International Mammalian Genome Society

The 13th International Mouse Genome Conference
October 31-November 3, 1999

Table of Contents * Structure * Bioinformatics * Sequence * Mapping * New Tools * Gene Discovery * Developmental * Mutagenesis * Functional Genomics

E48 Relative Roles of p16INK4a and p19ARF as the Mouse Plasmacytoma Susceptibility Gene, Pctr1

Shuling Zhang, Edward S. Ramsay, Valery Bliskovsky and Beverly A. Mock. Laboratory of Genetics, National Cancer Institute, Bethesda, Maryland 20892

Mouse plasmacytomas are a model for human B cell malignancies. Cdkn2a was identified as a candidate locus for the plasmacytoma (PCT) susceptibility/resistance gene, Pctr1 . The Cdkn2a gene has two products, p16INK4a and p19ARF. p16INK4a acts as an inhibitor of cyclin-D-dependent kinases, preventing them from phosphorylating the retinoblastoma (Rb) protein and thus inhibiting S-phase entry during the cell division cycle; p19ARF, encoded in part by an alternative reading frame of Cdkn2a exon 2, is completely unrelated in its primary structure and regulates a p53 dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals. Allelic variants were observed in genetically susceptible and resistant strains of mice. The BALB/c variants of p16INK4a were inefficient in their ability to inhibit the activity of cyclin D2/CDK4 in kinase assays with retinoblastoma (Rb) protein. Overexpression of allelic variants did not lead to G1 arrest after 48 hours transfection in two PCT cell lines. In the current studies, the role of p19ARF in plasmacytomagenesis was investigated. To determine whether p19ARF has an effect on PCT growth, wild-type p19ARF and the G257A variant of p19ARF, specific to BALB/c, were transiently transfected into the same two PCT cell lines. Forty-eight hours after transfection, DNA content was assayed by flow cytometry to determine cell cycle distribution, with gating to restrict analysis to cells expressing a co-transfected marker, membrane targeted-GFP. There were different cell cycle profiles between p19ARF and p16INK4a in the two cell lines. Wild-type p19ARF and the G257A variant of p19ARF induced both G1 and G2 phase arrest in TEPC1165 cells, as well as in NIH3T3 cells. In contrast, MOPC460 cells did not exhibit G1 or G2 arrest following transfection with p19ARF. This result suggested that the p53 signaling pathway might be defective. A 21-base pair deletion of p53 was found in the MOPC460 cells. Western blots showed that p53 is still expressed in both cell lines. Apoptosis experiments are being done to check p53 function in the two cell lines. These transfection experiments continue to support an important role of the p16INK4a exon 1 allele in the genetic susceptibility of BALB/c mice for PCT induction. These results also confirm that p16INK4a and p19ARF utilize different pathways in cell cycle arrest.


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