Functional Analysis of p53 in the JMW Myeloma Cell Line.

Background: The inactivation of p53 by deletion of 17p13 is observed in 10% of multiple myeloma patients and is associated with shorter survival. In this study we were interested in assessing p53 function in the JMW myeloma cell line established from a patient with aggressive disease and circulating plasma cells. We characterized the plasma cells derived from the patient with the disease from the initial stage of procurement ofpatient tumor cells to permanence as a cell line.

Results: At diagnosis, 95% of the cells had monosomy 17 and 4% of the cells had a FISH pattern consistent with interstitial deletion of p53 (17p13). However, we observed clonal evolution in that at 3 months from diagnosis, 70% of the patient cells exhibited a deletion of the p53 locus while 28% of the cells had a monosomy 17. This may be attributed to favored clonal expansion of the cells with the deletion of p53 (17p13). To resolve this issue we performed further interphase and metaphase FISH and found an unbalanced complex translocation resulting in LOH of 13q and 17p (der13 t(13;17)(q?;p?). In addition, we screened the remaining p53 gene for mutations by conformation-sensitive gel electrophoresis (CSGE). PCR fragments showing abnormal CSGE-banding patterns (in exons 5 and 8) were further characterized by DNA sequence analysis and we identified a G to A splice site mutation generating an alternate splice site 21 bp into exon 5. This mutation results in a mutant protein lacking amino acids 126–132 in the DNA binding domain of p53. We observed no differences in the expression level of p53 mRNA as determined by RT-PCR analysis. We determined the effect of the deletion on p53 protein expression and function. We examined the effect of DNA damage induced by UV radiation on p53 protein levels in JMW cells, using the myeloma cell line H929 (expressing wildtype p53) as a positive control for expression. The JMW cells were exposed to UV radiation (20 J/m²) and the cells were harvested for western blot analysis 5 hours after UV damage. An increase in the level of p53 protein was observed in JMW as well as the H929 cells following UV treatment. We then investigated the activation of p53 downstream targets. The CDK inhibitor p21^WAF1/CIP1 is a well-characterized p53 target that mediates G1 cell cycle arrest. Thus, we examined the levels of p21 protein following UV treatment of JMW and H929 cells. Strikingly, JMW cells lacked p21 expression in untreated cells as well as UV exposed cells suggesting that the characterized deletion in the DNA binding domain is an inactivating mutation.

Conclusion: Abrogation of p53 function was observed in the JMW cells. Further analysis of the p53 pathway in JMW and other myeloma cell lines will provide an in depth understanding of the role of p53 in disease progression. The study of the p53 pathway in myeloma is highly relevant, as we have shown it to be associated with relapsing and aggressive disease, particularly with extramedullary myeloma. Our studies suggest that malignant plasma cells have the ability to survive outside of the bone marrow microenvironment but a p53 dependent checkpoint results in cell death. In clones with p53 defects the cells survive and result in extramedullary disease, including plasma cell leukemia.