ORIGINAL ARTICLE Human and mouse cyclin D2 splice variants: transforming activity and subcellular localization C Denicourt 1 , P Legault 1 , F-AC McNabb and E Rassart Laboratoire de Biologie Mole´culaire, De´partement des Sciences Biologiques, Universite´du Que´bec a` Montre´al, Que´bec, Canada We have previously reported the identification of a novel 17 kDa truncated isoform of the cyclin D2 activated in 13% of the leukemias induced by the Graffi murine leukemia retrovirus. Retroviral integration in the Gris1 locus causes an alternative splicing of the mouse cyclin D2 gene and expression of a truncated protein of 159 amino acids that is detected at high levels in the Gris1 tumors and also in normal mouse tissues mainly the brain and ovaries. A truncated form of the cyclin D2 was also found in human. We show here that both mouse- and human- truncated cyclin D2 are able to transform primary mouse embryo fibroblasts (MEF) when co-expressed with an activated Ras protein. The truncated cyclin D2 localizes only to the cytoplasm of transfected cells. It has retained the ability to interact with cyclin-dependent kinases (CDKs), although it is a poor catalyst of pRb phosphor- ylation. Interestingly, the presence of a similar, alterna- tively spliced cyclin D2 mRNA was also detected in some human brain tumors. Oncogene (2008) 27, 1253–1262; doi:10.1038/sj.onc.1210750; published online 17 September 2007 Keywords: truncated cyclin D2; transformation; splice variant Introduction Slow-transforming murine retroviruses can induce leukemia in their host by insertional mutation of cellular proto-oncogenes or tumor suppressor genes. Following the retroviral integration, cellular genes may become aberrantly expressed through activation by viral pro- moter or enhancer sequences. The abnormal expression of a certain proto-oncogene may then provide a growth advantage to the target cell and contribute to malignant transformation. During the past years an important number of critical cancer genes have been identified in these leukemias by proviral tagging. We have recently characterized Gris1 as a novel common integration site in 13% of Graffi murine retrovirus-induced leukemias (Denicourt et al., 2002). This novel locus was located on mouse chromosome 6, 75–85 kbp upstream of the cyclin D2 gene. Integrations in Gris1 were shown to activate the expression of the 6.5 kb major transcript of the cyclin D2 gene (CCND2) and also a smaller 1.1 kb transcript that we have shown to represent an alter- native transcript encoding a 17 kDa truncated cyclin D2 protein. The protein is detected at high level in the Gris1 tumors and also in smaller quantities in normal tissue mainly in the brain and ovaries (Denicourt et al., 2002). We also have reported the existence of several expressed sequence tags (ESTs) in the databases encoding a similar truncated cyclin D2 in humans. Moreover, such a transcript was also reported in Xenopus laevis cloned from an ovary cDNA library (Taı¨eb and Jessus, 1996). Cyclin D2 is a G 1 cyclin that belongs to the family of three closely related D-type cyclins, namely, D1, D2 and D3. These cyclins have been identified as positive regulators of the cell cycle and have a well-established role in the progression through the G 1 phase of the cell cycle. D-type cyclins assemble with their cyclin-depen- dent kinase (CDK) partners CDK4 and CDK6 to phosphorylate target proteins such as pRb (reviewed in Sherr and Roberts, 1999; Ortega et al., 2002). Mitogenic signaling ultimately leads to the upregulated expression of D-type cyclins as well as their kinase-associated activity. Cyclin D2 is a well-defined human proto-oncogene and, when illegitimately activated or overexpressed, it can contribute to cellular transformation (reviewed in Malumbres and Barbacid, 2001). Indeed, deregulated expression of the cyclin D2 gene has been reported for the first time in murine leukemia virus-induced T-cell leukemias by the identification of the common viral inte- gration site vin1 (Tremblay et al., 1992; Hanna et al., 1993). In collaboration with an activated Ras (H-RasV12), overexpressed cyclin D2 can transform primary rat embryo fibroblasts (REF) (Kerkhoff and Ziff, 1995). In human, overexpression of cyclin D2 is associated with different types of lymphoid malignancy, testicular carcinoma and ovarian cancer (Sicinski et al., 1996; Bartkova et al., 1999; Teramoto et al., 1999). In this article, we present evidence that the over- expression of the truncated cyclin D2 can transform primary MEF in conjunction with activated Ras. We also present evidence for a human homolog transcript of Received 4 June 2007; revised 25 July 2007; accepted 26 July 2007; published online 17 September 2007 Correspondence: Dr E Rassart, De´ partement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, Case Postale 8888 Succ. Centre- ville, Montre´al, Canada H3C-3P8. E-mail: Rassart.Eric@UQAM.ca or ericrassart@gmail.com 1 These authors contributed equally to this work. Oncogene (2008) 27, 1253–1262 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc