Differential Regulation of D-Type Cyclins by Insulin-Like Growth Factor-I and Serum in Multiple Myeloma Cells.

Abstract

Dysregulation of a D-type cyclin gene is an early and universal event in multiple myeloma (MM), but given the low proliferative activity in this disease, the functional significance of these genetic lesions is unclear. In this study we first examined the expression and regulation of D-type cyclins and other cell-cycle regulatory proteins in a panel of human myeloma cell lines (HMCLs) and in primary normal and malignant CD138+ plasma cells by Western blotting. D-type cyclins, cyclin dependent kinase-4 (CDK4), CDK6, p27, the retinoblastoma protein (pRb) and proliferating cell nuclear antigen (PCNA) were absent in normal bone marow (BM) CD138+ cells (n=5) and heterogeneously expressed in both HMCLs (n=11) and in primary CD138+ MM cells (n=20; 16 from BM aspirates and 4 extra-medullary). Furthermore, expression of these proteins was positively associated with disease progression. Nine of the primary malignant samples were, like normal CD138+ cells, negative for D-type cyclins, their associated kinases or pRB. Of these, one was from a newly diagnosed patient, six were from patients with stable disease and two from patients on treatment. The remaining eleven MM patient samples had heterogeneous expression of D-type cyclins and other cell cycle regulators, four of which had detectable CDK 4/6-phosphorylated pRb. All four of these were from patients with progressive disease, and three were extra-medullary. Furthermore, we demonstrate for the first time in both HMCLs and primary CD138+ MM cells, that cyclins D1 and -D2 are positively regulated by IGF-I and foetal calf serum leading to increased phosphorylation of pRb on CDK4/6 specific sites and an increase in cells in S (DNA synthesis) phase of the cell cycle. In addition, p27 was down-regulated by IGF-I and FCS in some HMCLs suggesting that this cyclin dependent kinase inhibitor also contributes to cell cycle regulation in myeloma cells. However, at low stoichiometric concentrations observed in primary malignant plasma cells, p27 was up-regulated by mitogenic stimuli, consistent with its role in stabilising cyclin D/CDK complexes. Immunoprecipitation analysis revealed that cyclins D1 and -D2 were present in complexes with both CDKs −4 and −6, suggesting that both of these kinases mediate the effect of cyclin D1 or cyclin D2 on pRb phosphorylation and cell cycle entry in these cells. Unlike HMCLs in which cyclin D2 was the primary controller of cell cycle exit/entry (MM1-S and NCI-H929), HMCLs harbouring an 11q13 IgH translocation (KMS12-BM and U266) were refractory to serum withdrawal, suggesting that 11q13 may confer growth factor independent expression of cyclin D1. To determine if cyclin D1 expression via 11q13 was sufficient to promote cell cycle progression, we functionally inactivated it using siRNA in KMS12-BM. Knock-down of cyclin D1 coincided with decreased CDK4/6-specific pRb phosphorylation and an increase in cells arrested in G1 phase of the cell cycle, confirming that cyclin D1 expression via 11q13 leads to cell cycle progression in MM cells, independent of exogenous growth factors. In summary, we show that D-type cyclins are functional in MM, differentially responsive to exogenous growth factors and that their expression is positively associated with aggressive disease.