Inhibition of HDM4 Induces p21 Expression in Mantle Cell Lymphoma
M Liang, X Han, M Fernandez, M Nguyen, GZ Rassidakis, I Drakos, LJ Medeiros, CE Bueso-Ramos. University of Texas, M.D. Anderson Cancer Center, Houston, TX
Background: Alterations of the p53-murine double minute 2 (MDM2)/MDM4-pathway are thought to contribute to the pathogenesis of a variety of malignant neoplasms. Haploinsufficiency of MDM4 reduced lymphomagenesis in E-myc transgenic mice, a model of non-Hodgkin's lymphoma. p53 expression leads to increased expression of p21, a negative cell cycle regulatory protein that inhibits cyclin D1 activity. In mouse model and cell line studies, MDM2 and MDM4 have been shown to synergistically promote proteosomal mediated degradation of p21 and p53. MDM4 also inhibits p53-mediated transcriptional activation of p21. The aim of this study was to assess for Human homolog of MDM4 (HDM4) expression and its effect on p21 in mantle cell lymphoma (MCL).
Design: We assessed HDM4 expression using immunohistochemical methods in reactive lymph nodes and MCL. To characterize the differential upregulation of HDM4 in MCL, HDM4 mRNA expression was assessed by quantitative real-time polymerase chain reaction (RT-PCR) in MCL cell lines (Granta 519, Z-138, SP-53, and Mino) and leukemia patient peripheral blood samples. After using small interfering RNA (siRNA) to inhibit HDM4 in MCL cell lines, p21 protein levels were measured by immunocytochemical and Western blot analysis. Activation of the p53-apoptotic pathway was assessed by caspase-3 expression by immunocytochemical staining.
Results: HDM4 nuclear expression was observed in 18 of 19 MCL, but was negative in mantle zone B-cells of reactive lymph nodes (n=19) (p<0.01). HDM4 overexpression was confirmed by RT-PCR in all 4 MCL cell lines and 6 MCL patient specimens. Both splicing variants, HDM4-S (containing only p53 binding domain) and full length (FL)-HDM4, were increased compared with normal CD19+ B-cells. The HDM4-S to HDM4-FL ratio was significantly increased in MCL compared with normal CD19+ B-cells (p<0.05). After introducing siRNA inhibition of HDM4 in two MCL cell lines, SP53 and Mino, p21 protein levels were induced as shown by Western blot analysis. Immunocytochemical analysis also showed increased p21 expression and active caspase-3, the latter indicating increased apoptosis.
Conclusions: HDM4 is overexpressed in MCL and, at least in part, may exert its effect by suppressing p21 expression thereby enhancing cell cycle progression. Inhibition of HDM4 may serve as a potential therapeutic remedy in MCL by inhibiting cell cycling and inducing apoptosis.
Tuesday, March 10, 2009 9:30 AM
Poster Session III # 119, Tuesday Morning