Sequestration of p27protein by cyclin D1 in typical and blastic variants of mantle cell lymphomas (MCL): Implications for pathogenesis

P27 is a cyclin dependent kinase inhibitor that plays a critical role in regulating G1/S progression, and whose activity is, in part, regulated through interactions with D type cyclins. Mantle cell lymphoma (MCL) is characterized by the t(11;14) translocation resulting in deregulated cyclin D1. We previously showed that p27 expression, as assessed by immunohistochemistry (IHC) in MCL, does not show the usual inverse relationship to proliferate seen in most other lymphomas that do not overexpress cyclin D1. This suggested that the normal expression and/or control of p27 activity on cell growth might be altered through potential interactions with cyclin D1. Using Western blot and coimmunoprecipitation studies, we assessed the interrelationship between cyclin D1 and p27 in several cyclin D1+ cell lines and primary MCL cases. Similar to our previous results by IHC, typical MCL showed lower expression of p27 when compared to the more highly proliferative blastic cases or cell lines (mean arbitrary units: 58 Vs 236 Vs 120). Cyclin D1 was expressed at variable levels in both typical and blastic MCL. P27 protein could be consistently coimmunoprecipitated with cyclin D1 from both cell lines and cases. Using techniques of exhaustive immunoprecipitation, we could demonstrate that most p27 protein was sequestered into cyclin D1 containing complexes. We hypothesize that mantle cell lymphomagenesis results not only from direct consequences of inappropriate cyclin D1 expression, but also from the ability of overexpressed cyclin D1 to buffer physiologic changes in p27 levels, thereby rendering p27 ineffective as an inhibitor of cellular growth. Email:[email protected] only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Quintanilla-Martinez, et al 3 INTRODUCTION: Progression through the various phases of the cell cycle depends upon the orderly activation of specific cyclin-cyclindependent kinase (CDK) complexes.1 Specific inhibitory proteins, as well as, positive and negative phosphorylation events play critical roles in regulating the activation of cyclin/CDK complexes during the cell cycle. 1-7 P27 kip1 is a cyclin dependent kinase inhibitor that plays a critical role in regulating G1/S progression, at a stage called the restriction point, preceding the onset of DNA synthesis.8-11 P27 kip1 binds to both cyclin D/CDK4 complexes and cyclin E/CDK2 complexes with very different effects.5, 6 While p27 kip1 binds more efficiently with cyclin D/CDK4 complexes,3 this interaction does not inhibit the cyclin D1/CDK4 kinase activity but rather results in the stabilization of the complex.5 On the contrary, the interaction of p27 kip1 with cyclin E/CDK2 complexes inactivates the kinase activity that is essential for cell-cycle progression.12, 13 As cells progress through G1, cyclin D levels increase and compete with cyclin E for free p27 kip1 , while CAK kinase begins to activate CDK2.5 These two events respectively result in decreased levels of free p27 kip1 potentially available to inhibit the cyclin E/CDK2 kinase, and to the degradation of p27 kip1 bound to the cyclin E/CDK2 complex. Activated CDK2 phosphorylates p27 kip1 at Thr 187,14 triggering its subsequent ubiquitination and degradation by the ubiquitin ligase F-box protein Skp2.15-18 In contrast, activated cyclin D1/CDK4 complexes do not seem to be involved in p27 kip1 degradation.5, 12 We recently reported19 that p27 kip1 expression in normal lymphoid tissue and in lymphoid neoplasms is inversely proportional to the proliferation index as measured by Ki-67, confirming the important role of p27 kip1 as a negative regulator of cell cycle progression. However, in typical mantle cell lymphomas (MCL), we could detect no or very low levels of p27 kip1 , despite their generally low proliferation rate. Paradoxically, the blastic variant of MCL, only. For personal use at PENN STATE UNIVERSITY on February 23, 2013. bloodjournal.hematologylibrary.org From Quintanilla-Martinez, et al 4 with a much higher proliferation rate, expressed higher levels of p27 kip1 . The lack of immunologically detectable expression of p27 kip1 in typical MCL was not due to p27 kip1 gene deletions, because none of the 25 MCL cases examined showed gross rearrangement or deletion of the gene. This finding was consistent with previous studies in a variety of human primary tumors and cancer cell lines that have shown only rare mutations of the p27 kip1 gene.20-