Human and Mouse Cyclin D2 Splice Variants: Transformingactivity and Subcellular Localization
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Oncogene (2008) 27, 1253–1262 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Human and mouse cyclin D2 splice variants: transformingactivity and subcellular localization C Denicourt1, P Legault1, 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 characterized Gris1 as a novel common integration site 17 kDa truncated isoform of the cyclin D2 activated in in 13% of Graffi murine retrovirus-induced leukemias 13% of the leukemias induced by the Graffi murine (Denicourt et al., 2002). This novel locus was located on leukemia retrovirus. Retroviral integration in the Gris1 mouse chromosome 6, 75–85 kbp upstream of the cyclin locus causes an alternative splicingof the mouse cyclin D2 D2 gene. Integrations in Gris1 were shown to activate gene and expression of a truncated protein of 159 amino the expression of the 6.5 kb major transcript of the acids that is detected at high levels in the Gris1 tumors and cyclin D2 gene (CCND2)and also a smaller 1.1 kb also in normal mouse tissues mainly the brain and ovaries. transcript that we have shown to represent an alter- A truncated form of the cyclin D2 was also found in native transcript encoding a 17 kDa truncated cyclin D2 human. We show here that both mouse- and human- protein. The protein is detected at high level in the Gris1 truncated cyclin D2 are able to transform primary mouse tumors and also in smaller quantities in normal tissue embryo fibroblasts (MEF) when co-expressed with an mainly in the brain and ovaries (Denicourt et al., 2002). activated Ras protein. The truncated cyclin D2 localizes We also have reported the existence of several expressed only to the cytoplasm of transfected cells. It has retained sequence tags (ESTs)in the databases encoding a similar the ability to interact with cyclin-dependent kinases truncated cyclin D2 in humans. Moreover, such a (CDKs), although it is a poor catalyst of pRb phosphor- transcript was also reported in Xenopus laevis cloned ylation. Interestingly, the presence of a similar, alterna- from an ovary cDNA library (Taı¨ eb and Jessus, 1996). tively spliced cyclin D2 mRNA was also detected in some Cyclin D2 is a G1 cyclin that belongs to the family of human brain tumors. three closely related D-type cyclins, namely, D1, D2 and Oncogene (2008) 27, 1253–1262; doi:10.1038/sj.onc.1210750; D3. These cyclins have been identified as positive published online 17 September 2007 regulators of the cell cycle and have a well-established role in the progression through the G1 phase of the cell Keywords: truncated cyclin D2; transformation; splice cycle. D-type cyclins assemble with their cyclin-depen- variant 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 Introduction of D-type cyclins as well as their kinase-associated activity. Cyclin D2 is a well-defined human proto-oncogene Slow-transforming murine retroviruses can induce and, when illegitimately activated or overexpressed, it leukemia in their host by insertional mutation of cellular can contribute to cellular transformation (reviewed in proto-oncogenes or tumor suppressor genes. Following Malumbres and Barbacid, 2001). Indeed, deregulated the retroviral integration, cellular genes may become expression of the cyclin D2 gene has been reported for aberrantly expressed through activation by viral pro- the first time in murine leukemia virus-induced T-cell moter or enhancer sequences. The abnormal expression leukemias by the identification of the common viral inte- of a certain proto-oncogene may then provide a growth gration site vin1 (Tremblay et al., 1992; Hanna et al., advantage to the target cell and contribute to malignant 1993). In collaboration with an activated Ras (H-RasV12), transformation. During the past years an important overexpressed cyclin D2 can transform primary number of critical cancer genes have been identified in rat embryo fibroblasts (REF)(Kerkhoff and Ziff, 1995). these leukemias by proviral tagging. We have recently In human, overexpression of cyclin D2 is associated with different types of lymphoid malignancy, testicular Correspondence: Dr E Rassart, De´ partement des Sciences Biologiques, carcinoma and ovarian cancer (Sicinski et al., 1996; Universite´ du Que´ bec a` Montre´ al, Case Postale 8888 Succ. Centre- Bartkova et al., 1999; Teramoto et al., 1999). ville, Montre´ al, Canada H3C-3P8. In this article, we present evidence that the over- E-mail: [email protected] or [email protected] 1These authors contributed equally to this work. expression of the truncated cyclin D2 can transform Received 4 June 2007; revised 25 July 2007; accepted 26 July 2007; primary MEF in conjunction with activated Ras. We published online 17 September 2007 also present evidence for a human homolog transcript of Characterization of mouse and human truncated cyclin D2 C Denicourt et al 1254 a truncated cyclin D2 that is overexpressed in some types of primary human brain tumors. As opposed to 12 3 the subcellular localization of the cyclin D2 protein, this truncated cyclin D2 was never observed in the nucleus of NIH/3T3 fibroblasts but was predominantly localized to the cytoplasm. Despite the fact that this truncated cyclin represents half of the full-length cyclin D2, this isoform still retains the ability to interact with CDK4, but the 45 6 complex is unable to phosphorylate its target pRb. Results Ectopic expression of the truncated cyclin D2 and activated Ras causes transformation of primary MEF Since the truncated cyclin D2 transcript was found over- expressed in the leukemias induced by Graffi retrovirus, b we hypothesized that the protein could probably function as an oncogene. MEF were transfected with a truncated cyclin D2 expressing construct and with an activated H-Ras to determine if it was able to trans- form in vitro. As controls, MEF were also transfected with H-Ras alone, H-Ras þ c-Myc and H-Ras þ cyclin D2. The truncated cyclin D2 was not able to induce transformation alone or with c-Myc (not shown and Figure 1a). Very few foci were obtained with H-Ras alone and no foci at all were seen in the mock-trans- fected MEF. As expected, the co-transfection of the cyclin D2 in combination with an activated Ras gave some foci. It has been shown that cyclin D2 is able to trans- form primary REF in co-expression with an activated Ras (Kerkhoff and Ziff, 1995). In that study, the oncogenic activity of cyclin D2 and H-Ras was much lower than the combination of c-Myc and H-Ras (Kerkhoff and Ziff, 1995). However, when the MEF Figure 1 Transformation properties of the truncated cyclin D2 on were co-transfected with an activated Ras and a primary mouse embryo fibroblasts (MEF). (a)For the focus truncated cyclin D2 expression vector, they presented formation assay, primary MEF were mock transfected (1), a large number of foci at a density comparable to that transfected with Ras EJ 6.6 construct (2), truncated cyclin D2 obtained with H-Ras þ c-Myc (Table 1). The density of and pSV2-Myc constructs (3), cyclin D2 and Ras EJ 6.6 constructs (4), truncated cyclin D2 and Ras EJ 6.6 constructs (5), pSV2-Myc foci induced with the truncated cyclin D2 was much and Ras EJ 6.6 constructs (6). Fourteen days after transfection, higher than that obtained with the full-length cyclin D2 cells were fixed with methanol and stained with methylene blue. suggesting that the truncated cyclin D2 is a more potent (b)MEF transfected with the truncated cyclin D2 and activated inducer of transformation. Also, the size of each foci Ras were also treated with G418 for selection of stable was larger with the truncated cyclin D2. Similar transfectants. The colonies showing a transformed phenotype were isolated and replated for observations. Upper panel (magnification transformation results were obtained with the human  40)shows typical foci obtained with truncated cyclin D2 and truncated cyclin D2 (results not shown). In contrast, the activated Ras. Arrows in lower panel (magnification  100) combination of the normal and the truncated cyclins D2 indicate transformed multinucleated cells. did not produce foci (results not shown). MEF transfected with the truncated cyclin D2 and activated Ras were also treated with G418 for selection of stable transfectants. The colonies showing a trans- with the combination of cyclin D2 and activated Ras formed phenotype were selected and replated. These stable transfectants (not shown; Kerkhoff and Ziff, cells were dependent on the presence of serum for their 1995). The majority of the truncated cyclin D2 and proliferation and showed no contact inhibition and H-Ras stable transfectants were attached very weakly to continued to proliferate even when confluency was the plates and only stable transfectants expressing low reached (Figure 1b). These cells were also morphologi- level of the truncated cyclin D2 were obtained (not cally different from the primary MEF as they were rod shown). It is possible that high levels of expression of the shaped and highly refractile. An important number of truncated cyclin D2 in transformed MEF interfere with cells presenting multinucleation were also observed cell adhesion. This property was also observed in the (Figure 1b, lower panel). Similar results were obtained cyclin D2 transformed REF (Kerkhoff and Ziff, 1995). Oncogene Characterization of mouse and human truncated cyclin D2 C Denicourt et al 1255 Truncated cyclin D2 expression is detected in human to normal brain tissue, an increased expression of the brain tissues truncated human cyclin D2 was observed in certain We previously reported the identification of human tumor types (Figure 2).