Mdm2 and MdmX are structurally related p53-binding proteins that function as critical negative regulators of p53 activity in embryonic and adult tissue. The overexpression of Mdm2 or MdmX inhibits p53 tumor suppressor functions in vitro, and the amplification of Mdm2 or MdmX is observed in human cancers retaining wild-type p53. We now demonstrate a surprising role for MdmX in suppressing tumorigenesis that is distinct from its oncogenic ability to inhibit p53. The deletion of MdmX induces multipolar mitotic spindle formation and the loss of chromosomes from hyperploid p53-null cells. This reduction in chromosome number, not observed in p53-null cells with Mdm2 deleted, correlates with increased cell proliferation and the spontaneous transformation of MdmX/p53-null mouse embryonic fibroblasts in vitro and with an increased rate of spontaneous tumorigenesis in MdmX/p53-null mice in vivo. These results indicate that MdmX has a p53-independent role in suppressing oncogenic cell transformation, proliferation, and tumorigenesis by promoting centrosome clustering and bipolar mitosis.Although genetic and biochemical studies clearly indicate that Mdm2 and MdmX are key regulators of p53 activity, there are distinct differences in their mechanisms of p53 inhibition. Mdm2 forms a complex with p53 and functions as an E3 ligase to target p53 for ubiquitination and proteosomal degradation (16,18,25), thereby inhibiting p53's transactivation of genes whose products are involved in the regulation of cell growth and apoptosis (47). MdmX complexes with p53 and inhibits p53 transactivation without altering p53 stability (11,46), and in contrast to that of Mdm2, MdmX expression is not regulated by p53. Regardless of these differences, Mdm2 and MdmX act as critical negative regulators of p53 function in development. The developmental block imposed by the loss of Mdm2 or MdmX can be relieved by the deletion of p53 (9,21,28,30,32) or by Mdm2 amplification (20,22) or, in the case of MdmX, partially rescued by the deletion of the p53 downstream effector p21 (43). These data indicate that the primary role of Mdm2 or MdmX in development is to regulate p53.The amplification and overexpression of either Mdm2 (31) or MdmX (7, 36) have been observed in a variety of human cancers, including sarcoma, glioma, and, in the case of MdmX, retinoblastoma (24), suggesting that either Mdm2 or MdmX can function as an oncogene to inhibit p53 activity and promote tumorigenesis. Since many of these Mdm-overexpressing tumors retain wild-type p53 alleles, the reactivation of p53 by small-molecule inhibition of the Mdm2-p53 or MdmX-p53 interaction is an attractive strategy for treating these cancers (26,27).The results of experiments in vitro or in vivo involving the forced overexpression of Mdm proteins suggest that Mdm2 and MdmX may also have p53-independent roles in promoting cell growth (13,23,29,38,41). However, molecular targets for Mdm2 or MdmX activity other than p53 have yet to be confirmed. Furthermore, it remains unclear if physiologic levels of eit...

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 inEscherichia coli, and its formation may be a carcinogenic event in higher organisms. To investigate the repair ofN2,3-ethenoguanine, we have prepared an N2,3-etheno[14Clguanine-containing DNA substrate by nick-translating DNA with [14CldGTP 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 cells might protect them from exposure to metabolites of vinyl chloride. gated the activity of this enzyme towards an EG-containing DNA substrate.Both glycosylase II and 06-alkylguanine-DNA alkyltransferase are synthesized by E. coli in increased amounts as part of the adaptive response to methylating carcinogens (17). 06-alkylguanine-DNA alkyltransferase removes alkyl groups from the Q6 position of guanine and from phosphotriesters, thereby restoring the original structure. Glycosylase II acts in a different way, releasing modified bases from the DNA and leaving apurinic sites which are subject to further repair.Glycosylase II releases 3-and 7-alkylated purines as well as 02-methylcytosine and 02-methylthymine from DNA (17). The recent finding that N2,3-ethanoguanine is also released extends the specificity of this enzyme to DNA bases that bear exocyclic groups. The studies reported here show that the enzyme has activity towards a base that has been associated with vinyl chloride carcinogenicity.The industrial chemical vinyl chloride is carcinogenic in animal tests and is associated with an increased incidence of hepatic angiosarcomas in exposed workers (1). Vinyl chloride is metabolized by the cytochrome P450 system in the liver to the unstable electrophile chloroethylene oxide, which rearranges spontaneously to the more stable alkylating agent chloroacetaldehyde (CAA) (2, 3). Chloroethylene oxide and CAA react with DNA to form a variety of adducts, many of which have been identified in vivo (4-10). The question has arisen, therefore, as to which DNA modification or modifications may be responsible for initiating the carcinogenic process.The modified base that is found in the greatest quantity in livers of animals exposed to vinyl chloride is 7-(2-oxoethyl)-guanine (oeG) (4, 6, 7). This base does not cause misincorporation when it is present in a DNA template strand (11), but it does form apurinic sites, which could be mutagenic or carcinogenic. On the other hand, N2,3-ethenoguanine (EG) has been shown to mispair in in vitro transcription systems (12, 13) and causes G --A transitions in Escherichia coli (14).These results...

The cellular homologues Mdm2 and MdmX play critical roles in regulating the activity of the p53 tumor suppressor in damaged and non-damaged cells and during development in mice. Recently, we have utilized genetically defined primary cells and mice to reveal that endogenous levels of MdmX can also suppress multipolar mitosis and transformation in hyperploid p53-deficient cells and tumorigenesis in p53-deficient mice. These MdmX functions are not shared by Mdm2, and are distinct from the well-established ability of MdmX to complex with and inhibit p53 activity. Here we discuss some of the ramifications of MdmX loss in p53-deficient cells and mice, and we explore further the fate of MdmX/ p53-double null embryonic fibroblasts undergoing multi-polar cell division using time-lapse video microscopy. We also discuss the relationship between chromosomal loss, cell proliferation, and the tumorigenic potential of p53-deficient cells lacking MdmX.

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 afecting 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, abrDl, reduces the expression of aidB observed when aeration is ceased and oxygen becomes limiting. Genetic and phenotypic analysis of the abrDI mutation demonstrates that it is an allele of rpoS. Thus, aidB is a member of the family of genes that are transcribed by a crs-directed RNA polymerase holoenzyme. Examination ofaidB expression in an rpoS insertion mutant strain indicates that both rpoSl3::TnlO and abrDl 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 elect on the adadependent alkylation induction of aidB.The Escherichia coli aidB gene is one of several genes induced in response to alkylation damage caused by treatments with agents that methylate DNA. The aidB gene encodes a protein that has a high degree of homology to several mammalian coenzyme A dehydrogenases (12). In agreement with this homology, it has been shown that the aidB gene product has isovaleryl coenzyme A dehydrogenase activity (12), an activity known to be required for leucine metabolism in mammalian cells (8,9). Overexpression of aidB protein has also been shown to reduce the mutagenic effects of the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, although the mechanism responsible for this resistance is unclear (12). Thus, aidB has roles in basic metabolism as well as defenses against methylation damage.In E. coli, the Ada protein mediates the response to alkylation damage, which has been called the adaptive response. Adaptive-response induction occurs when the Ada protein transfers a methyl group from methylphosphotriesters formed in DNA by methylating agents to its own Cys-69 residue (for reviews, see references 12, 15, and 36). Methylation of Ada converts this protein to a positive regulatory factor that binds sequences upstream of the ada, alkA, and aidB promoters, stimulating their transcription (12,15,36). Unlike the ada-alkB operon and the alkA gene, aidB is also subject to a second form of regulation (18,37,39). This second aidB induction pathway is activated when cells are grown in the absence of aeration and does not require a functional ada gene (37). Thus, there are two independent aidB regulatory pathways: an ada-dependent alkylation induction pathway and an ada-independent pathway induced when aeration is ceased and oxygen becomes limiting.To examine further the regulatory pathway triggered in unaerated cultures, random mini-T...

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.