Defective DNA repair genes in a primary culture of human renal cell carcinoma.

PURPOSE: Genomic stability is maintained by error-free DNA replication, repair, and recombination. To determine if repair genes contribute to genomic instability, we used a newly established cell line RCC-AJR (from clear-cell renal cell carcinoma) to examine hMSH2 (a mismatch-repair gene) and the gene encoding DNA beta polymerase (polbeta; a known contributor to base-excision repair). METHODS: Coding sequences of hMSH2 and polbeta were amplified by the polymerase chain reaction (PCR) using RNA from RCC-AJR cells and matched normal kidney (NK) cells from the same patient. Nucleotide sequences of the PCR products were determined by the dideoxy-DNA method and direct sequencing. Expressions of repair genes were assayed by Western blotting. Microsatellite stability in RCC-AJR cells was assayed by alteration in (CA)n repeats. RESULTS: In the RCC-AJR cells, we detected (a) a deletion of 1476 bp encoding 492 amino acids of hMSH2 cDNA, (b) an 87-bp deletion in the polbeta coding sequence, (c) truncated forms of hMSH2 and polbeta proteins, and (d) microsatellite instability. CONCLUSIONS: This study provides evidence of alterations in hMSH2 and polbeta in the homogeneous cell population of an RCC-AJR tumor culture. The data indicate that repair genes may help preserve genomic stability in this cell line. We believe that this new primary RCC-AJR cell line will prove a useful model for investigating the cascade of genetic events in renal cells that leads to renal carcinogenesis.

The adenovirus E1A 289R and 243R proteins inhibit the phosphorylation of p300.

A protein of 300 kDa (p300) associates with the adenovirus E1A proteins and has been implicated in the control of cell cycle progression. In mammalian cells, p300 is actively phosphorylated in both quiescent and proliferating cells and its level of phosphorylation increases as it travels from late G1 into M phase. E1A requires p300 for the induction of cellular DNA synthesis and the repression of enhancer mediated transcription, suggesting that p300 may be involved in pathways that are important to cell proliferation and gene expression. Since the activities of most cell cycle regulatory proteins depend on their phosphorylation state, the possibility exists that certain activities of p300 might also be controlled by phosphorylation and that E1A might in fact be affecting these events. We show here by in vitro analysis that E1A inhibits the phosphorylation of p300 by decreasing the rate of incorporation of phosphate into p300. We also show that p300 can be used as a substrate for the cyclin-dependent p33cdk2 and p34cdc2 kinases, and propose that E1A might be antagonistic to these enzymes in phosphorylating p300. Thus, these results indicate a possible novel function by which E1A can interfere with cellular pathways.

Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain.

DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.