Original Article

Oncogene (2008) 27, 1253–1262; doi:10.1038/sj.onc.1210750; published online 17 September 2007

Human and mouse cyclin D2 splice variants: transforming activity and subcellular localization

C Denicourt1,2, P Legault1,2, F-A C McNabb1 and E Rassart1

1Laboratoire de Biologie Moléculaire, Département des Sciences Biologiques, Université du Québec à Montréal, Québec, Canada

Correspondence: Dr E Rassart, Département des Sciences Biologiques, Université du Québec à Montréal, Case Postale 8888 Succ. Centre-ville, Montréal, Canada H3C-3P8. E-mail: Rassart.Eric@UQAM.ca or ericrassart@gmail.com

2These authors contributed equally to this work.

Received 4 June 2007; Revised 25 July 2007; Accepted 26 July 2007; Published online 17 September 2007.



We have previously reported the identification of a novel 17kDa 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 phosphorylation. Interestingly, the presence of a similar, alternatively spliced cyclin D2 mRNA was also detected in some human brain tumors.


truncated cyclin D2, transformation, splice variant



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 promoter 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, 75–85kbp upstream of the cyclin D2 gene. Integrations in Gris1 were shown to activate the expression of the 6.5kb major transcript of the cyclin D2 gene (CCND2) and also a smaller 1.1kb transcript that we have shown to represent an alternative transcript encoding a 17kDa 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 (Taïeb and Jessus, 1996).

Cyclin D2 is a G1 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 G1 phase of the cell cycle. D-type cyclins assemble with their cyclin-dependent 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 integration 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 overexpression of the truncated cyclin D2 can transform primary MEF in conjunction with activated Ras. We also present evidence for a human homolog transcript of a truncated cyclin D2 that is overexpressed in some types of primary human brain tumors. As opposed to 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 complex is unable to phosphorylate its target pRb.



Ectopic expression of the truncated cyclin D2 and activated Ras causes transformation of primary MEF

Since the truncated cyclin D2 transcript was found overexpressed in the leukemias induced by Graffi retrovirus, 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 transform in vitro. As controls, MEF were also transfected with H-Ras alone, H-Ras+c-Myc and H-Ras+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-transfected 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 transform 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 were co-transfected with an activated Ras and a truncated cyclin D2 expression vector, they presented a large number of foci at a density comparable to that obtained with H-Ras+c-Myc (Table 1). The density of foci induced with the truncated cyclin D2 was much higher than that obtained with the full-length cyclin D2 suggesting that the truncated cyclin D2 is a more potent inducer of transformation. Also, the size of each foci was larger with the truncated cyclin D2. Similar transformation results were obtained with the human truncated cyclin D2 (results not shown). In contrast, the combination of the normal and the truncated cyclins D2 did not produce foci (results not shown).

Figure 1.
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Transformation properties of the truncated cyclin D2 on primary mouse embryo fibroblasts (MEF). (a) For the focus formation assay, primary MEF were mock transfected (1), transfected with Ras EJ 6.6 construct (2), truncated cyclin D2 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 and Ras EJ 6.6 constructs (6). Fourteen days after transfection, cells were fixed with methanol and stained with methylene blue. (b) MEF transfected with the truncated cyclin D2 and activated Ras were also treated with G418 for selection of stable transfectants. The colonies showing a transformed phenotype were isolated and replated for observations. Upper panel (magnification × 40) shows typical foci obtained with truncated cyclin D2 and activated Ras. Arrows in lower panel (magnification × 100) indicate transformed multinucleated cells.

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MEF transfected with the truncated cyclin D2 and activated Ras were also treated with G418 for selection of stable transfectants. The colonies showing a transformed phenotype were selected and replated. These cells were dependent on the presence of serum for their proliferation and showed no contact inhibition and continued to proliferate even when confluency was reached (Figure 1b). These cells were also morphologically different from the primary MEF as they were rod shaped and highly refractile. An important number of cells presenting multinucleation were also observed (Figure 1b, lower panel). Similar results were obtained with the combination of cyclin D2 and activated Ras stable transfectants (not shown; Kerkhoff and Ziff, 1995). The majority of the truncated cyclin D2 and H-Ras stable transfectants were attached very weakly to the plates and only stable transfectants expressing low level of the truncated cyclin D2 were obtained (not shown). It is possible that high levels of expression of the truncated cyclin D2 in transformed MEF interfere with cell adhesion. This property was also observed in the cyclin D2 transformed REF (Kerkhoff and Ziff, 1995).

Truncated cyclin D2 expression is detected in human brain tissues

We previously reported the identification of human ESTs encoding the homolog of the murine truncated cyclin D2 (Denicourt et al., 2002). Interestingly, these ESTs were isolated from human brain tumor cDNA libraries. Also, in mouse, the truncated cyclin D2 is mainly expressed in the brain and ovaries (Denicourt et al., 2002). To assess if the truncated cyclin D2 expression could be detected in primary brain tumors, we screened the RNA from a panel of different types of brain tumors by semi-quantitative RT–PCR. Compared to normal brain tissue, an increased expression of the truncated human cyclin D2 was observed in certain tumor types (Figure 2). Oligodendrogliomas, gliomas and glioblastomas multiforme (GBM) showed several fold increase in expression. In contrast, the expression levels in meningiomas, schwannomas and ependymomas remained as in normal brain tissue. The variation of expression observed in the GBMs could be due to some difference in the degree of malignancy in each sample or different form of tumor. GBMs can be classified as primary or secondary and there are evidences that primary and secondary glioblastomas are distinct diseases that evolve through distinct genetic pathways and may require different therapeutic approaches (Watanabe et al., 1996).

Figure 2.
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Expression of the truncated cyclin D2 in human brain tumors. Semi-quantitative RT–PCR analysis of the human truncated cyclin D2 in different brain tissues: normal (normal brain), GBM (glioblastoma multiforme), glio (mixed glioma), oligo (oligodendroglioma), epen (ependymoma), schw (schwannoma), men (meningioma). Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as a control for the integrity of RNA samples. The fold increase indicates the relative level of the truncated human cyclin D2 amplification product over the HPRT internal control after normalization to the control sample (normal brain).

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The expression of the normal cyclin D2 was also measured. No correlations were found between the expression levels of the two cyclins in the tumors nor between the expression levels of the full-length cyclin D2 and the different tumor types.

Truncated cyclin D2 displays a unique cytoplasmic localization

To determine the subcellular localization of both the human and mouse truncated cyclin D2, we generated constructs containing, respectively, the truncated and the full-length cyclin D2 coding sequences fused to the EGFP or Myc epitope at the N-terminus. A mouse cyclin D2 mutant (D2Δ157–289) was generated by the deletion of amino acids 157–289 (Figure 3a). The constructs were transfected into NIH/3T3 cells and the localization of the proteins was examined by fluorescence confocal microscopy. As shown in Figure 3b, EGFP alone shows a diffused generalized localization. EGFP-cyclin D2 protein was localized to the nucleus as expected. The mouse truncated cyclin D2, on the contrary, localizes only to cytoplasmic region. The mutant D2Δ157–289 who only differs from the D2Trc in the last 20 amino acids of the C-terminal end is observed in the nucleus. The human homolog also shows the same cellular distribution (Figure 3b). Markers of the early and late endosomes (Rab 5a and Rab7) as well as a marker of the Golgi complex (BODIPY-Tr ceramids) did not show significant co-localization with the EGFP-truncated cyclin D2 (not shown). Treatment of transfected cells with leptomycin B revealed that the truncated cyclin D2 was not imported to the nucleus (Supplementary data). Cells transfected with Myc-tagged truncated cyclin D2 construct followed by immunocytochemistry with a Myc epitope antibody showed the same cytoplasmic distribution suggesting that the fused EGFP moiety does not alter the localization of the fusion protein (not shown).

Figure 3.
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Cellular localization of human, mouse truncated and mutant cyclin D2. (a) Schematic representation of the normal, truncated and mutant mouse cyclin D2. The first 136 amino acids are identical. The letters in bold indicate conserved amino acids in the new C-terminal region of the cyclin D2Trc. (b) These three constructs were fused to the C-terminus of enhanced GFP using the pEGFP-C2 vector (mD2, mD2Trc, mD2Δ157–289). Similar constructions were made with the human normal (hD2) and truncated (hD2Trc) cyclin D2. The resulting constructs were transfected into NIH/3T3 cells, and the cells were examined by confocal microscopy.

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Truncated cyclin D2 retains the ability to interact with CDK4 in NIH/3T3 fibroblasts and forms an inactive complex

In fibroblasts, CDK4 appears to be the most prominent partner of cyclin D2 (Matsushime et al., 1992; Xiong et al., 1992). To test whether the truncated cyclin D2 can also interact with CDK4, NIH/3T3 fibroblasts were transfected with the Myc-tagged truncated and full-length cyclin D2 (positive interaction control) constructs. Cell lysates were immunoprecipitated with an anti-CDK4 antibody. Western blot analysis with an anti-Myc epitope antibody showed that both cyclin D2 and truncated cyclin D2 could be co-immunoprecipitated in complex with CDK4 (Figure 4a). To evaluate if the truncated cyclin D2 is able to form an active complex with CDK4 or CDK6, we performed immunoprecipitation kinase assays using pRb C-terminus protein as a substrate. Myc-tagged cyclin D2 and truncated cyclin D2 constructs were transfected in NIH/3T3 cells and assayed for kinase activity after immunoprecipitation with Myc epitope antibody. Interestingly, we were not able to detect any activity above background for the truncated cyclin D2 (Figure 4b). This suggests that the truncated cyclin D2 may not form complex with CDK under physiologic conditions or that the D2Trc–CDK4 complex that we detected may have activity toward another substrate. Because phosphorylation of CDK4 by CAK (CDK-activating kinase) is required for the activation of the assembled complex prior to the phosphorylation of pRb (Kato et al., 1994), we verified if the assembled D2Trc–CDK4 complex was phosphorylated by CAK. Myc-tagged cyclin D2 and D2Trc constructs were transfected in NIH/3T3 cells, isolated by immunoprecipitation with Myc epitope antibodies, and the immunoprecipitates were suspended in a kinase reaction buffer containing recombinant CAK (CDK7/cyclin H/MAT1) and [γ32P]ATP. CDK4 associated with full-length cyclin D2 was efficiently phosphorylated by CAK, but the CDK4 associated to D2Trc was a very poor substrate (Figure 4c, upper panel). The inability of CAK to phosphorylate the CDK4–D2Trc complex is not due to a decreased association of CDK4 with D2Trc compared to the full-length cyclin D2 since equivalent amounts of CDK4 were assembled with the two cyclin proteins, as confirmed by blotting of the D2 immunoprecipitates with anti-CDK4 antibodies (Figure 4c, lower panel). Moreover, in vitro kinase assay using the total cellular lysate as substrate was also performed and no significant phosphorylation from the D2Trc–CDK4 complex was observed (results not shown).

Figure 4.
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Analysis of the Myc-tagged truncated cyclin D2 binding with cyclin-dependent kinase 4 (CDK4) and phosphorylation of pRb. (a) For co-immunoprecipitations, NIH/3T3 mock transfected (NIH/3T3), transfected with the pCMV-Myc vector, the Myc-truncated cyclin D2 construct (Myc-D2Trc) and the Myc-cyclin D2 construct (Myc-D2) were used. Immunoprecipitation (IP) was performed with anti-CDK4 antibodies and 1mg of protein from the whole-cell lysate. The upper panel shows the precipitated Myc-tagged truncated cyclin D2 and the Myc-tagged cyclin D2 detected by western blot analysis with a Myc epitope antibody. The lower panel shows the endogenous expression levels of CDK4 in the different transfected cells used for IP. (b) For kinase assays, NIH/3T3 cells were transfected with pCMV-Myc vector, Myc-cyclin D2 and Myc-truncated cyclin D2. Lysates were prepared and subjected to immune precipitation using anti-Myc antibody. Precipitates were tested for in vitro kinase activity using glutathione S-transferase (GST)-Rb C′-terminus as substrate. Kinase reactions were analysed by SDS–PAGE followed by exposure of the gel on X-ray film (top panel). The gel was stained with Coomassie Brilliant Blue to visualize the substrate (GST-Rb) in each lane. Whole-cell lysates corresponding to each lane were analysed by immunoblot with anti-CDK4 and c-Myc antibody. (c) In vitro CAK phosphorylation of cyclin D2–CDK4 complexes. NIH/3T3 cell lysates containing CDK4 assembled with either Myc-cyclin D2 or Myc-D2Trc were precipitated with anti-Myc antibodies. Suspended immune complexes were used as substrates for recombinant CAK (CDK7/cyclin H/MAT1 produced in Sf21 cells), and 32P-labeled complexes were denaturated and separated on polyacrylamide gels. The position of phosphorylated CDK4 is indicated on the left. Autoradiographic exposure time was 3h (top panel). A portion of each cell lysate used in the CAK assay was precipitated with anti-Myc antibodies, separated on gel, blotted to PVDF membrane, probed with anti-CDK4 antibodies and visualized by enhanced chemiluminescence.

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Expression of the truncated cyclin D2 in 32Dcl3 cells

In the hematopoietic cell line 32Dcl3, cyclin D2 is expressed in proliferating cells and its expression is dependent on interleukin-3 (IL-3). The expression declines rapidly in the absence of this growth factor (Ando et al., 1993). To examine if the mouse truncated cyclin D2 presents the same profile of expression in this cell line, we analysed its expression in the presence and absence of IL-3. To arrest the cells in G0/G1, IL-3 was removed from the culture medium for 16h. After, IL-3 was reintroduced in the medium to induce re-entry in the cell cycle. Samples of cells were collected at different time points and the expression of the cyclin D2 transcripts was analysed by northern blot. Figure 5a shows that the truncated cyclin D2 transcript declined rapidly in the absence of IL-3. When the IL-3-deprived cells were retreated back with IL-3, the 1.1kb transcript was rapidly induced within 3h. We also observed the same kinetic for the cyclin D2 6.5kb transcript. The presence of the proteins was also analysed and the profiles of both the 17kDa truncated and 32kDa cyclin D2 proteins coincide with the observed induction of the mRNAs (Figure 5b). These results suggests that the induction of the truncated cyclin D2 synthesis is controlled in the same fashion as the induction of the cyclin D2 protein, both expression being dependent on the presence of mitogenic factors.

Figure 5.
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Expression analysis of the truncated cyclin D2 in 32Dcl3 cells. (a) Northern blot analysis of the full-length cyclin D2 (6.5kb transcript upper panel) and the truncated cyclin D2 (1.1kb transcript lower panel) in 32Dcl3 cells. Cells were starved of interleukin-3 (IL-3) for 16h (0) and restimulated by adding back IL-3. Samples of cells were removed at 1, 3, 8, 10, 12 and 24h after IL-3 stimulation. RNA (10μg) was loaded in each lane. The exposure period for the lower panel was twice as much as that for the top panel. (b) The 32Dcl3 cells were treated as in (a) and samples of cells were analysed by western blot with specific antibodies against the cyclin D2 (top panel), the truncated cyclin D2 (middle panel) or GAPDH (bottom panel). Thirty micrograms of protein was loaded per lane.

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The truncated cyclin D2 was initially identified as a proto-oncogene that becomes activated upon retroviral insertion of the Graffi murine leukemia virus (Denicourt et al., 2002). The results presented in this study also support the notion that the truncated cyclin D2 may play an important role in tumorigenesis. Indeed, we have shown that ectopic expression of the truncated cyclin D2, with an activated Ras, is able to induce a transformed phenotype to primary MEF. Moreover, the truncated cyclin D2 seems to be a more potent oncogene than the full-length cyclin D2 since the number of foci was much more important and comparable to that observed with the co-expression of c-Myc and activated Ras (Figure 1a).

Cells derived from transformed foci after selection for the expression of the truncated cyclin D2 showed a high percentage of multinucleation, which is a characteristic of abnormal mitosis. These cells also presented loss of contact inhibition and grew as aggregates under subconfluent conditions suggesting that the ability of the truncated cyclin D2 to transform primary MEF may be due to alterations in cell adhesion (Figure 1b). It was suggested that primary REF transformed by both activated Ras and full-length cyclin D2 possibly presented an altered expression of cell adhesion proteins since these cells attached very weakly to tissue culture plate and that this property necessitated their passage on collagen-coated plates (Kerkhoff and Ziff, 1995). We have subcloned cell lines from individual foci of primary MEF and found that the cyclin D2 was poorly expressed. This is probably due to a selection against the establishment of transformed cell lines expressing high levels of the truncated cyclin D2. It is also possible that the high expression of the truncated cyclin D2 completely inhibits the adhesion of these cells. Consistent to our observations, rodent fibroblasts were found to have a strong selection against the establishment of cell lines that overexpress D-type cyclins (Quelle et al., 1993). Integrin-mediated cell adhesion to the extracellular matrix is required for normal cell growth. It was shown that integrin signaling through focal adhesion kinase (FAK) plays a role in the regulation of the cell cycle progression by regulating the expression and activities of D-type cyclins (Assoian, 1997; Schwartz and Assoian, 2001). Roovers et al. (1999) have demonstrated that integrin-mediated cell adhesion is required for sustained ERK activation and induction of cyclin D1 expression by growth factors. Also, ectopic overexpression of cyclin D1 induced anchorage-independent cell cycle progression of NIH/3T3 and Rat1 cells (Schulze et al., 1996; Zhu et al., 1996; Resnitzky, 1997). These observations suggest that the truncated cyclin D2 is able to influence cellular adhesion.

We also showed that the truncated cyclin D2 is expressed in a subset of primary brain tumors (oligodendrogliomas, mixed gliomas and glioblastomas multiforme) supporting again its role in tumorigenesis. It is interesting to note that the truncated cyclin D2 is abundant in the most aggressive brain tumor types. This overexpression does not seem to be correlated with the expression of the normal cyclin D2 (data not shown) but could be related with the degree of malignancy of these specific tumors. Buschges et al. (1999) also reported that the expression of normal cyclin D2 was not statistically significant between different tumor grades. We have already shown that the truncated cyclin D2 is present in the brain of normal mouse (Denicourt et al., 2002).

We have shown that the truncated cyclin D2 localizes to a distinct cytoplasmic substructure in NIH/3T3 cells. D-types cyclins are known to accumulate in the cell nucleus during G1 phase and disappear from the nuclei of cells undergoing DNA synthesis (Baldin et al., 1993). However, in our study, the truncated cyclin D2 was never observed in the cell nucleus of NIH/3T3 cells grown at different density. This was confirmed by leptomycin B treatment of cells transfected with an EGFP-truncated cyclin D2 construct (Supplementary data). It was shown that a cyclin D1 T156A mutant, which retains its ability to interact with CDK4, localizes uniquely to the cytoplasm of NIH/3T3 cells (Diehl and Sherr, 1997). On the cyclin D2 protein, the T156 residue of cyclin D1 corresponds to T154. This residue is not conserved on the truncated cyclin D2 and this could explain its cytoplasmic localization. This observation is also supported by the nuclear localization of mutant D2Δ157–289 who still retains the T154 residue. It is interesting to note that mutant D2Δ157–289 that contains the first 156 amino acids of the normal cyclin D2 remains to the nucleus albeit the D2Trc is cytoplasmic. This indicates that the last 20 amino acids are responsible of the localization.

We have also demonstrated that the truncated cyclin D2 can interact with CDK4 when expressed ectopically in NIH/3T3 cells. The cyclins present a globular structure containing two sequential 90 amino acid repeats, known as the cyclin folds, each consisting of five super imposable α-helical bundles (Jeffrey et al., 1995; Kim et al., 1996; Russo et al., 1996; Andersen et al., 1997). The two cyclin folds are connected via a short linker peptide of 10–15 amino acids. The first cyclin fold corresponds to what is defined as the cyclin box that makes contact with the interacting CDK. The second repeat on the cyclin does not contribute to the cyclin–CDK interface. The truncated cyclin D2 contains an almost intact cyclin box that could be responsible and sufficient for the interaction with CDK4. We examined if the truncated cyclin D2–CDK4 complex was catalytically active. It appears that the complex was unable to phosphorylate recombinant pRb protein in vitro (Figure 4b). This lack of kinase activity is due to the inability of CAK to recognize and phosphorylate the D2Trc–CDK4 complex, which thus remains catalytically inactive (Figure 4c). Cyclin D binding must induce a conformational change in the structure of CDK4 to make it available for phosphorylation. The fact that CAK could not phosphorylate CDK4 when complexed to the D2Trc suggests that the truncated form could not induce the conformational change but instead give rise to abortive complexes that are no longer recognized by CAK. The integrity of Thr-156 on cyclin D1 is required for CAK activity on the CDK4–cyclin complex (Diehl and Sherr, 1997). The loss of Thr-154 on the D2Trc could be responsible for the inability of the CDK4 complex to be phosphorylated. Interestingly, a splice variant of cyclin D1 (cyclin D1b) harboring a different C′-terminus and lacking T286 was found to be a potent nuclear oncogene compared to the normal cyclin D1 (Lu et al., 2003). This isoform has retained its ability to bind CDK4, but was unable to catalyse efficiently pRb phosphorylation and inactivation (Solomon et al., 2003). This suggests that critical residues in the C-terminus of cyclin D proteins are important for substrate specificity. Despite its inability to phosphorylate Rb, the truncated cyclin D2 isoform demonstrated enhanced transforming activity compared with cyclin D2. The ability of the truncated cyclin D2 to promote cellular transformation is probably attributed to its constitutively cytoplasmic localization. Truncated cyclin D2 could sequester p27 (a cyclin D/CDK inhibitor) in the cytoplasm where it is degraded by the cytoplasmic ubiquitin ligase complex KPC, this step being essential for the progression of the cell cycle into S phase (Susaki et al., 2007). An upregulated cell cycle caused by the presence of the truncated cyclin D2 in the cytoplasm could be the cause of the enhanced transformation in presence of an activated Ras.

Expression of D-type cyclins is dependent on continuous mitogenic stimulation. In the hematopoietic cell line 32Dcl3, cyclin D2 is expressed in proliferating cells and this expression is dependent on the presence of IL-3. It was also shown in the same cell line that the expression and the protein levels of cyclin D2 declined rapidly upon withdrawal of IL-3 (Ando et al., 1993). To investigate if the expression of the truncated cyclin D2 was also regulated in the same fashion in 32Dcl3 cells, we have reproduced some of the experiments by Ando et al. (1993). Our results show that the truncated cyclin D2 expression and protein stability are dependent on the presence of mitogenic stimuli (IL-3) as already shown for the cyclin D2. This suggests that the expression of both transcripts (6.5 and 1.1kb) of the cyclin D2 gene is controlled via the same regulatory elements, which are activated by growth factors. Indeed, the two transcripts most likely depend on the same promoter as they share the same 5′ end and the first two exons (Denicourt et al., 2002). Site-specific phosphorylation by GSK-3B of cyclins D1 and D2 on a single threonine residue (T286) is critical for maintaining their rapid turnover rates throughout the cell cycle and also upon mitogen signaling withdrawal (Diehl and Sherr, 1997; Diehl et al., 1998). Although the truncated cyclin D2 does not harbor this T286 residue, our results showed that the protein level declined rapidly in the absence of IL-3. Also, we have demonstrated that the stability of the truncated cyclin D2 was dependent on proteasomal degradation (data not shown). It is possible that the turnover of the truncated cyclin D2 is dependent on the phosphorylation of another residue.

Taken together, these data demonstrate the activity of the truncated cyclin D2 in cellular transformation and a role in tumor formation. Because of its cytoplasmic localization, we propose that the truncated cyclin D2 may contribute to transformation by sequestering p27 in the cytoplasm, causing its degradation and, therefore, upregulating the cell cycle. It will, therefore, be important to identify the protein partners of this truncated cyclin D2.


Materials and methods

Cell lines, cell culture conditions and transfections

The 32Dcl3 diploid, non-leukemic, IL-3-dependent murine myeloid cell line (Greenberger et al., 1983) was obtained from Alan Friedman (Johns Hopkins University, Baltimore, MD, USA). These cells were maintained in RPMI 1640 medium (GIBCO, Burlington, ON, Canada) supplemented with 10% of heat-inactivated fetal calf serum (GIBCO) and 10% of WEHI-3B cells conditioned medium (WEHI–CM) as a source of IL-3. NIH/3T3 murine fibroblasts (ATCC, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% of calf serum (GIBCO). All transfections were made with Polyfect regent (QIAGEN, Mississauga, ON, Canada) according to the manufacturer's protocol.

Mouse embryo fibroblasts transformation assay

Primary fibroblasts were isolated from 15 days BALB/c mouse embryos. The cells were transfected with 4μg of the pCMV-D2Trc construct or the pCMV-D2 construct made by cloning the complete coding sequence of the truncated cyclin D2 or cyclin D2 obtained by PCR. The fragments were cloned in the pRcCMV vector (Invitrogen, Burlington, ON, Canada). The human truncated cyclin D2, obtained by PCR, and normal cyclin D2, Image clone ID: 4133621 (ATCC), were cloned in the pCMV5 vector. The cells were also transfected with 4μg of the RAS EJ 6.6 construct (Tabin et al., 1982) and the pSV2-Myc construct. Transfections were made with Polyfect (QIAGEN) according to the manufacturer's instructions.

Primary human brain tumors

Samples of primary human brain tumors were obtained from The Arthur and Sonia Labatt Brain Tumour Research Center (Toronto, Canada).

RNA extraction and northern blot analysis

Total RNA from frozen human brain tumor tissue or 32Dcl3 cells was extracted with the TRIzol reagent (Invitrogen) according to the manufacturer's instructions.

Reverse transcription–PCR

Total RNA (2μg) was reverse transcribed into cDNA by using an oligo-dT primer (10μM), dNTP mix (5mM each dNTP), 10 × reverse transcription buffer (500mM Tris–HCl pH 8.3, 250mM KCl, 50mM MgCl2 and 50mM DTT), 10U RNase inhibitor (GE Healthcare, Baie d’Urfe, Quebec, Canada) and 4U of Omniscript Reverse Transcriptase (QIAGEN). The mix was incubated at 37°C for 60min. PCR was performed in 100μl with 5μl of the cDNA template, 0.2mM dNTP, 2mM MgCl2, 0.25μM of each primer and 2.5U of Taq DNA polymerase. The cycling conditions were 94°C for 3min, followed by 94°C for 30s, 60°C for 30s and 72°C for 1min for 25 cycles and a final extension at 72°C for 10min. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) primers were used as an internal control under the same conditions. The primer sequences were as follows: normal cyclin D2, cyc1 (5′-GGCTGGGGTCCCGACTCCGAAG-3′) and cyc2 (5′-GCAGCTCAGTCAGGGCATCACA-3′), truncated cyclin D2, cyc1 (5′-GGCTGGGGTCCCGACTCCGAAG-3′) and tcyc (5′-TGGCCAGGGGAAAGCGGGAATC-3′). The HPRT primers were HPS1 (5′-GTGATGAAGGAGATGGGAGGCC-3′) and SPH1 (5′-CTTCGTGGGGTCCTTTTCACC-3′). The resulting products were separated on a 1% agarose gel and the individual bands were quantified using the volume Rect tool from the Quantity one 4.4.0 software (Bio-Rad, Mississauga, ON, Canada).

Immunoprecipitation and immunoblotting

NIH/3T3 cells (1 × 106 per 100cm dish) transfected with the Myc-tagged truncated cyclin D2 (Myc-D2Trc) and Myc-tagged full-length cyclin D2 (Myc-D2) constructs were lysed 24h after transfections in immunoprecipitation buffer (IP buffer) containing 50mM Tris–HCl (pH 7.5), 150mM NaCl, 1% Nonidet P-40 and complete protease inhibitor cocktail (Roche, Laval, Quebec, Canada) and sonicated on ice (Fisher cell dismembrator, 80% microtip power 10s). Lysates were clarified by centrifugation at 10000g for 5min. The supernatants were precipitated for 4h at 4°C with protein A-Sepharose beads (GE Healthcare) alone or precoated with saturating amounts of the anti-CDK4 antibody (Santa Cruz sc-601, Santa Cruz, CA, USA). The proteins immunoprecipitated on beads were washed five times with 1ml of IP buffer. The beads were resuspended in 2 × loading buffer (30% glycerol, 4% SDS, 160mM Tris–HCl (pH 6.8), 10% B-mercaptoethanol and 0.02% bromophenol blue) and boiled for 10min at 95°C. Immunoprecipitated samples were separated on 12% SDS–PAGE and electrophoretically transferred to Immobilon-P PVDF membranes (Millipore, Billerica, MA, USA).

For immunoblotting analysis, proteins were extracted in RIPA buffer and the protein concentration in the different extracts was measured using the Bio-Rad protein assay. Equal amounts of lysate proteins (30–80μg) were diluted in 2 × sample buffer (30% glycerol, 4% SDS, 160mM Tris–HCl, pH 6.8, 10% β-mercaptoethanol and 0.02% bromophenol blue) and boiled for 3min. Protein were separated on 12% SDS–PAGE and electrophoretically transferred to Immobilon-P PVDF membranes (Millipore). Membranes were blocked in PBS containing 0.2% Tween-20 and 4% skimmed milk powder (PBS-Tween-milk) for 1h at room temperature. The antibodies were diluted in PBS-Tween-milk at the following dilutions: anti-truncated cyclin D2 peptide antibody (1:100) (Denicourt et al., 2002), the anti-cyclin D2 (1:1000) (Cell Signaling, Danvers, MA, USA), the anti-Myc epitope antibody (1:5000) (Santa Cruz) or the anti-GAPDH antibody (1:300) (Chemicon, Temecula, CA, USA). The blots were incubated for 1h at room temperature, washed in PBS-Tween and further incubated for 1h with either anti-rabbit or anti-mouse IgG-peroxidase (BD Pharmingen, Mississauga, ON, Canada). The immune complexes were revealed using the ECL plus chemiluminescence regents (GE Healthcare).

Kinase assay

NIH 3T3 cells were transfected with Myc-tagged cyclin D2 and truncated cyclin D2 using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were lysed in buffer containing 50mM HEPES (pH 7.5), 10mM MgCl2, 0.1% Tween 20, 1mM dithiothreitol, 25μM ATP and protease inhibitors. Lysates were precleared with protein G-Sepharose beads (Zymed, Burlington, ON, Canada) on a rotating wheel at 4°C for 1h. Two micrograms of anti-Myc epitope (9E10 SantaCruz) antibodies was used for immunoprecipitation. Kinase reactions were performed in 10mM MgCl2 and 50mM HEPES (pH 7.2) with 2μg of bacterially isolated glutathione S-transferase (GST)-Rb C′-terminus, 50μM cold ATP and 10μCi of [γ-32P]ATP (Amersham, Baie d'Urfe, Quebec, Canada). Reactions were performed at 30°C for 30min.

CAK activation assay

NIH 3T3 cells were transfected with Myc-tagged cyclin D2 and truncated cyclin D2 using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, cells were lysed and immunoprecipitated as described above. The immunoprecipitates were washed three times with IP buffer and then three times with CAK buffer (80mM β-glycerophosphate (pH 7.3), 15mM MgCl2, 20mM EGTA and 5mM dithiothreitol) (Matsuoka et al., 1994). The beads were resuspended in 50μl of CAK buffer containing protease, phosphatase inhibitors and 1.4μg of recombinant CDK7–cyclin H–MAT1 complex (Upstate, Charlottesville, VA, USA). After addition of 40μCi of [γ-32P] ATP in the presence of 50μM ATP, the suspensions were incubated at 30°C for 30min. The precipitates were washed six times in Tween 20 lysis buffer (50mM HEPES (pH 7.5), 150mM NaCl, 1mM EDTA, 2.5mM EGTA, 1mM dithiothreitol, 0.1% Tween 20 and 10% glycerol), followed by denaturation by boiling in electrophoresis sample buffer for 5min. Phosphorylated products were separated on denaturing polyacrylamide gels and visualized by autoradiography.

IL-3 starvation, stimulation and cell cycle analysis of 32Dcl3 cells

IL-3 starvation was performed by washing the cells twice with PBS and reseeding them in culture medium without WEHI–CM for 16h. After the IL-3 starvation, the cells were centrifuged and reseeded in culture medium containing WEHI–CM. Samples of cells were collected at different time points after IL-3 stimulation for northern and western blot analyses.


NIH/3T3 cells were seeded on coverslips 16h prior to transfection. The cells were fixed 8–24h after transfection in 4% paraformaldehyde for 10min and permeabilized in 0.2% Triton X-100. Fixed cells were washed in PBS, and then blocked in 10% normal goat serum for 1h at room temperature. Immunostaining was subsequently performed with an anti-Myc epitope antibody (Santa Cruz) (1:50) for 1h at 25°C. The cells were then incubated with Cy3-conjugated anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) for 1h at 25°C. The cells were washed extensively, mounted and imaged using a Zeiss Axioskop fluorescence microscope.

Construction of GFP fusion protein expression vectors

The cyclin D2 and the truncated cyclin D2 coding sequences for both the human and mouse were PCR amplified and cloned into in pEGFP-C2 vector (BD Biosciences Clontech, Mississauga, ON, Canada). The constructs were transfected in NIH/3T3 cells with QIAGEN Polyfect transfection reagent as recommended by the supplier. Cells were plated on Permanox Plastic Chamber Slide System (Fisher Scientific, Ottawa, ON, Canada). Twenty-four hours later, cells were visualized by confocal microscopy. For control, cells were transfected with pEGFP-C2 vector alone.

Construction of cyclin D2 mutant

The cyclin D2 mutant (D2Δ157–289) was produced by a PCR-based site-directed mutagenesis method as previously described by Fisher and Pei (1997). The following oligonucleotide primers were used: 5′-PTTAGTGAGGGGTGACTGCGGCCAGG-3′ (stop codon is underlined) and 5′-GCCACCGCGGTGGAGCGCCAATTC-3′. Cyclin D2 cloned into pBluescript II KS+ was used as PCR template.



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We thank the Arthur and Sonia Labatt Brain Tumour Research Center (http://www.sickkids.ca/BTRC/ Toronto, Canada) and Dr Abhijit Guha for the gift of human tumor tissues. This work was supported by grant FRN 37994 from the Canadian Institutes of Health Research and by La Société de recherche sur le Cancer. CD is a recipient of a FCAR PhD scholarship.

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).



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