Oncogene (2007) 26, 1723–1730 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE D2 and play opposite roles in mouse skin carcinogenesis

P Rojas1,4, MB Cadenas2,4, P-C Lin3, F Benavides1, CJ Conti1 and ML Rodriguez-Puebla2

1Department of Carcinogenesis, Science Park Research Division, MD Anderson Cancer Center, Smithville, TX, USA; 2Center for Comparative Medicine and Translational Research, Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA and 3Department of Molecular and Cellular Oncology, MD Anderson Cancer Center, Houston, TX, USA

D-type are components ofthe cell-cycle engine a family of key cell-cycle regulators, as they are the that link cell signaling pathways and passage throughout regulatory subunits of the CDK4 and CDK6, which . We previously described the effects of over- phosphorylate pRb family of that are critical expression , D2 or D3 in mouse epidermis and substrates for cell-cycle progression (Matsushine et al., tumor development. We now asked whether 1994; Meyerson and Harlow, 1994; Sherr and Roberts, and/or cyclin D3 play a relevant role in ras-dependent 1999; Farkas et al., 2002; Leng et al., 2002). Initially, tumorigenesis. Here, we described the effect of cyclin D3 several reports assigned redundant roles to the three and cyclin D2 overexpression in mouse skin tumor members of D-type cyclins, but in the last few years, it development. Notably, overexpression ofcyclin D3 results has become evident that each member is differentially in reduced tumor development and malignant progression expressed in various tissues playing specific roles (Sherr, to squamous cell carcinomas (SCC). Biochemical analysis 1995; Ciemerych et al., 2002). ofkeratinocytes shows that overexpression ofcyclin D3 The cyclin D1 is rearranged or amplified in breast results in strong reduction ofcyclin D2 and its associated carcinomas, squamous cell carcinomas of head and neck kinase activity. Furthermore, we found that reinstatement and several other cancers (Dickson et al., 1995; Cheung ofcyclin D2 level in the cyclin D3/cyclin D2 bigenic mice et al., 2001; Fujii et al., 2001). Abnormalities in the cyclin results in a complete reversion ofthe inhibitory action of D2 gene have also been demonstrated, especially in cyclin D3. Supporting these results, ablation ofcyclin D2 testicular tumors (Sicinski et al., 1996; Houldsworth results in reduced tumorigenesis and malignant progres- et al., 1997), as well as in a wide range of B-cell sion. On the other hand, overexpression ofcyclin D2 malignancies (Delmer et al., 1995). Cyclin D3 was also results in an increased number ofpapillomas and found overexpressed in several human cancers (Ito et al., malignant progression. We conclude that cyclin D3 and 2001; Wong et al., 2001; Hedberg et al., 2002; Pruneri cyclin D2 play opposite roles in mouse skin tumor et al., 2005) such as malignancies of the thymus (Filipits development and that the suppressive activity ofcyclin et al., 2002). Consistent with these results Sicinska et al. D3 is associated with cyclin D2 downregulation. (2003) established that cyclin D3-deficient mice displayed Oncogene (2007) 26, 1723–1730. doi:10.1038/sj.onc.1209970; specific defects in the development of T lymphocytes. In published online 18 September 2006 addition to their growth-promoting functions, it was suggested that cyclin D3 plays a unique, nonredundant Keywords: cyclin D3; cyclin D2; skin; DMBA; kerati- tissue-specific role such as in muscle differentiation (Kiess nocytes; papillomas et al., 1995; Mariappan and Parnaik, 2005). Cyclin D3 was also immunolocalized in quiescent cells during advanced stages of differentiation such as in the surface epithelia of the stomach, intestine, gallbladder and ureter (Bartkova et al., 1998). Introduction In the last few years, we and others have used the mouse skin model to study the role of D-type cyclins in The mammalian is a sequential and ordered normal and neoplastic proliferation (Zhang et al., 1997; process that requires formation and activation of cyclin- Rodriguez-Puebla et al., 1998a, 1999, 2002; Miliani de dependent kinase complexes (CDKs). D-type cyclins are Marval et al., 2004). These studies showed that cyclin D1 and D2 are overexpressed in mouse skin papillomas and squamous cell carcinomas (SCC), whereas cyclin D3 Correspondence: Assistant Professor ML Rodriguez-Puebla, Center for Comparative Medicine & Translational Research, Molecular levels remain constant in normal epidermis and Biomedical Sciences, College of Veterinary Medicine, North Carolina skin tumors (Rodriguez-Puebla et al., 1998b). State University – CVM, 4700 Hillsborough Street, Raleigh, NC In this paper, we have studied the role of cyclin D2 27606, USA. and cyclin D3 in skin tumorigenesis. Surprisingly, E-mail: [email protected] 4These authors contributed equally to this work. overexpression of cyclin D3 in mouse epidermis results Received 23 November 2005; revised 11 July 2006; accepted 28 July 2006; in decreased number of skin tumors, whereas over- published online 18 September 2006 expression of cyclin D2 increased the number of Role of cyclin D2 and D3 in tumor development P Rojas et al 1724 papillomas compared to wild-type mice. Biochemical Protein lysates from epidermis of transgenic mice analysis show that forced expression of cyclin D3 results overexpressing cyclin D2 (K5-D2) showed that the in a significant reduction of cyclin D2 protein levels cyclin D3 status was not affected by forced expression correlating well with the reduced number of tumors of cyclin D2 (Figure 1a, lanes 1 and 2). In addition, observed in K5-cyclin D3 mice. We found that Western blot analysis of keratinocyte from both K5-D2 reinstatement of cyclin D2 levels in the K5-cyclin D3/ and K5-D3 mice did not show changes in the cyclin D1 cyclin D2 bigenic mice overturned the reduced number level (Figure 1a and b). Collectively, all of these of tumors, suggesting that the decreased cyclin D2 observations suggest that a negative feedback loop protein level is essential for the inhibitory activity exists in mouse epidermis in which cyclin D3 normally observed in K5-cyclin D3 mice. Supporting this represses the expression of cyclin D2, but not cyclin D1. observation, ablation of cyclin D2 results in reduced number of papillomas and SCC. Thus, whereas over- expression of the three D-type cyclins produces similar Cyclin D3-overexpressing mice exhibit reduced skin epidermal phenotype, each D-type cyclin appears to tumor development play a unique role in skin carcinogenesis (Rodriguez- According to the current model of cell proliferation, Puebla et al., 1999, 2000). These observations and our aberrant levels of a positive regulator of cell cycle previous studies suggest that cyclin D1 and cyclin D2 provides a growth advantage that can result in increased play a preponderant role in ras-induced tumor develop- tumor development. Thus, we performed a two-stage ment, whereas deregulation of cyclin D3’s ability to chemical carcinogenesis protocol on the skin of K5-D3 control cyclin D2 levels plays a central role in tumor mice. Tumor latency and incidence were similar in resistance. K5-D3 and wild-type mice, which began to develop papillomas at 7 weeks of promotion, and by 20weeks, 80% of the mice had tumors (Figure 2b). Although, a mild difference in the incidence of K5-D3 and wild-type Results mice was observed between weeks 7–11, both group reaches 95% of incidence when the experiment was Molecular analysis of keratinocytes overexpressing ended (Figure 2b). Surprisingly, K5-D3 mice averaged a cyclin D3 significantly lower number of tumors per mice (2.5 The expression of murine cyclin D3 was targeted to papillomas/mouse) compared with wild-type siblings stratified epithelial by the bovine keratin 5 gene as (four papillomas/mouse) (P 0.05) (Figure 2a). previously described (Ramirez et al., 1994; Rodriguez- o We previously observed that cyclin D2 and cyclin D1 Puebla et al., 2000). As expected, cyclin D3 protein are aberrantly expressed in mouse skin papillomas levels increased 30-fold when compared with normal (Rodriguez-Puebla et al., 1998a), suggesting that these sibling mice (Figure 1a, lanes 1 and 3). In addition, we cyclins play an important role in tumor development. determined the status of the other D-type cyclins Thus, we asked whether the reduction of cyclin D2 levels and found that cyclin D2 protein levels decreased is in part responsible for the diminished tumorigenesis 2- and 2.5-fold in epidermis and primary keratinocyte observed in K5-D3 mice. Therefore, we interbred from K5-D3 mice (Figure 1a, lanes 1 and 3 and transgenic mice to generate the K5-D3/K5-D2 bigenic Figure 1b). Consistent with the cyclin D2 protein mice. Analysis of protein lysates from epidermis of decline, cyclin D3 overexpression resulted in threefold bigenic mice confirms that transgenic expression of reduction of cyclin D2 messenger levels (Figure 1c). cyclin D2 compensates for the reduction of endogenous cyclin D2 observed in K5-D3 (Figure 1a, lane 4). We next challenged the double transgenic and K5-D2 mice to the two-stage carcinogenesis protocol. Transgenic expression of cyclin D2 resulted in an increased number of tumors (five tumors/mice) compared with wild-type mice (3.8 tumors/mice) (Po0.05). More importantly, restoration of cyclin D2 levels in the bigenic mice resulted in increased numbers of papillomas as observed in K5-D2 mice (Figure 2a). We conclude that cyclin D2 expression abrogates the tumor suppressive effect of cyclin D3, highlighting the importance of cyclin D2 in Figure 1 D-type cyclin levels in epidermis and keratinocytes skin tumorigenesis. from transgenic mice. (a) Protein homogenates from epidermis of Biochemical analysis of D-type cyclin levels in dorsal mouse skin corresponding to K5-D2, K5-D3, K5-D2/K5-D3 (D2/D3) and wild-type sibling mice were immunoblotted with papillomas demonstrated that cyclin D2 was also antibodies against cyclin D1, cyclin D2 and cyclin D3. (b) Lysates downregulated in tumors from K5-D3 mice were prepared from primary keratinocytes culture from K5-D3 and (Figure 2d, lanes 1 and 3). Interestingly, the increased wild-type newborn mice and immunoblotted as above. (c) mRNA cyclin D2 protein levels in papillomas from K5-D3/K5- expression of cyclin D2 and cyclin D3 was determined in primary keratinocytes from K5-D3 and wild-type mice. b-Actin and D2 mice (twofold) were lower than that observed in K5- glyceraldehydes 3-phosphate dehydrogenase (GAPDH) were used D2 papillomas (10-fold) (Figure 2d, lanes 1, 2 and 4). as loading controls. Because a transcriptional mechanism does not likely

Oncogene Role of cyclin D2 and D3 in tumor development P Rojas et al 1725 operate on an exogenous promoter in transgenic mice, a Cyclin D2 overexpression also results in increased post-transcriptional mechanism may be responsible for malignant progression to SCC. Figure 2c shows that the reduced level of cyclin D2 in mice overexpressing 60% of the papillomas developed by both K5-D2 and cyclin D3. In this case, the putative post-transcriptional K5-D2/K5-D3 bigenic mice progress to SCC at 30 mechanism would result in a reduction of the endoge- weeks of tumor promotion, whereas only 40% of K5-D3 nous and transgenically expressed cyclin D2 as observed and 30% of wild-type mice progress to SCCs (Po0.05) in K5-D3/K5-D2 bigenic mice. (Figure 2c). In addition, SCC latency was reduced in K5-D2 mice, where the first SCC appeared at 14 weeks of promotion, whereas a delay of 5–10weeks was observed in the other genotypes (Figure 2c). Also, an increased number of SCC per mouse (SCC multiplicity) was observed in K5-D2 (1.4 SCC/mouse) compared to wild-type mice (0.45 SCC/mouse) (Figure 3A). We studied the histopathological aspect of SCCs at 30 weeks of promotion taking into account the dysplastic and anaplastic changes that included disturbed cell polarity, differentiation, abnormal , nuclear hyperchromatism and nuclear/cytoplasmic ration. Strik- ingly, 67% of the SCCs from K5-D2 mice presented marked atypia with areas of microinvasion and were classified as poorly differentiated SCCs (Figure 3B). In contrast, only 17% of SCCs from wild-type mice showed similar histopathological changes, whereas 83% were classified as moderate and well differentiated. In addition, SCCs from K5-D2/K5-D3 bigenic mice showed the same pattern of atypia observed in K5-D2 SCCs (data not shown). We conclude that cyclin D2 overexpression enhances tumor development and malignant progression under conditions where ras is constitutively activate (ras induced by 7,12-dimethylbenz[2]nthracene (DMBA) treatment). In sharp contrast, cyclin D3 overexpression results in decreased levels of cyclin D2, which in turn leads to a significantly lower number of papillomas.

Cyclin D2 ablation reduces skin tumor development Our results show that expression of cyclin D2 enhances tumor development in mouse skin. Whereas a positive role of cyclin D1 was clearly established in mouse skin tumors (Robles et al., 1998), the question remained whether cyclin D2 is a mediator of keratinocyte transformation by oncogenic ras or is induced as a nonessential consequence of the proliferative response generated by ras transformation. To address these possibilities, we studied skin tumor development in cyclin D2-null mice. At 15 weeks of promotion, 100% of wild-type mice had tumors, whereas only 45% of the cyclin D2-null mice developed tumors. By the end of the

Figure 2 Kinetic of papilloma formation in K5-D2 and K5-D3 transgenic mice. Groups of K5-D2, K5-D3, bigenic K5-D2/K5-D3 (D2/D3) and wild-type sibling mice were initiated with DMBA and promoted with multiple applications of TPA on dorsal mouse skin. (a) Average number of papillomas per mouse as a function of weeks of study (multiplicity). (b) Percentage of mice with papillomas (incidence). (c) Percentage of mice with SCC (SCC incidence). (d) Western blot analysis of D-type cyclins in papillomas from K5-D2, K5-D3, K5-D2/K5-D3 (D2/D3) and wild-type (wt) mice. b-Actin was used as loading control.

Oncogene Role of cyclin D2 and D3 in tumor development P Rojas et al 1726 wave of keratinocyte proliferation that accelerates entry of K5-D3 keratinocytes in (Supplementary material, Figure S1). Epidermis lysates from K5-D3 and wild-type mice were immunoprecipitated with antibo- dies against cyclin D1, D2 and D3, followed by Western blot analysis to determine association with CDK4 and CDK6. We found increased cyclin D3/CDK4 as well as cyclin D3/CDK6 complex formation in K5-D3 epider- mis; although in our experimental conditions the later complex was hardly detected (Figure 5a, lanes 8 and 9). As anticipated, downregulation of cyclin D2 in K5-D3 epidermis resulted in reduced cyclin D2/CDK4 complex formation compared to wild-type mice (Figure 5a, lanes 4 and 5). Analysis of CDK4/cyclin D1 showed that this complex was not affected by cyclin D3 overexpression and the consequent reduction of cyclin D2 (Figure 5a, lanes 1 and 2). It is worth mentioning that CDK6/cyclin D1 and CDK6/cyclin D2 complexes were undetectable in our experimental setting. We next asked whether changes in the level of CDK complexes results in changes in the cyclin Ds-associated kinase activities. K5-D3 mice showed fivefold increase of the cyclin D3-associated kinase activity (Figure 5b, lane 7) and, consistent with the reduced level of cyclin D2, we observed a 50% decrease of cyclin D2-associated kinase activity (Figure 5b, lane 5). Unexpectedly, a sevenfold increase in the cyclin D1-associated kinase activity was observed in K5-D3 mice (Figure 5b, lane 2), which is not due to changes in complex formation (Figure 5a, lanes 1 and 2). Thus, we hypothesized that mechanisms other than the simple CDK/cyclin complex formation are involved in the overactivation of cyclin D1-associated kinase activity. In order to establish whether the elevated cyclin D3- associated kinase activity was derived from CDK4 and/ Figure 3 Histopathological analysis of SCC. (A) Average number of SCC per mouse (multiplicity) in K5-Cyclin D2 (K5-D2) and or CDK6 subunits, we also determined their in vitro wild-type sibling mice at 30weeks of promotion. ( B) Representa- kinase activities. Figure 5c shows that overexpression tive paraffin sections of papillomas at 30weeks of promotion from of cyclin D3 results in a fourfold increase in both CDK4 wild-type (a) and K5-D2 (b) sibling mice. Magnification  20. and CDK6 kinase activities (Figure 5c, lanes 1, 2, 4 and 5).

observation period, 60% of the cyclin D2-null mice showed tumors (Figure 4a). Moreover, cyclin D2-null mice average a significantly lower number of tumors per Discussion mice (4.8 papillomas/mouse) compared to wild-type sibling (9.5 papillomas/mouse) (Po0.05) (Figure 4a). In addition to its growth-promoting function, cyclin D3 Analysis of SCC latency showed that malignant expression has been associated with terminal differentia- progression was delayed 5 weeks in cyclin D2-null mice tion in muscle cells (Kiess et al., 1995; Skapek et al., compared to wild-type sibling. In addition, the SCC 1995; Mariappan and Parnaik, 2005). Bartkova et al. incidence reached 45% in wild-type mice and 30% (1998) have shown that cyclin D3 expression is in cyclin D2-null mice at 38 weeks of promotion associated with the maintenance of terminal differentia- (Figure 4b). tion in epithelium. Therefore, an unresolved question is whether cyclin D3 promotes cell proliferation and/or differentiation in diverse tissues. Cyclin D3 overexpression decreases cyclin D2/CDKs Surprisingly, overexpression of cyclin D3 results in an complex formation and its kinase activity inhibitory function in mouse skin tumor development. We analyzed the CDKs complex formation in hyper- On the other hand, overexpression of cyclin D2 results proliferative epidermis from K5-D3 and wild-type in elevated numbers of tumors and increased malignant siblings (16 h after tetradecanoyl phorbol acetate progression to SCC. Thus, it is evident that deregulated (TPA) application). In this model, one topical applica- expression of each member of the D-type cyclin family tion of TPA on the mice’s back induces a synchronized affects papilloma development in a different way.

Oncogene Role of cyclin D2 and D3 in tumor development P Rojas et al 1727

Figure 4 Kinetic of papilloma and SCC formation in cyclin D2À/À mice. Groups of cyclin D2-null and wild-type sibling mice were initiated with DMBA and promoted with multiple applications of TPA on dorsal mouse skin. (a) Average number of papillomas per mouse (multiplicity) and percentage of mice with papillomas as a function of weeks of study (incidence). (b) Percentage of mice with SCC as function of weeks of study (SCC incidence).

Our biochemical analysis revealed that overexpression cyclin D2. Supporting the oncogenic activity of cyclin of cyclin D3 results in a strong reduction of cyclin D2 D2, depletion of this cyclin result in decrease number of level in mouse epidermis, primary keratinocyte and skin papillomas and strong reduction of malignant progres- tumors. Notably, restitution of cyclin D2 levels, in the sion to SCCs. Thus, using a model of ras-mediated bigenic mice K5-D3/K5-D2, results in increased num- tumorigenesis of mouse skin in a cyclin D2-deficient bers of papillomas as observed in K5-D2 mice. We background, we have provided genetic evidence of interpret this result as an indication that a mild elevation the role of cyclin D2 as a downstream mediator of in the protein level of cyclin D2 is enough to overpass oncogenic ras. the tumor suppression effect of cyclin D3. Macroscopic Analysis of epidermis from K5-D3 mice shows not and histological analysis of papillomas from K5-D3 and only reduction of cyclin D2 protein levels but also a wild-type mice indicate no changes in the size of the reduction of cyclin D2 mRNA. These data suggest that tumors and/or the degree of differentiation (data non- a transcriptional mechanism is responsible for the shown). Consistent with this data, we did not observe negative feedback loop that regulates cyclin D2 levels. significant differences in the levels of keratinocyte However, a more detailed analysis of skin tumors proliferation and/or when K5-D3 tumors showed that the level of cyclin D2 was elevated 10-fold were compared with wild type (data not shown). These in papillomas from K5-D2 mice, whereas a mere two data indicate that reduced level of cyclin D2 affects the times increase was observed in papillomas from K5-D3/ initiation stage (clonal expansion of Ha-ras-mutated K5-D2 mice (Figure 2d). These results suggest that cells) but not the promotion stage. In addition, these overexpression of cyclin D3 in tumors from the bigenic observations strongly support the notion that cyclin D2 mice results in reduction not only of the endogenous but induces tumor development and the tumor suppressive also the transgenically expressed cyclin D2. In this effect of cyclin D3 is associated with the reduced level of scenario, the level of the transgenically expressed cyclin cyclin D2. D2 is probably not regulated at the transcriptional level, It is important to note that overexpression of cyclin as an exogenous promoter was used to drive its D2 also promotes malignant transformation to SCC and expression, and it is unlikely that the mechanism it is not affected by reduction of the endogenous cyclin involved in the reduction of cyclin D2 mRNA acts D2 in K5-D3/K5-D2 bigenic mice. Also, the reduction through an external promoter. Hence, a post-transcrip- of cyclin D2 levels in K5-D3 epidermis does not decrease tional mechanism is likely involved in the negative the rate of malignant transformation that is similar to feedback loop. the percentage observed in wild-type mice. These In vivo kinetic analysis demonstrated that overexpres- findings support the notion that the level of cyclin D2 sion of cyclin D3, and the consequent reduction of cyclin strongly affects papilloma development, but malignant D2 levels, does not decrease the rate of keratinocyte transformation to SCC required a threshold level of proliferation. In fact, the entry into S phase is

Oncogene Role of cyclin D2 and D3 in tumor development P Rojas et al 1728

Figure 5 CDKs complex formation and kinase activities in epidermis from transgenic mice. At 16 h after TPA treatment, fresh protein lysates from epidermis of K5-D3 and wild-type mice were immunoprecipitated with antibodies against cyclin D1 (D1), cyclin D2 (D2), cyclin D3 (D3), CDK4, CDK6 or normal rabbit serum (NR). (a) D-type cyclins’ immunoprecipitates were immunoblotted with antibodies against CDK4, CDK6 and the corresponding D-type cyclin. Protein lysates from CDK4-null mice (CDK4 KO) was used as control to determine the specificity of the CDK antibodies. (b and c), D-type cyclins, CDK4 and CDK6 kinase activities were determined in the immunoprecipitates using a pRb peptide as substrate. Kinase activities were quantified with a densitometer.

accelerated in K5-D3 keratinocytes compared to wild- Altogether, we interpreted our results as an indication type mice (Supplementary material, Figure S1). that cyclin D2 appears to play an important role in Our biochemical analysis established that the de- skin tumorigenesis, although the reduced level is well creased cyclin D2/CDK complexes and cyclin D2- tolerated by normal keratinocytes. Indeed, the genera- associated kinase activities are compensated by an tion of cyclin D1, cyclin D2 and cyclin D3 knockout increase in cyclin D3 and cyclin D1 kinase activities. mice confirmed that keratinocytes stand very well the Whereas the increased cyclin D3 kinase activity corre- ablation of any of these (Sicinski et al., 1996, 1995, lated well with the elevated cyclin D3/CDK complexes, 2003). In addition, ablation of two members of the increased cyclin D1-associated kinase activity is not due D-type cyclin family results in normal development of to increased complex formation. It is plausible that the the ‘single-cyclin’ mice until late gestation (Ciemerych elevated cyclin D3/CDK complexes bind to p27 and/or et al., 2002), without defects in skin development releasing cyclin D1/CDK of these inhibitors and (Sicinski, personal communication). consequently increasing the kinase activities without It is worth mentioning that cyclin D1 protein is more increasing the level of cyclin D1/CDK complexes. stable in cell lines derived from SCC compared to

Oncogene Role of cyclin D2 and D3 in tumor development P Rojas et al 1729 keratinocytes and papillomas cell lines. On the other electrophoresed and transferred onto nitrocellulose mem- hand, cyclin D3 stability is increased in non-malignant branes. Western blot analysis was performed with the cell lines (Supplementary material, Figure S2). following antibodies: cyclin D1 (C-20), cyclin D2 (M-20), Ultimately, these results demonstrate that inhibition cyclin D3 (C-16), CDK4 (C-22), b-actin (I-19) (Santa Cruz or activation of D-type cyclins will affect the outcome of Biotechnology Inc., Santa Cruz, CA, USA), and CDK6 Ab-2 (K6.90) (Labvision-NeoMarkers Co., Fremont, CA, USA). tumors in different ways. Mouse skin tumor experiments demonstrate that whereas cyclin D2 overexpression increase tumor development, cyclin D1 does not affect Immunoprecipitation assay tumorigenesis (Rodriguez-Puebla et al., 1999), and The skin of TPA-treated mice (16 h after TPA treatment) cyclin D3 results in reduction of the number of tumors was scraped off with a razor blade, and homogenized in associated to decrease the cyclin D2 level. Therefore, it is lysis buffer (25 mM HEPES (pH 7), 5 mM EDTA, 50m M necessary to determine whether positive and negative NaCl, 50m M NaFl, 10% glycerol, 1% Triton X-100, anti- feedback loops exist in different tissues regulating the protease and anti-phosphatase cocktails). The epidermal homogenate was centrifuged at 10 000 g and supernatant was D-type cyclin levels. In the final analysis, these feedback used directly for immunoprecipitation assay with protein loops could represent possible targets for future cancer A-agarose beads and specific antibodies. Immunoprecipitates therapies. were electrophoresed and transferred onto nitrocellulose membranes.

Materials and methods Kinase activity assay Experimental animals Protein lysates were obtained as described above, and 1 FVB/N-K5-Ccnd2 (K5-D2) and FVB/N-K5-Ccnd3 (K5-D3) centrifuged at 10 000 g for 30min at 4 C. The supernatant transgenic mice generation was previously described (Rodriguez- was collected and used for a kinase assay. Puebla et al., 2000). Bigenic mice K5-D2/K5-D3 were Protein lysates (250 mg) were immunoprecipitated with obtained by interbreeding K5-D2 and K5-D3 transgenic mice. antibodies against CDK4, CDK6, cyclin D1, cyclin D2 or Cyclin D2-null mice were provided by Dr Piotr Sicinski cyclin D3. Antibody-precoated beads were incubated with the 1 (Sicinski et al., 1996). lysates for 1 h at 4 C, washed twice with Tween-20buffer and kinase buffer and further incubated with pRb substrate and [g-32P]ATP (Rodriguez-Puebla et al., 2000). Sodium dodecyl Two-stage carcinogenesis protocol sulfate (SDS) sample buffer was added and electrophoresed Initiation was performed by a single topical application of through acrylamide gels. DMBA (3.9 mM in 50 ml of acetone) on the dorsal skin of newborn mice. At 3 weeks after DMBA initiation, TPA (32.4 mM in 200 ml of acetone) was applied twice a week on each Statistical analysis mouse’s dorsal skin. Mouse skin and tumors were grounded in Statistical analysis was performed using GraphPad Software homogenization buffer (60m M Tris-HCl, pH 8.6, 5 mM (GraphPad Software, San Diego, CA, USA). ethylenediaminetetraacetate (EDTA), 5 mM ethylene glycol- bis(2-aminoethyl ether)-N,N,N0,N0-tetraacetic acid (EGTA), 300 mM sucrose, anti-protease and anti-phosphatase cocktails). Acknowledgements The homogenates were sonicated and centrifuged at 10 000 g for 10min. The supernatants were collected and used directly We thank Dr Paula L Miliani de Marval and Dr Jonathan for Western blot analysis. Horowitz for helpful reading and discussion of this paper, Dr Piotr Sicinski for providing the cyclin D2-null mice; Juan C Western blot analysis Santiago for technical support, the Laboratory Animal Skin proteins were obtained as previously described (Rodriguez- Resources (LAR), and the CVM histology service personnel. Puebla et al., 1998a). Protein lysates (25–50 mg) were This study was supported by NIH Grant CA116328.

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

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