Malignant transformation of human fibroblast cell strain MSU-1.1 by N-methyl-N-nitrosourea: evidence of elimination of p53 by homologous recombination.

To determine whether N-methyl-N-nitrosourea (MNU) can induce malignant transformation of human fibroblasts and whether O6-methylguanine (O6-MeG) is involved, two populations of infinite life span cell strain MISU-1.1, differing only in level of O6-alkylguanine-DNA alkyltransferase, were treated with MNU and assayed for focus formation. MNU caused a dose-dependent increase in the frequency of foci in both groups, but the dose required was significantly lower in the cells lacking O6-alkylguanine-DNA alkyltransferase, indicating that O6-MeG was causally involved. Of 35 independent focus-derived strains assayed for p53 transactivating abilily, one was heterozygous, and 15 had lost all activity, 1 of 7 from untreated cells and 14 of 27 from MNU-treated cells. These results indicate that loss of p53 is not required for focus formation but may permit cells to form foci. Of 35 strains assayed for tumorigenicity, 10 formed malignant tumors with a short latency, all 10 lacked wild-type p53. The p53 heterozygous strain also formed tumors after a long latency, and the cells from those tumors lacked p53 transactivating ability. None of the 19 strains with wild-type p53 formed tumors. These results indicate that although loss of p53 is not sufficient for malignant transformation of MSU-1.1 cells, it may be necessary. Analysis of the p53 cDNA from several focus-derived strains lacking p53 activity revealed that each contained the same mutation, an A to G transition at codon 215, resulting in a change from serine to glycine. Because p53 can be inactivated by mutations at any one of a large number of sites, finding the same mutation in each strain assayed strongly suggests that the target population included a subpopulation of cells with this codon 215 mutation in one allele. Further analysis showed that all 15 focus-derived cells strains that lacked p53 transactivating activity contained two alleles, each with the same codon 215 mutation, and that the mutant allele in the heterozygous strain also had that mutatation. Analysis of the p arm of chromosome 17 of the focus-derived cell strains containing the codon 215 mutation revealed seven patterns of loss of heterozygosity, evidence of mitotic homologous recombination. Similar analysis of a separate series of cell strains, derived from foci induced by cobalt-60, revealed four patterns of loss of heterozygosity, only two of which had been found with those induced by MNU. These data suggest that homologous mitotic recombination, induced by O6-MeG in a subpopulation of cells heterozygous for p53 mutation, rendered the cells homozygous for loss of p53 activity, that this allowed the cells to form foci, and that although loss of p53 is not sufficient for malignant transformation, it predisposes cells to acquire the additional changes needed for such transformation.

The function of the human homolog of Saccharomyces cerevisiae REV1 is required for mutagenesis induced by UV light.

In Saccharomyces cerevisiae, most mutations induced by a wide range of mutagens arise during translesion replication employing the REV1 gene product and DNA polymerase zeta. As part of an effort to investigate mammalian mutagenic mechanisms, we have identified cDNA clones of the human homologs of the yeast REV genes and examined their function in UV mutagenesis. Previously, we described the isolation of a human homolog of yeast REV3, the catalytic subunit of pol zeta, and here report the identification and sequence of a human homolog of yeast REV1. This gene was isolated by identifying an expressed sequence tag encoding a peptide with similarity to the C terminus of yeast Rev1p, followed by sequencing of the clone and retrieval of the remaining cDNA by 5' rapid amplification of cDNA ends. The human gene encodes an expected protein of 1,251 residues, compared with 985 residues in the yeast protein. The proteins share two amino-terminal regions of approximately 100 residues with 41% and 20% identity, a region of approximately 320 residues with 31% identity, and a central motif in which 11 of 13 residues are identical. Human cells expressing high levels of an hREV1 antisense RNA grew normally, and were not more sensitive to the cytotoxic effect of 254 nm UV radiation than cells lacking antisense RNA. However, the frequencies of 6-thioguanine resistance mutants induced by UV in the cells expressing antisense hREV1 RNA were significantly lower than in the control (P = 0.01), suggesting that the human gene has a function similar to that of the yeast homolog.

Vanadium affects macrophage interferon-gamma-binding and -inducible responses.

Mouse WEHI-3 cells were exposed overnight to vanadium [V; ammonium metavanadate (NH4VO3) or vanadium pentoxide (V2O5)] to determine whether documented V-induced immunomodulation might arise from altered macrophage (M phi) interactions with interferon-gamma (IFN gamma) or altered IFN gamma-inducible responses. Binding studies performed at 22 degrees C indicated that although NH4VO3-pretreated cells had approximately 48% fewer actively-binding Class I IFN gamma receptors, binding affinities were 1.5-fold greater than that of control cell receptors; Class II expression was unaffected but affinities were reduced 2-fold. Postbinding IFN gamma-receptor complex internalization was unaffected by V pretreatment. Spontaneous production of both hydrogen peroxide and superoxide anion was significantly increased by treatment with both V compounds. Total hydrogen peroxide and superoxide production was increased by stimulation of IFN gamma-primed cells with zymosan, but relative increases in primed V-treated cells were lower than that in controls. Vanadium-treated cells also displayed decreased rates of IFN gamma-induced changes in [Ca2+]i levels secondary to increased resting [Ca2+]i levels. Although V-treated cells did not display significant increases in I-A expression after IFN gamma treatment, increased numbers of I-A+ cells (irrespective of priming) and lower maximal antigen densities than observed on I-A+ control cells were evident. Results from this study show that V exposure may produce alterations in M phi-mediated functions, in part, by modifying cell interactions with IFN gamma and subsequent IFN gamma-dependent functional parameters.