescence from cells transfected with undamaged plasmid, is increased by host cell repair activity. The results show that NER-competent cells have a higher reactivation capacity than NER-deficient cells for plasmids damaged by either SM or CEES. Significantly, NER-competent cells are also more resistant to the toxic effects of SM and CEES, indicating that NER is not only proficient in repairing DNA damage caused by either agent but also in decreasing their toxicity. This host cell repair assay can now be used to determine what other cellular mechanisms protect cells from mustard toxicity and under what conditions these mechanisms are most effective.

Role of base excision repair in protecting cells from the toxicity of chloroethylnitrosoureas.

The chloroethylnitrosoureas react extensively with cellular DNA to produce a variety of DNA adducts, including a deoxycytidine-deoxyguanosine (dC-dG) cross-link that is clearly cytotoxic. It is now well established that O6-alkylguanine-DNA-alkyltransferase can prevent formation of this dC-dG cross-link and thereby diminish the toxicity of the chloroethylnitrosoureas. Besides alkyltransferase, DNA glycosylases from various species can also contribute to cellular resistance to the chloroethylnitrosoureas, but the mechanism for this increased resistance has not been established. It is known, however, that several chloroethylnitrosoureas-modified DNA bases, including the exocyclic adduct, N2,3-ethanoguanine, are released by Escherichia coli 3-methyladenine DNA glycosylase II. In the study described here, we examined the possibility that this enzyme might act on the exocyclic intermediate in dC-dG formation, 1,O6-ethanodeoxyguanosine, and prevent-dC-dG cross-linking in this way. However, the presence of E. coli 3-methyladenine DNA glycosylase II does not decrease the amount of dC-dG cross-link formed when chloroethylnitrosourea reacts with DNA, and we conclude that this enzyme does not recognize 1,O6-ethanodeoxyguanosine. Therefore, its contribution to resistance probably resides in its action on other nitrosourea-induced DNA modifications.

Hypothermia causes a reversible, p53-mediated cell cycle arrest in cultured fibroblasts.

Normal human fibroblasts grown in cell culture undergo a reversible growth arrest when incubated at 28 degrees C. During incubation at 28 degrees C, levels of p53 and p21 rise in these cells and cell cycle analysis shows that they have undergone a cell cycle arrest. To examine the importance of p53 in mediating this arrest, mouse embryo fibroblasts that are either wild-type or that are defective in p53 were also subjected to hypothermia. Only those cells with wild-type p53 undergo a cell cycle arrest, indicating that p53 has a role in mediating this response. Because many tumor cells have defective p53, this suggests that hypothermia may increase the selective toxicity of chemotherapeutic agents for tumor cells.

Release of sulfur mustard-modified DNA bases by Escherichia coli 3-methyladenine DNA glycosylase II.

The toxic effects of sulfur mustard have been attributed to DNA modification with the formation of 7-hydroxyethylthioethyl guanine, 3-hydroxyethylthioethyl adenine and the cross-link, di-(2-guanin-7-yl-ethyl)sulfide. To investigate the action of bacterial 3-methyladenine DNA glycosylase II (Gly II) on these adducts, calf thymus DNA was modified with [14C]sulfur mustard and used as a substrate for Gly II. Gly II releases both 3-hydroxyethylthioethyl adenine and 7-hydroxyethylthioethyl guanine from this substrate. In comparison with the activity of Gly II towards methylated DNA, 3-hydroxyethylthioethyl adenine is released somewhat more slowly than 3-methyladenine, while 7-hydroxyethylthioethyl guanine is released much more readily than 7-methylguanine. Glycosylase action may play a role in protecting cells from the toxic effects of sulfur mustard.

A 32P-postlabeling method for the detection of adducts in the DNA of human fibroblasts exposed to sulfur mustard.

Since the toxicities of sulfur mustard are attributed to DNA alkylation, levels of DNA modification in exposed cells should correlate with the intensity of exposure. We have found that 32P-postlabeling can be used successfully to detect the major adduct, 7-hydroxyethylthio- ethyldeoxyguanosine 5'-phosphate (HETEpdG), that is formed in DNA by sulfur mustard. This method has been used to establish a correlation between exposure and adduct formation in human fibroblasts grown in cell culture and exposed to sulfur mustard concentrations between 2.5 and 15 microM. DNA was recovered from these cells using a salt precipitation method to remove proteins and was found to have an HETEpdG content which increased linearly with SM concentration. This relationship shows that one HETEpdG per 10(6) nucleotides is produced at a SM concentration of 2.3 microM. Growth of fibroblast cells, assayed by trypan-blue exclusion, is somewhat inhibited by 2 microM SM, indicating that 32P-postlabeling has the requisite sensitivity to detect adducts at levels of SM that are minimally toxic.

Induction of the Escherichia coli aidB gene under oxygen-limiting conditions requires a functional rpoS (katF) gene.

The Escherichia coli aidB gene is regulated by two different mechanisms, an ada-dependent pathway triggered by methyl damage to DNA and an ada-independent pathway triggered when cells are grown without aeration. In this report we describe our search for mutations affecting the ada-independent aidB induction pathway. The mutant strain identified carries two mutations affecting aidB expression. These mutations are named abrB (aidB regulator) and abrD. The abrB mutation is presently poorly characterized because of instability of the phenotype it imparts. The second mutation, abrD1, reduces the expression of aidB observed when aeration is ceased and oxygen becomes limiting. Genetic and phenotypic analysis of the abrD1 mutation demonstrates that it is an allele of rpoS. Thus, aidB is a member of the family of genes that are transcribed by a sigma S-directed RNA polymerase holoenzyme. Examination of aidB expression in an rpoS insertion mutant strain indicates that both rpoS13::Tn10 and abrD1 mutations reduce aidB expression under oxygen-limiting conditions that prevail in unaerated cultures, reduce aidB induction by acetate at a low pH, but have little or no effect on the ada-dependent alkylation induction of aidB.

The effect of glucocorticoids on the activity of monoamine oxidase and superoxide dismutase in the rat interscapular brown adipose tissue.

The effect of dexamethasone (DEX) and corticosterone (COR) on the activity of monoamine oxidase (MAO), copper-zinc superoxide dismutase (CuZn SOD) and manganese superoxide dismutase (Mn SOD) in the rat interscapular brown adipose tissue (IBAT) were studied. DEX (1 mg/kg, i.p. for two days) significantly increased MAO activity in the IBAT as compared to the corresponding controls. On the contrary, COR, in the corresponding dose (5 mg/kg), did not affect MAO activity in the IBAT. DEX also markedly enhanced the activity of both SODs in the tissue studied, while COR was ineffective. The results suggest that there exist the differences in the effect between the synthetic glucocorticoid, such as DEX, and COR, which is a natural glucocorticoid in the rat, on the activity of IBAT enzymes studied.

Protection against chloroethylnitrosourea cytotoxicity by eukaryotic 3-methyladenine DNA glycosylase.

A eukaryotic 3-methyladenine DNA glycosylase gene, the Saccharomyces cerevisiae MAG gene, was shown to prevent N-(2-chloroethyl)-N-nitrosourea toxicity. Disruption of the MAG gene by insertion of the URA3 gene increased the sensitivity of S. cerevisiae cells to N-(2-chloroethyl)-N-nitrosourea, and the expression of MAG in glycosylase-deficient Escherichia coli cells protected against the cytotoxic effects of N-(2-chloroethyl)-N-nitrosourea. Extracts of E. coli cells that contain and express the MAG gene released 7-hydroxyethylguanine and 7-chloroethylguanine from N-(2-chloroethyl)-N-nitrosourea-modified DNA in a protein- and time-dependent manner. The ability of a eukaryotic glycosylase to protect cells from the cytotoxic effects of a haloethylnitrosourea and to release N-(2-chloroethyl)-N-nitrosourea-induced DNA modifications suggests that mammalian glycosylases may play a role in the resistance of tumor cells to the antitumor effects of the haloethylnitrosoureas.

Release of N2,3-ethenoguanine from chloroacetaldehyde-treated DNA by Escherichia coli 3-methyladenine DNA glycosylase II.

The human carcinogen vinyl chloride is metabolized in the liver to reactive intermediates which form N2,3-ethenoguanine in DNA. N2,3-Ethenoguanine is known to cause G----A transitions during DNA replication in Escherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair of N2,3-ethenoguanine, we have prepared an N2,3-etheno[14C]guanine-containing DNA substrate by nick-translating DNA with [14C]dGTP and modifying the product with chloroacetaldehyde. E. coli 3-methyladenine DNA glycosylase II, purified from cells which carry the plasmid pYN1000, releases N2,3-ethenoguanine from chloroacetaldehyde-modified DNA in a protein- and time-dependent manner. This finding widens the known substrate specificity of glycosylase II to include a modified base which may be associated with the carcinogenic process. Similar enzymatic activity in eukaryotic cell might protect them from exposure to metabolites of vinyl chloride.

Anaerobic induction of the alkylation-inducible Escherichia coli aidB gene involves genes of the cysteine biosynthetic pathway.

The Escherichia coli aidB gene is a component of the adaptive response to alkylation damage. This gene is subject to two different forms of induction: an ada-dependent alkylation induction and an ada-independent induction that occurs when cells are grown anaerobically (M. R. Volkert, L. I. Hajec, and D. C. Nguyen, J. Bacteriol. 171:1196-1198, 1989; M. R. Volkert, and D. C. Nguyen, Proc. Natl. Acad. Sci. USA 81:4110-4114, 1984). In this study, we isolated and characterized strains bearing mutations that specifically affect the anaerobic induction pathway. This pathway requires a functional cysA operon, which encodes sulfate permease. Mutations in cysA block this pathway of aidB induction. In contrast, mutations in either cysH, cysD, cysN, or cysC result in elevated levels of aidB expression during aerobic growth. These results indicate that the sulfate transport genes perform a role in anaerobic induction of the aidB gene and suggest that growth under anaerobic conditions may modify either the function or the expression of gene products encoded by the cysA operon.

The formation and enzymatic repair of DNA modifications caused by the haloethylnitrosoureas and related compounds.

The haloethylnitrosoureas are both useful antitumor agents and known carcinogens. These biological activities are believed to be associated with DNA modification, and some biologically significant lesions have been identified in DNA exposed to these agents. At the same time, DNA repair is a cause of resistance to treatment by these agents, and may also serve as protection against their carcinogenic effects.

The influence of the nucleotide excision-repair system on mutagenesis in Salmonella typhimurium LT2 after exposure to low doses of monofunctional alkylating agents.

The role of nucleotide excision repair in the mutagenicity of the monofunctional alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methyl methanesulfonate (MMS), N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), and N-ethyl-N-nitrosourea (ENU) in Salmonella typhimurium was examined. The mutagenic potential of the mutagenic agents used increased in the following order: MMS less than ENU less than ENNG less than MNNG. The results obtained confirm the involvement of nucleotide excision repair in the removal of mutagenic lesions from the DNA of S. typhimurium cells exposed to high doses of methylating as well as ethylating agents. At the low doses of all the alkylating agents used, the nucleotide excision repair-proficient strain was mutagenized more efficiently than the uvrB mutant. This phenomenon, a consequence of competition between nucleotide excision-repair enzymes and constitutive O6-methylguanine-DNA methyltransferase, is discussed.

3-Methyladenine DNA glycosylase activity in a glial cell line sensitive to the haloethylnitrosoureas in comparison with a resistant cell line.

Extracts of a glial cell line (SF-126) which is sensitive to the cytotoxic effect of the haloethylnitrosoureas and of a cell line (SF-188) which is resistant to these agents have been tested for their ability to release methylated bases from a DNA substrate which has been modified with [3H]dimethyl sulfate. In comparison with the sensitive cell line, extracts from the resistant cell line have 2-3-fold higher enzymatic activity. High performance liquid chromatography profiles of the bases which are released by these extracts show that the activity is specific for 3-methyladenine, suggesting that the resistant cells contain elevated levels of 3-methyladenine DNA glycosylase. Previous studies have shown that these cells also contain elevated levels of O6-alkylguanine-DNA alkyl-transferase, suggesting that both enzyme activities may be involved in the resistance of this cell line to the haloethylnitrosoureas.

Genotoxicity of nitrosated ranitidine.

The genotoxicity of ranitidine, widely used in the therapy of peptic ulcers, and of nitrosated ranitidine was examined in test systems with the bacteria Salmonella typhimurium for gene mutations, and with the yeast Saccharomyces cerevisiae D7 for reverse mutations and gene conversion. Under the experimental conditions applied, ranitidine was negative in both systems, while the product obtained by nitrosation in vitro was mutagenic for Salmonella strains TA100 and TA98 with and without metabolic activation. The largest increase of his+ revertants, 3 times greater than the control, was obtained in strain TA100 in the absence of S9 fraction. Nitrosated ranitidine was also recombinogenic for the yeast S. cerevisiae.

DNA binding and mutagenicity of ethyl methanesulfonate in wild-type and uvrB cells of Salmonella typhimurium.

The extent of DNA ethylation and the influence of excision repair on ethyl methanesulfonate (EMS) mutagenesis of Salmonella typhimurium were examined. The relationship between the dose to DNA and the exposure concentration of EMS was linear. EMS induction of his+ revertants followed exponential kinetics and did not parallel the increase in total DNA ethylation. Mutant induction was influenced by the cells' nucleotide excision repair ability. Although mutagenized to a larger extent than the wild-type (uvr+) strain at high doses, the uvrB strain was more resistant to the mutagenic effect of low doses of EMS.

Mutagenicity of pyrene in Salmonella.

Pyrene was tested for mutagenicity in Salmonella typhimurium strains TA97, TA98, TA100 and TA1537. Mutagenicity was seen in all strains when S9 was present.