ALBERT

Description: The murine double minute 2 (MDM2) protein facilitates G1 to S phase transition by activation of E2F-1 and can enhance cell survival by suppressing wild-type p53 (wtp53) function. In this study, we examined MDM2 expression and function in multiple myeloma (MM) cells. MDM2 is strongly and constitutively expressed in MM cell lines (ARH-77, RPMI 8226, and OCI-My5) and in the cells of plasma cell leukemia (PCL) patients, but is not expressed in normal bone marrow mononuclear cells (BM MNCs). Treatment of MM cells with MDM2 antisense, but not sense, nonsense, or scrambled, oligodeoxyribonucleotides (ODNs) decreased DNA synthesis and cell viability; it also induced G1 growth arrest, as evidenced by propidium iodide (PI) staining and induction of retinoblastoma protein (pRB) to E2F-1 binding. Moreover, inhibition of MDM2 using antisense ODNs also triggered MM cell apoptosis as evidenced by acridine orange–ethidium bromide staining. We next studied the association of MDM2 with wtp53 and/or mutant p53 (mtp53), E2F-1, CDK4, and p21. MDM2 constitutively binds to E2F-1 in all MM cells, to both wtp53 and mtp53, and to p21 in tumor cells lacking p53. These data suggest that MDM2 may enhance cell-cycle progression in MM cells both by activating E2F-1 and by downregulating cell-cycle inhibitory proteins (wtp53 and p21). Overexpression of MDM2 may therefore contribute to both growth and survival of MM cells, suggesting the potential utility of treatment strategies targeting MDM2 in MM.

Description: Homozygous p16INK4A (p16) gene deletion is frequent in primary tumor cells from acute lymphoblastic leukemia (ALL), suggesting that loss of p16 may be an important precursor to transformation in ALL. We have previously described JKB, a human ALL cell line, that contains homozygous deletion of the p16 gene. Because ectopic expression of p16 suppresses cell growth, we created a temperature sensitive p16 mutant to develop a system for inducible p16 function in human ALL. JKB cells were transfected either with a p16 gene mutated at position 119 (E119G) to confer temperature sensitivity (JKB p16MT) or with control vector. The percentage of cells in G1 phase was similar in JKB control cells or in JKB p16MT cells cultured at restrictive conditions (40°C). However, with lowering of temperature from 40°C to permissive conditions (31°C), the percentage of JKB p16MT cells in G1 phase and binding of p16 to CDK4 and CDK6 increased, with associated decreases in CDK4 and CDK6 kinase activities, and dephosphorylation of retinoblastoma protein (pRB). Culture of JKB p16MT cells at 31°C for ≥3 days irreversibly inhibited growth. Moreover, JKB p16MT cells cultured under these permissive conditions showed a less transformed morphology and more differentiated phenotype than did these cells cultured under restrictive temperatures. Finally, dexamethasone (Dex) induced apoptosis of JKB p16MT cells cultured at 40°C, but did not trigger death of these cells cultured at 31°C. These results suggest that deletion of p16 gene in JKB human ALL cells is associated with dysregulated growth of less differentiated tumor cells, which nonetheless remain susceptible to apoptosis triggered by Dex.

Description: Interleukin-6 (IL-6) mediates autocrine and paracrine growth of multiple myeloma (MM) cells and inhibits tumor cell apoptosis. Abnormalities of retinoblastoma protein (pRB) and mutations of RB gene have been reported in up to 70% of MM patients and 80% of MM-derived cell lines. Because dephosphorylated (activated) pRB blocks transition from G1 to S phase of the cell cycle whereas phosphorylated (inactivated) pRB releases this growth arrest, we characterized the role of pRB in IL-6-mediated MM cell growth. Both phosphorylated and dephosphorylated pRB were expressed in all serum-starved MM patient cells and MM-derived cell lines, but pRB was predominantly in its phosphorylated form. In MM cells that proliferated in response to IL-6, exogenous IL-6 downregulated dephosphorylated pRB and decreased dephosphorylated pRB-E2F complexes. Importantly, culture of MM cells with RB antisense, but not RB sense, oligonucleotide (ODN) triggered IL- 6 secretion and proliferation in MM cells; however, proliferation was only partially inhibited by neutralizing anti-IL-6 monoclonal antibody (MoAb). In contrast to MM cells, normal splenic B cells express dephosphorylated pRB. Although CD40 ligand (CD40L) triggers a shift from dephosphorylated to phosphorylated pRB and proliferation of B cells, the addition of exogenous IL-6 to CD40L-treated B cells does not alter either pRB or proliferation, as observed in MM cells. These results suggest that phosphorylated pRB is constitutively expressed in MM cells and that IL-6 further shifts pRB from its dephosphorylated to its phosphorylated form, thereby promoting MM cell growth via two mechanisms; by decreasing the amount of E2F bound by dephosphorylated pRB due to reduced dephosphorylated pRB, thereby releasing growth arrest; and by upregulating IL-6 secretion by MM cells and related IL-6- mediated autocrine tumor cell growth.