Potential role for concurrent abnormalities of the cyclin D1, p16^CDKN2 and p15^CDKN2B genes in certain B cell non-Hodgkin’s lymphomas. Functional studies in a cell line (Granta 519)

Abstract

Abnormalities of several cell-cycle regulatory genes including cyclin D1, p16^CDKN2 and p15^CDKN2B have been described in B cell non-Hodgkin’s lymphoma (B-NHL). We describe a new B-NHL cell line (Granta 519), with concurrent abnormalities of the cyclin D1, p16^CDKN2 and p15^CDKN2B genes. An independent clinical case of mantle cell NHL (Mc-NHL) with concomitant overexpression of cyclin D1, and deletion of the p16^CDKN2 gene was also identified, suggesting that this combination of oncogenic aberration is a pathophysiologic contribution to a subset of NHL cases. More in-depth functional studies of this concept were facilitated by the availability of the cell line Granta 519 which was derived from a case of high-grade NHL and has a mature B cell immunophenotype. Cytogenetic analysis identified translocation t(11;14)(q13;q32) and complex rearrangements involving chromosomes 9p22, 13p21, 17p11, and 18q21. Molecular analysis identified overexpression of cyclin D1 mRNA and biallelic deletion of the p16^CDKN2 and p15^CDKN2B genes. To elucidate the effect of these genetic abnormalities on the G1 control of Granta 519 cells, the level and function of the major components of the cyclinD/retinoblastoma (RB) pathway were investigated. Cyclin D1 was dominant among the D-type cyclins, formed abundant complexes with cyclin-dependent kinase (Cdk) Cdk4 rather than Cdk6, and the immunoprecipitated cyclin D1/Cdk4 holoenzyme was active as a pRB kinase. Electroporation of wild-type p16^CDKN2 arrested the Granta 519 cells in G1, consistent with the p16^CDKN2 loss as a biologically relevant event during multistep evolution of the tumor, and with the expression of functional pRB. Direct cooperation of these distinct abnormalities to cell-cycle deregulation in NHL cells was suggested by G1 acceleration upon inducible overexpression of cyclin D1 in a control breast cancer cell line lacking p16^CDKN2 , an effect which could be prevented by ectopic expression of p16^CDKN2. Taken together, these data suggest that concurrent overexpression of cyclin D1 and functional elimination of p16^CDKN2 and p15^CDKN2B may characterize certain cases of mantle cell NHL, and that cooperation of the abnormalities is likely to provide a growth advantage of the tumour cells through more efficient inactivation of the RB tumor suppressor. Further clinicopathologic studies of this possibility are warranted.