Cyclin D1 Overexpression Increases the Susceptibility of Human U266 Myeloma Cells to CDK Inhibitors through a Process Involving p130-, p107- and E2F-Dependent S-Phase Entry

Abstract

Dysregulation of cyclin D1 expression is one of the most common genetic aberrations found in hematopoietic malignancies, including multiple myeloma. To address the effects of cyclin D1 overexpression might have on the response of malignant hematopoietic cells to CDK inhibitors, the impact of ectopic cyclin D1 overexpression on the response of human multiple myeloma U266 cells to various cyclin-dependent kinase (CDK) inhibitors was examined. Cyclin D1 overexpression markedly increased the apoptotic response of cells to the CDK inhibitors flavopiridol, roscovitine, and R-roscovitine. Ectopic expression of cyclin D1 resulted in p21CIP1 accumulation, an effect that was diminished by CDK inhibitor exposure. In pRb-null U266 cells, enforced overexpression of cyclin D1 diminished CDK inhibitor-mediated dephosphorylation of the pocket proteins p130 and p107, reduced binding of E2F1 and E2F4 to p130 and p107, and attenuated inhibition of E2F activity. Notably, CDK inhibitors failed to reduce the S-phase fraction in cyclin D1/U266 cells in contrast to effects in their wild-type counterparts. Finally, cyclin D1/U266 cells exhibited diminished basal NF-?B activity compared to controls, which was essentially completely abrogated by CDK inhibitor exposure. Together, these findings suggest that dysregulation of cyclin D1 sensitizes human myeloma cells to the actions of CDK inhibitors through mechanisms involving interference with p21CIP1 expression, dephosphorylation of pocket proteins and inactivation of E2Fs culminating in S-phase entry, as well as inactivation of NF-?B, leading to apoptosis rather than growth arrest.