Multiple mutations and frameshifts are the hallmark of defective hPMS2 in pZ189-transfected human tumor cells.

Two HeLa variants defective in the mismatch repair protein hPMS2 were isolated by selection for methylation tolerance. Neither variant expressed detectable hPMS2 protein as determined by western blotting. Cell extracts were defective in correcting a single base mispair and were unable to perform mismatch repair-dependent processing of a methylated DNA substrate. Correction of the repair defect and restoration of sensitivity to a methylating agent was achieved by introducing a wild-type copy of chromosome 7 on which the hPMS2 gene is located. Loss of hPMS2 function in the HeLa variants was associated with a 5-fold increase in mutation frequency in the supF gene of the pZ189 shuttle vector. Wild-type levels of mutagenesis were restored by the transferred chromosome 7. Comparisons of mutational spectra identified multiple base substitutions, frameshifts and, to a lesser extent, single base pair changes as the types of mutation which are selectively increased in a hPMS2-defective background. The location of multiple mutations and frameshifts indicates that misalignment-mediated mutagenesis could underlie most of these events. Thus the mutator phenotype associated with loss of hPMS2 most likely arises because of the failure to correct replication slippage errors. Our data also suggest that a considerable fraction of mutagenic intermediates are recognized by the hMutSbeta complex and processed via the hMLH1/hPMS2 heterodimer.

Combined mismatch and nucleotide excision repair defects in a human cell line: mismatch repair processes methylation but not UV- or ionizing radiation-induced DNA damage.

Interaction between long patch mismatch repair (MMR) and persistent DNA O6-methylguanine or 6-thioguanine (6-TG) is implicated in the cytotoxicity of methylating agents and 6-TG, respectively. Human cells with defective MMR tolerate DNA methylation damage and are cross-resistant to 6-TG. To determine whether MMR contributes to the lethal effects of persistent UV-induced DNA lesions, MMR deficiency was introduced into nucleotide excision repair (NER)-defective XP12RO cells. The doubly repair-defective cells, designated XP12ROB4, did not express detectable hMSH2 protein. They had the mutator phenotype, N-methyl-N-nitrosourea and 6-TG resistance typical of MMR-defective cells. Active MMR was not required for the cytotoxicity of UV light, and the hMSH2 defect did not detectably alter the survival of XP12ROB4. The level of spontaneous or UV-induced SCE was also similar in XP12RO and XP12ROB4, indicating that hMSH2 is not required for this recombination process. The combined deficiency in MMR and NER did not confer a significant degree of tolerance to ionizing radiation, and the survival of XP12RO and XP12ROB4 after gamma-radiation was similar. Although it recognizes and processes some persistent damaged or modified DNA base pairs, MMR is unlikely to serve as a general sensor of DNA damage.

Increased somatic recombination in methylation tolerant human cells with defective DNA mismatch repair.

We have studied whether spontaneous intrachromosomal recombination is altered in methylation tolerant human cells with a defect in mismatch repair. Somatic recombination was analysed in HeLaMR cells containing the vector pTPSN, which carries two copies of the gene for hygromycin resistance. The hygromycin genes are both inactivated by an inserted HindIII linker but hygromycin-resistant clones can arise by recombination. The spontaneous rate of recombination in a clone of HeLaMR cells containing a single integrated copy of pTPSN (HeLaG1) was 3.1x10(-6)/cell per generation. Two methylation tolerant variants from HeLaG1 cells (clone 12 and clone 15) were isolated by exposure to MNNG. Clone 12 cells exhibited a 16-fold increase in spontaneous mutation rate at the HPRT gene and extensive microsatellite instability at both mono- and dinucleotide repeats. Microsatellite instability limited to mononucleotide repeats was found in clone 15, whereas the mutation rate at HPRT was not significantly affected. A mismatch binding defect in extracts of clone 15 could be complemented by exogenous GTBP but not by purified hMSH2 protein. These data suggest that clone 15 is defective in GTBP. Extracts of clone 12 were unable to correct a single C:T mispair and complementation by extracts of human colorectal carcinoma cells with known deficiencies in mismatch repair indicated a defect in hMutLalpha. Western blotting with antibodies against different human mismatch repair proteins showed that clone 12 cells did not express hPMS2 protein, but expression of hMLH1, hMSH2 and GTBP appeared normal. The spontaneous recombination rate of clone 12 was 19-fold higher than the parental HeLaG1 cells, whereas no increase was observed in clone 15. Analysis of individual recombinants showed that hygromycin resistance arose exclusively by gene conversion. Our data indicate that mismatch correction regulates somatic recombination in human cells.

Normal rat intestinal cells IEC-18: characterization and transfection with immortalizing oncogenes.

IEC-18 cells, a cell line derived from the ileum of rat intestine, have the characteristics of normal cells since they have a contact inhibited cell growth, do not form colonies in soft agar and are not tumorigenic when injected in nude mice. IEC-18 cells were transfected with nuclear oncogenes, c-myc, v-myc and SV40 T antigen in order to obtain immortal cell lines. Independent clones were isolated and characterized for the growth properties. Expression of v-myc altered the morphology of the cells and shortened the doubling time. A slow growth together with a low cloning efficiency was associated with the expression of SV40 T antigen. No changes either in growth or in morphology were observed in c-myc-expressing IEC-18 cells. Expression of these nuclear oncogenes did not result in the neoplastic transformation of the IEC-18 cells, since none of the clones lost the anchorage dependence or were able to form tumors in vivo. The c-myc-containing IEC-18 cells were unable to secrete in the growth medium TGF α and exposure to TGF ß inhibited the growth rate by 30%. All these observations are consistent with the conclusion that the expression of nuclear oncogenes does not lead to the neoplastic transformation of these cells.

Mutation analysis in two newly identified rat p53 pseudogenes.

We have analysed the genomic organization of the rat p53 gene in normal intestinal rat cells. Exons 5-8 of the p53 gene were amplified by PCR from rat genomic DNA using primers complementary to stretches of nucleotides identical in rat and human cDNAs. Two amplification products of 727 and 339 bp were obtained from the rat DNA. The PCR products were subcloned into M13mp18 and sequenced. By comparison with the rat cDNA sequence the 727 bp band was identified as the functional p53 gene containing exons 5, 6 7 and 8 and introns 5 and 7. The sequences corresponding to human intron 6 are absent from the rat p53. The second amplification product was a mixture of two different 'processed' pseudogenes, in which 42 mutations accumulated in a sequence corresponding to the cDNA between exons 5 and 8. Analysis of these mutations shows that in both pseudogenes the vast majority is constituted by base substitutions, with transitions being more frequent than transversions. The most prominent mutational class is formed by G-->A transitions which are predominantly located at CpG sites. Since a high level of homology is present between the rat and the human cDNA, the type and the positions where mutations occur in the two rat pseudogenes is discussed in relation to the possible origin of these mutations in human tumors.

Genetic tests to reveal TAT homodimer formation and select TAT homodimer inhibitor.

The HIV Tat protein is essential for productive infection and is a potent activator of viral gene expression. By constructing a genetic fusion between the amino-terminal DNA-binding domain of the lambda repressor (as a reporter for dimerization) and Tat, we show that Tat forms dimers in vivo. By deletion analysis and site-directed mutagenesis, we show that (i) the peptide encoded by exon-1 of Tat is sufficient to promote dimerization and (ii) cys37 is essential for homo-dimerization of Tat protein. Furthermore, by using a new E. coli strain in which the expression of beta-galactosidase is under the negative control of the cl::Tat repressor, we select a protein (CD10/Nep) expressed by human Jurkat T-cells which inhibits Tat dimerization.

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo.

Evidence is presented that the pR bat gene is essential for plasmid replication and for spontaneous induction of the SOS response in Escherichia coli. Mutations preventing single-stranded DNA production, needed for pR plasmid replication, also prevent the induction of the SOS system. The following experimental design was used. Firstly, we identified the minima rep region, defined as the minimal DNA sequence necessary for pR plasmid replication and, secondly, analyzed the nucleotide sequence of this region. This identified structures and functions (ori-plus, ori-minus and Rep protein) homologous to those found in phages and plasmids replicating by the rolling-circle mechanism. Finally, mutations were introduced either in the replication protein catalytic site or in the nick site consensus sequence, which caused the pR plasmid to lose its ability to induce the SOS system. We conclude that, in this system, the in vivo SOS-inducing signal appears to be the single-stranded DNA produced during pR replication.

Growth properties, differentiation capacity and oncogene expression in metastatic and nonmetastatic Friend leukemia cell variants.

The aims of this study were: (1) to characterize the biologic properties of the WGA-resistant (WR) Friend leukemia cells (FLC) as compared to the original nonmetastatic or highly metastatic FLC; (2) to investigate the possible correlations between the expression of some oncogenes (i.e., c-myc, H-ras and K-ras) and the in vitro and in vivo behavior of FLC. The tumorigenic behavior of the different FLC types strongly depended on the site of tumor injection. Both WR FLC and in vitro passaged FLC did not grow as ascites (when injected intraperitoneally) and developed large solid tumors (when injected subcutaneously), without forming any spleen or liver metastasis. In contrast, in vivo passaged FLC rapidly formed hemorrhagic ascites when injected intraperitoneally; the subcutaneous injection of these cells resulted in the development of solid tumors, which were smaller than the other FLC tumors, but capable of metastasizing to the liver and to the spleen. No significant differences were observed in the in vitro growth characteristics and cell cycle parameters among the different FLC types under various experimental conditions (i.e., FCS concentration or cell seeding densities). Similarly to the metastatic in vivo passaged parental cells, WR FLC exhibited a much lower erythroid differentiation after in vitro addition of either dimethyl sulfoxide or hexamethylene bisacetamide than the in vitro passaged FLC. High levels of c-myc oncogene mRNA were expressed in all FLC variants; no major variations in the c-myc expression were observed in FLC cultivated in medium supplemented with different FCS concentrations and/or seeded at various cell densities. In addition, no changes in the expression of H-ras or K-ras were observed between the different FLC types.

cis-acting sequences that control the level of viral DNA synthesis in the polyomavirus late region.

A deletion in the polyomavirus late region results in a drastic reduction of viral replication, as shown after transfection of viral DNA into 3T6 cells. This mutation is cis acting, since cotransfection with wild-type DNA did not restore the normal phenotype. Viral DNA synthesis returned to normal levels only after reintroduction of the authentic sequences in either orientation. The data presented here suggest that these sequences are involved in the binding of a factor(s) that controls the level of viral replication.