ABSTRACT
Previous studies have shown that UV-induced binding of p21(WAF1) to PCNA through the PCNA-interacting protein (PIP) domain in p21(WAF1) promotes a switch from DNA replication to DNA repair by altering the PCNA protein complex. Here we show that the p33(ING1b) isoform of the ING1 candidate tumour suppressor contains a PIP domain. UV rapidly induces p33(ING1b) to bind PCNA competitively through this domain, a motif also found in DNA ligase, the DNA repair-associated FEN1 and XPG exo/endonucleases, and DNA methyltransferase. Interaction of p33(ING1b) with PCNA occurs between a significant proportion of ING1 and PCNA, increases more than tenfold in response to UV and is specifically inhibited by overexpression of p21(WAF1), but not by p16(MTS1), which has no PIP sequence. In contrast to wild-type p33(ING1b), ING1 PIP mutants that do not bind PCNA do not induce apoptosis, but protect cells from UV-induced apoptosis, suggesting a role for this PCNA-p33(ING1b) interaction in eliminating UV-damaged cells through programmed cell death. These data indicate that ING1 competitively binds PCNA through a site used by growth regulatory and DNA damage proteins, and may contribute to regulating the switch from DNA replication to DNA repair by altering the composition of the PCNA protein complex.
Subject(s)
Apoptosis/physiology , Proliferating Cell Nuclear Antigen/metabolism , Proteins/genetics , Proteins/metabolism , Binding Sites/physiology , Cell Cycle Proteins , Cell Line , Cell Nucleus/metabolism , DNA Repair/physiology , DNA Replication/physiology , DNA-Binding Proteins , Fibroblasts/cytology , Gene Expression , Genes, Tumor Suppressor , Humans , Inhibitor of Growth Protein 1 , Intracellular Signaling Peptides and Proteins , Nuclear Proteins , Protein Binding/physiology , Protein Binding/radiation effects , Protein Structure, Tertiary , Proteins/chemistry , RNA Splicing , Tumor Suppressor Proteins , Ultraviolet RaysABSTRACT
Nine monoclonal antibodies (MAbs) against human and rodent ING1 protein have been generated using an IL6-secreting mouse myeloma line. These antibodies are all effective in recognizing ING1 protein in ELISAs, Western blot assays, and by indirect immunofluorescence. Combining different CAb monoclonal antibodies in a Western blot assay also allows detection of the very low levels of endogenous ING1 found in fibroblast cells in culture and the identification of at least two isoforms of ING1 in normal human diploid fibroblasts and established brain cancer cell lines.
Subject(s)
Antibodies, Monoclonal/immunology , Proteins/immunology , Animals , Antibodies, Monoclonal/chemistry , Blotting, Western , Cell Cycle Proteins , DNA-Binding Proteins , Enzyme-Linked Immunosorbent Assay , Fibroblasts/cytology , Fluorescent Antibody Technique, Indirect , Genes, Tumor Suppressor , Humans , Immunoglobulin Isotypes , Inhibitor of Growth Protein 1 , Intracellular Signaling Peptides and Proteins , Mice , Mice, Inbred BALB C , Nuclear Proteins , Protein Isoforms/immunology , Transfection , Tumor Cells, Cultured , Tumor Suppressor ProteinsABSTRACT
Involvement of the retinoblastoma susceptibility (RB-1), p16INK4, p53 and telomerase genes in immortalisation was examined by determining their status in 15 human cell lines representing four immortalisation complementation groups. No abnormalities of RB-1, p53 and p16INK4 were detected in cell lines containing DNA tumour virus proteins known to bind to the protein products of the RB-1 and p53 genes. In contrast, in all other cell lines from each of the four groups either RB-1 was mutant or p16INK4 protein was undetectable and there were cell lines containing p53 mutations in three of the groups. Telomerase activity was detected in 12/15 lines, including some of the virally immortalised lines and in some lines from each group. Since none of these changes correlated with complementation group, other genetic changes must be required for immortalisation.
