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Cell Growth & Differentiation Vol. 10, 467-472, July 1999
© 1999 American Association for Cancer Research

Cyclin D1 Overexpression in Mouse Epidermis Increases Cyclin-dependent Kinase Activity and Cell Proliferation in Vivo but Does Not Affect Skin Tumor Development1

Marcelo L. Rodriguez-Puebla, Margaret LaCava and Claudio J. Conti2

The University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In a previous study, we showed that synchronized proliferation of mouse epidermis was induced by topical application of 12-O-tetradecanoyl-phorbol 13-acetate. Here, we used this system to study modifications in the cell cycle regulation and kinetics of proliferation in transgenic mice that overexpress cyclin D1 (K5D1 mice). Overexpression of cyclin D1 corresponded with an increase of proliferation in the epidermis of these transgenic mice. After proliferation reached its peak, the labeling index remained high in the transgenics, but not in the wild-type animals. In addition, cyclin D1/cyclin-dependent kinase (CDK) complex formation increased in the transgenic mice and was correlated with elevated CDK4 and CDK6 kinase activities. However, the increased CDK activities were not sufficient to effect mouse skin tumor development. In summary, these results show that cyclin D1 has a unique growth-promoting role in tumor development, but does not act as an oncogene independent of ras activity.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In mammalian cells, proliferation is under the control of factors that regulate the transitions between different cell cycle stages at two main checkpoints (1) . The better characterized checkpoint is at the G1-S transition, which initiates DNA replication in S phase. The other checkpoint is at the G2-M transition, which controls mitosis and cell division (2) . The cyclins are a family of key cell cycle regulators that function by association with and activation of CDKs3 at specific points in the cell cycle to phosphorylate various proteins that are important during cell cycle progression (3) . Three D-type cyclins (D1, D2, and D3) and cyclin E are expressed in G1. Depending on cell lineage, various combinations of D-type cyclins are induced by mitogens in the middle of G1 (3) , whereas cyclin E expression is maximal at the G1-S transition (4 , 5) . D-type cyclins form complexes with CDK4 and CDK6 during G1, whereas cyclin E forms complexes with CDK2 and becomes active during the G1-S transition (4 , 5) . Cyclin A synthesis is also initiated during G1-S transition, and cyclin A kinase activity is first detected in S phase (6) . In addition to being activated by cyclins, CDK activity is also modulated by phosphorylation and by CKIs that can bind and inactivate cyclin/CDK complexes. Two families of CKIs have been described. p21Cip1, p27Kip1, and p57Kip2 are members of one family. These proteins can interact with and inhibit a wide variety of cyclin/CDK holoenzymes, and their overexpression blocks cells in G1. The other CKI family is Ink4 and its members (p16INK4a, p15INK4b, p18INK4c, and p19INK4d) interact only with cyclin D/CDK4 and cyclin D/CDK6 complexes (7, 8, 9) . The expression of cyclins, CDKs, and CKIs varies in different cell types (3 , 10) , and regulation by both positive and negative factors ensures that the cell cycle phases occur in the correct order. A key substrate for G1 cyclin/CDK complexes is the pRb. The phosphorylation of pRb, a tumor suppressor gene product, has been attributed to cyclin/CDK complexes and implicated in the regulation of proliferation in keratinocytes and other cell types (9 , 11) . Thus, phosphorylation of pRb blocks its ability to suppress the activity of S phase-promoting transcription factors, such as E2F (11 , 12) . Furthermore, abnormalities in cell cycle regulation, such as increased activity of cyclin/CDK complexes due to p53 mutation (13 , 14) and overexpression of cyclin D1 (15 , 16) , have been reported in experimentally produced squamous cell carcinomas originating from mouse keratinocytes. K5D1 mice, transgenic mice that express cyclin D1 under the control of the bovine keratin 5 promoter, have normal epidermal differentiation, although they develop a basal cell hyperplasia (17) . No spontaneous development of epithelial tumors was perceived during the first 8 months of life (17) . Recently, we reported that cyclin D1-null mice have reduced skin tumor development in the two-stage carcinogenesis protocol and two other independent approaches (18) . The results from these three models of experimental carcinogenesis indicate that cyclin D1 has a unique role in promoting ras-mediated growth in mouse skin and that ras-mediated tumor development is dependent on pathways that favor cyclin D1 expression (18) . In addition, analysis of mouse skin tumors during premalignant progression showed that cyclin D1 and cyclin D2 overexpression and formation of cyclin D1/CDK4 complexes were detected at specific time points concomitant with the overexpression of the mutated ras allele (19) .4 These results led us to postulate that in the K5D1 mouse epidermis, cyclin D1 behaves like an oncogene or synergizes with oncogenic ras in the mouse skin two-stage carcinogenesis model. In this study, we test this hypothesis and also analyze the proliferative behavior of mouse epidermis treated with 12-O-tetradecanoyl-phorbol 3-acetate. Our results demonstrate that overexpression of cyclin D1 in mouse epidermis affects cell proliferation, cyclin D/CDK complex formation, and CDK activities, but does not influence skin tumor development.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Keratinocyte Proliferation in Transgenic Mice.
In previous work, we used the in vivo SENCAR mouse skin model to study the regulation of the cell cycle machinery in vivo (20) . We have used the same approach here with K5D1 mice to determine whether cyclin D1 overexpression affects epidermal cell proliferation. Overexpression of cyclin D1 in epidermal basal cells under the control of the bovine cytokeratin 5 promoter was previously described in K5D1 mice (17) . A significantly higher number of BrdUrd-positive cells or S phase basal cells is seen in the transgenic K5D1 mice compared with their normal siblings (17) . Therefore, our initial experiment was to measure the proliferative action of TPA in the epidermis of the transgenic and normal sibling mice by monitoring DNA synthesis by BrdUrd incorporation. Mouse dorsal skins were treated topically with TPA, and the BrdUrd labeling index was calculated at various times after TPA treatment. As previously reported, the labeling index reached a maximum peak at 16 h after TPA application when 80% of the cells were in S phase (Fig. 1Citation ; Ref. 20 ). In both transgenic and wild-type animals, the maximum levels were reached with similar kinetics, however, the labeling index of the K5D1 keratinocytes decreased more slowly than in wild type (Fig. 1)Citation . At 48 h, the labeling index in the K5D1 epidermis was 2.5-fold greater than in wild type. This difference in kinetics may be related to specific differences in the levels of cyclin D1 expression and its influence on cyclin/CDK kinase activities.



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Fig. 1. TPA-induced proliferation of epithelial cells. After TPA treatment for the times indicated, BrdUrd was injected into the mice. BrdUrd labeling was immunohistochemically detected in formalin-fixed, paraffin-embedded sections of dorsal skin. The numbers of BrdUrd-positive and total cells/200 µm of interfollicular epithelia were determined. The data are representative of two independent experiments.

 
Isolating proteins from the epidermis at various times after TPA treatment enabled us to evaluate the expression of cyclin D1 and other cell-cycle regulators. To extract epidermal proteins, the epidermis was separated from the dermis by mechanical methods and proteins were isolated from the epidermal lysate (20) . We found that cyclin D1 was overexpressed in K5D1 mice and, after TPA application, the level of expression increased 1.5-fold at 12 h, reached a peak at 16 h (2-fold), and remained constant up to 24 h. The level of expression in the wild-type animals reached a peak at 12 h (an increase of 3.5-fold) and subsequently decreased (Fig. 2)Citation . In untreated epidermis, cyclin D1 expression was 7.6-fold higher in transgenic mice than in normal siblings. Consistent with our previous study, the level of cyclin D1 decreased 4 h after TPA treatment in normal, but not transgenic, mice (Fig. 2)Citation (20) . On the other hand, the cyclin D3 protein level decreased 2-fold within 0–8-h in K5D1 mice compared with wild-type animals. We also observed that cyclin A protein levels increased in K5D1 mice at 8 h and 16 h (2.5-fold and 4-fold, respectively), then remained constant; however, in wild-type animals, cyclin A protein levels remained low early after TPA application and then increased 5-fold at 16 h. On the other hand, the CDK2 and CDK4 protein levels remained constant at all times in transgenic and normal sibling mice (Fig. 2)Citation . We also looked at the levels of CKIs in the transgenic mice. Consistent with our previous study, p21Cip1 was expressed at the end of S phase (20–24 h after treatment) in normal siblings, but in K5D1 mice, p21Cip1 was detected at all times, and the level increased 4-fold at 12 h (Fig. 2Citation ; Ref. 20 ). The p27Kip1 protein level was also higher in transgenic mice, although this CKI was also detected in normal siblings.



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Fig. 2. Western blot analysis of cyclin (Cyc), CDK, and CKI expression in epithelial cells at various times after TPA treatment. Protein lysates from epithelial cells of mice treated with TPA were electrophoresed and transferred onto a nitrocellulose membrane. Antibodies against the proteins indicated were used in Western blot analysis. K5D1, transgenic mice; WT, wild-type mice.

 
Cyclin D1/CDK Complex Formation and Kinase Activities.
Cyclin D1/CDK4 and cyclin D1/CDK6 complexes are detected in the untreated epidermis of SENCAR mice. Complex formation is elevated 8 h after TPA application and remains high up to 24 h (20) . We also studied cyclin D1/CDK4 and cyclin D1/CDK6 complex formations in epidermal protein lysates of normal skin. For this purpose, CDKs were immunoprecipitated with antibodies against CDK4 or CDK6 and analyzed by Western blot with an anticyclin D1 antibody. Fig. 3Citation shows that cyclin D1/CDK4 and cyclin D1/CDK6 complex formations increased in transgenic mice. Only the faster migrating band of cyclin D1 was observed in transgenic mice, which was consistent with the results shown in Figs. 2Citation and 3Citation . To evaluate whether cyclin D1 overexpression activates CDK4 and CDK6, we performed a kinase assay with pRb as the substrate. After immunoprecipitation of CDK4 and CDK6 in normal skin (untreated), pRb phosphorylation levels were determined by measuring the incorporation of 32P. The CDK6 kinase activity was 1.9-fold higher in K5D1 cells than in normal cells, and CDK4 activity showed a similar increase (2.1-fold) in K5D1 mice (Fig. 4)Citation . CDK4 and CDK6 activities at 16 h after TPA treatment were also elevated 2-fold in the K5D1 epidermis (data not shown). It is worth noting that CDK4 and CDK6 kinase activities were higher in wild-type and transgenic hyperplastic skin (3 weeks of TPA application) than in normal untreated skin. These activities were still higher in transgenic mice than in wild-type mice (data not shown). These results clearly demonstrated that overexpression of cyclin D1 in vivo increases the kinase activities of G1 CDKs in normal and TPA-treated skin.



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Fig. 3. Coimmunoprecipitation and immunoblot analysis of cyclin D1/CDK4 and cyclin D1/CDK6 complexes. Fresh protein lysates of untreated mouse epidermis were immunoprecipitated (IP) with polyclonal anti-CDK6 and anti-CDK4 antibodies and immunoblotted with polyclonal antibody for cyclin D1 (Cyc D1). The control was normal rabbit serum and lysates obtained from untreated normal epidermis (NR). K5D1, transgenic mice; WT, wild-type mice.

 


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Fig. 4. CDK4 and CDK6 kinase activities. Fresh protein lysates of untreated mice epidermis were immunoprecipitated (IP) with polyclonal anti-CDK6 and anti-CDK4 antibodies. An in vitro kinase assay, with the IP proteins and pRb as a substrate, was performed. pRb phosphorylation levels were determined by measuring 32P incorporation. K5D1, transgenic mice; WT, wild-type mice.

 
Cyclin D1 Expression Does Not Synergize with Oncogenic Ras.
Recently, we showed that cyclin D1-null mice have reduced skin tumor development in the two-stage carcinogenesis model (18) . Cyclin D1 expression is considered essential to mouse skin tumor development, and it is a downstream mediator of ras activity (21) . To determine whether cyclin D1 overexpression can influence the development of tumors in mouse skin, transgenic K5D1 and normal sibling mice were topically treated with a subcarcinogenic dose of the genotoxic carcinogen DMBA and then treated twice a week topically with the tumor promoter TPA. Tumors developed 5 weeks after the start of promotion in both wild type and transgenic animals (Fig. 5)Citation . Papilloma multiplicity was the same in the transgenic and normal siblings and reached an average of 17 papillomas/mouse at 15 weeks. No significant differences were observed in the penetrance, and all of the mice had developed skin tumors at 17 weeks of promotion (Fig. 5)Citation . Analysis of tumors at various durations of promotion showed that cyclin D1 overexpression did not affect the state of differentiation. In fact, immunohistochemical detection with antibody to cytokeratin 1 (a marker of epidermal differentiation) showed no difference between the papillomas of wild-type and transgenic mice (data not shown). We also analyzed the tumors for a point-mutated Ha-ras gene, as was described earlier (22, 23, 24, 25) . Mutation of the Ha-ras gene was identified in papillomas obtained from K5D1 and wild-type animals. This mutation can be easily detected by the appearance of a Xba I restriction fragment length polymorphism at codon 61 (23 , 24) . All the K5D1 and wild-type tumors studied contained the specific mutation at codon 61 (data not shown). These data demonstrate that tumors generated in the transgenic animals contain the same specific mutation described in wild-type animals. Spontaneous skin tumors did not develop in uninitiated and untreated K5D1 animals. On the other hand, transgenic mice that overexpress a transcription factor, which has a role downstream of cyclin D1 (K5-E2F1), develop spontaneous tumors (26) . These results show that cyclin D1 overexpression did not synergize with ras in mouse skin tumor development and cyclin D1 did not behave like an oncogene in this model.



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Fig. 5. Mouse skin tumor development. K5D1 mice were crossed onto a SENCAR background. Twenty female K5D1 mice and 20 normal sibling mice were used. Initiation was performed with DMBA, and, 2 weeks after initiation, TPA was applied twice a week on each mouse’s dorsal skin. K5D1, transgenic mice; WT, wild-type mice.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In a previous study, we reported that the absence of cyclin D1 has a significant effect on mouse skin tumor development. In fact, ras-mediated skin tumorigenesis is substantially reduced in a cyclin D1-deficient background (18) . On the other hand, we did not observe clear alterations in the kinetics of proliferation in cyclin D1-null keratinocytes.4 These results led us to hypothesize that overexpressed cyclin D1 could behave like an oncogene in mouse skin tumor development.

Here, we show that the kinetics of proliferation of K5D1 and normal sibling keratinocytes were different and that proliferation was higher in transgenic mice. Both normal and transgenic animals reach a peak of proliferation at 16 h, when 80% of cells were in S phase. However, the labeling index of untreated epidermis was higher in transgenic mice. After the peak of proliferation (20–48 h), the labeling index of K5D1 mice remained higher than that of their normal siblings. The higher labeling index in transgenic mice may be due to the fraction of keratinocytes that do not enter S phase in normal mice but do in transgenic mice, although in an unsynchronized fashion. However, other factors are needed for the cells to pass the commitment point and further promote the G1-S phase transition. This requirement is not compensated for cyclin D1 overexpression because G1 is not shorter in K5D1 than in wild-type keratinocytes (Fig. 1)Citation . This result conflicts with previous observations that expression of cyclin D1 produces shorter G1 phase (27, 28, 29) . However, previous studies were performed with cell lines and, as far as we know, ours is the first study of cyclin D1 overexpression in keratinocytes in vivo. Analysis of protein expression showed that cyclin D1 protein levels were 8-fold higher in K5D1 mice than in wild-type mice. This difference was observed at all time points and increased after 12–16 h. On the other hand, cyclin D3 protein levels were similar, but a small difference in the protein level was observed early in G1 phase, in which the wild-type mice had a higher level. These results are consistent with a model in which overexpression of one of the D-type cyclins is compensated by reduction in the level of another member of this protein family.

Cyclin A is another positive regulator of the cell cycle that participates in the G1-S phase transition and in S phase (30, 31, 32, 33) . The cyclin A protein level in transgenic mice also increased during the 48 h of the experiment, especially during G1 phase. This result and the BrdUrd labeling index showed that a larger fraction of K5D1 keratinocytes than wild-type keratinocytes are in S phase at the time of TPA application, which may account for the difference in the kinetics of proliferation and loss of synchronization that was observed 16 h after TPA application.

p21Cip1, a member of a general family of CKIs, was expressed at the end of S phase in wild-type mice, which is consistent with our previous study on SENCAR mouse epidermis (20) . As was previously reported, p21Cip1 may be involved in the inhibition of cyclin/CDK complexes in late S phase and in the exit from the cell cycle during the differentiation that follows basal cell division (20) . Consistent with our results, expression of p21Cip1 protein increases in postmitotic cells adjacent to the proliferative compartment, and then the protein level decreases in terminally differentiated primary keratinocytes (34) . Furthermore, in several cell lineages, p21Cip1 expression correlates well with terminal differentiation (35, 36, 37) . Thus, the increased p21Cip1 expression at the end of S phase observed in normal sibling mice may be related to the early function of p21Cip1 as an inducer of differentiation. In the transgenic mice, p21Cip1 was expressed in early G1 phase, and its levels increased at 12 h. Several functions have been assigned to p21Cip1, including that of a promoter of CDK/cyclin complex formation (38) . The presence of p21Cip1 in early G1 phase of K5D1 mice may be related to this function.

p27Kip1 is another member of the CKI family and has high homology with p21Cip1 and p57Kip2 in the NH2-terminal domain (10 , 39 , 40) . p27Kip1 was also overexpressed in the transgenic mice. The protein levels in normal siblings decreased after TPA application, whereas in K5D1 mice, the protein levels remained high, as in untreated skin. Our previous study showed that p27Kip1 forms complexes with CDK4, CDK6, and CDK2 during G1 and S (20) , and this is consistent with a model in which p27Kip1 is involved in the inhibition of CDK activity at different points of the cell cycle. However, the kinase activities of G1 CDKs were increased in the transgenic mice at 16 h (data not shown). Thus, p27Kip1 did not seem to inhibit the kinase activities in transgenic epidermis. A possible explanation for the high p27Kip1 protein level is the presence of a mechanism in which K5D1 keratinocytes compensate for the overexpression of a positive regulator.

Complex formation between cyclin D1 and G1 CDKs was elevated in transgenic mice. These differences correlated well with the increased kinase activity of CDK4 and CDK6 in the transgenic mice. Phosphorylation of pRb blocks its ability to bind and suppress the activity of S phase-promoting transcription factors such as E2F (12 , 41) , allowing the cells to enter S phase. Thus, increased kinase activities permit higher levels of proliferation or permit proliferation indepen-dent of external stimuli. Amplification or overexpression of cyclin D1 is also known to be involved in human and experimental tumor development (42, 43, 44) . Early studies of the two-stage carcinogenesis model showed that cyclin D1 is overexpressed in papillomas and squamous cell carcinomas (15 , 16 , 19) . We recently provided biochemical and genetic evidence that cyclin D1 is a critical target for oncogenic ras in mouse skin (18) . To determine whether overexpressed cyclin D1 behaves like an oncogene or synergizes with oncogenic ras, we analyzed the skin of K5D1 transgenic mice for tumor development. No significant differences were observed between transgenics and normal siblings. In this context, cyclin D1 overexpression in keratinocytes in vivo increased the G1 CDK kinase activities and the level of proliferation, but did not increase mouse skin tumor development. However, as mentioned above, cyclin D1-null mice had a reduced level of tumor development. Therefore, these data are consistent with a model in which cyclin D1 has a unique growth-promoting role in tumor development, but does not act like an oncogene, nor does it synergize with ras activity.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Experimental Animals.
SENCAR mice that overexpressed cyclin D1 (line 7111) were developed in our laboratory, as reported previously (17) . Before TPA treatment, the dorsal side of the young adult mice was shaved, and the mice were allowed to rest for 2 days. TPA (2 mg in 0.2 ml of acetone, Sigma Chemical Co., St. Louis, MO) or acetone was then topically applied to each mouse’s shaved dorsal skin. At 0, 4, 8, 12, 16, 20, 24, 30, and 48 h after TPA treatment, three mice were killed by cervical dislocation, and their dorsal skins were treated with a depilatory agent for 1 min and then washed. Each skin was excised, and epidermal tissue was scraped off with a razor blade into homogenization buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.1% Tween-20, 1 mM DTT, 0.1 mM phenyl methyl sulfonyl fluoride, 0.2 unit/ml aprotinin, 10 mM ß-glycerophosphate, 0.1 mM sodium vanadate, and 1 mM NaF] and homogenized with a manual homogenizer at 4°C. The epidermal homogenate was centrifuged at 10,000 x g for 5 min. The supernatant was collected and used directly for Western blot analysis or immunoprecipitation or stored at -70°C. Two mice from each group received injections of BrdUrd (Sigma Chemical Co.) 30 min before being killed, and their dorsal skins were fixed in formalin.

Western Blot Analysis, Immunoprecipitation, and Kinase Assay.
The protein concentration in each skin lysate was measured with the Bio-Rad protein assay system (Bio-Rad Laboratories, Richmond, CA). Protein lysates (25 µg from each sample) were electrophoresed through 10 or 12% acrylamide gels and electrophoretically transferred onto nitrocellulose membranes. After being blocked with 5% nonfat powdered milk in Dulbecco’s phosphate-buffered saline (Sigma Chemical Co.), the membranes were incubated with 1 µg/ml of specific antibodies. We used polyclonal antibodies against cyclin D1 (C-20), cyclin D3 (C-16), cyclin A (C-19), CDK4 (C-22), CDK2 (M-2), p21 (C-19), and p27 (C-19) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Horseradish peroxidase-conjugated secondary antibody (Amersham Corp., Arlington Heights, IL) and an enhanced chemiluminescense (ECL detection kit; Amersham Corp.) were used for immunoblotting detection.

To study cyclin D/CDK complex formations, we used polyclonal anti-CDK4 and anti-CDK6 antibodies conjugated with protein G-Sepharose beads (Life Technologies Inc., Grand Island, NY) to immunoprecipitate fresh protein lysates overnight at 4°C with constant rotation. After washing, Western blot analysis was performed, as described above, with polyclonal antibody against cyclin D1 (C20) (Santa Cruz Biotechnology, Inc.). Bio-image analysis was used to quantitate the expression levels of those proteins.

To study the kinase activities of CDK4 and CDK6, protein lysates were obtained as described above, but the homogenate was frozen on powdered dry ice, thawed in ice water, incubated on ice for 15 min and centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was collected and used for a kinase assay. Protein lysate (500 µg) was immunoprecipitated with antibodies against CDK4 or CDK6. 50 µl of precoated antibody beads (50 µl; Life Technologies, Inc.) were incubated with the lysate for 2 h at 4°C. The beads were washed four times with homogenization buffer and twice with kinase buffer [50 mM HEPES (pH 7.5) and 10 mM MgCl2]. Then, 30 µl of kinase buffer [0.5 µg of pRb substrate (Santa Cruz Biotechnology, Inc.), 5 µCi [{gamma}-32P]ATP (6000 Ci/mmol), 2.5 mM EGTA, 1 mM DTT, 20 µM ATP, 10 mM ß-glycerophosphate, 0.1 mM sodium vanadate, and 1 mM NaF] was added to the bead pellet and incubated for 30 min at 30°C. Sodium dodecyl sample buffer was added, and each sample was boiled for 5 min and electrophoresed through a 10% acrylamide gel.

Immunohistochemical Staining.
Epithelial cell proliferation was measured by i.p. injection of BrdUrd (60 mg/g body weight) 30 min before the mice were killed. BrdUrd incorporation was detected by immunohistochemical staining of paraffin-embedded sections with mouse anti-BrdUrd monoclonal antibody (Becton Dickinson Immunocytometry System; Becton Dickinson, San Jose, CA). The reaction was visualized with a biotin-conjugated antimouse antibody (Vector Laboratories, Inc., Burlingame, CA) and an avidin-biotin-peroxidase kit (Vectastain Elite; Vector Laboratories, Inc.) with diaminobenzidine as chromogen. Then, the numbers of BrdUrd-positive cells and total cells were determined per 200 µm of interfollicular epithelia in each section. Fifteen sections of 200 µm were counted/animal. The average and SD values were calculated for two animals per time point in independent experiments.

Two-Stage Carcinogenesis Protocol.
For chemical carcinogenesis studies, K5D1 mice were crossed into a SENCAR background. Twenty female mice of each group (transgenic and normal sibling mice) were identified by PCR, as reported previously (17) . Initiation was performed with DMBA (100 nmol in 0.2 ml of acetone; Sigma Chemical Co.), which was topically applied to each mouse’s shaved dorsal skin. Two weeks after DMBA initiation, 1 mg of TPA in 0.2 ml of acetone was applied twice a week on each mouse’s dorsal skin. Papillomas appeared after 6 weeks of continuous TPA treatment and were counted for 20 weeks.


    Acknowledgments
 
We thank April Ott and the Science Park animal facility personnel, Dr. Irma B. Gimenez-Conti and the Science Park histology service for assistance with the immunohistochemical staining, Cassie Smith Bigbee for technical assistance, Melissa Bracher for secretarial assistance, Sharon Stockman for preparation of the manuscript, and Dr. Maureen Goode for editing the manuscript.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by DHS Grants CA 42157 and CA 57596; by institutional support Grant CA 16672 to the M. D. Anderson Cancer Center, which funds the animal facility; and by Center Grant ES07784, which funds the hystology service. Back

2 To whom requests for reprints should be addressed, at The University of Texas M. D. Anderson Cancer Center, Science Park–Research Division, P. O. Box 389, Smithville, TX 78957. Phone: (512) 237-9428; Fax: (512) 237-2444, E-mail: sa83125{at}odin.mdacc.tmc.edu Back

3 The abbreviations used are: CDK, cyclin-dependent kinase; CKI, CDK inhibitor; DMBA, 7,12-dimethylbenz[a]anthracene; pRb, retinoblastoma protein; TPA, 12-O-tetradecanoylphorbol 13-acetate; BrdUrd, bromo-2'-deoxyuridine. Back

4 M. L. Rodriguez-Puebla and C. J. Conti, unpublished results. Back

Received for publication 12/21/98. Revision received 4/ 9/99. Accepted for publication 5/13/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

  1. Nurse P. Ordering S phase and M phase in the cell cycle. Cell, 79: 547-550, 1994.[Medline]
  2. Hartwell L. H., Weinert T. A. Checkpoints: controls that ensure the order of cell cycle events. Science (Washington DC), 246: 629-634, 1989.[Abstract/Free Full Text]
  3. Sherr C. J. Mammalian G1 cyclins. Cell, 73: 1059-1065, 1993.[Medline]
  4. Sherr C. J. G1 phase progression: cyclin on cue.. Cell, 79: 551-555, 1994.[Medline]
  5. Koff A., Giordano A., Desai D., Yamashita K., Harper W., Elledge S., Nishimoto T., Morgan D., Franza R., Roberts J. Formation and activation of a cyclin E-cdk2 complex during G1 phase of the human cell-cycle. Science (Washington DC), 257: 1689-1693, 1992.[Abstract/Free Full Text]
  6. Labbe J. C., Picard A., Peaucellier G., Cavadore J. C., Nurse P., Doree M. Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation. Cell, 57: 253-263, 1989.[Medline]
  7. Guan K. L., Jenkins C. W., Li Y., Nichols M. A., Wu X., O’Keefe C., Matera A. G., Xiong Y. Growth suppression by p18, a p16 and p15 related CDK6 inhibitor, correlates with wild-type pRb functions. Genes Dev., 8: 2939-2952, 1994.[Abstract/Free Full Text]
  8. Hannon G. J., Beanch D. p15INK4B is a potential effector of TGF-ß-induced cell cycle arrest. Nature (Lond.), 371: 257-261, 1994.[Medline]
  9. Demers G. W., Foster S. A., Halbert C. L., Calloway D. A. Growth arrest by induction of p53 in DNA damaged keratinocytes is bypassed by human papillomavirus 16 E7. Proc. Natl. Acad. Sci. USA, 91: 4382-4386, 1994.[Abstract/Free Full Text]
  10. Sherr C. J. D-type cyclins. Trends Biochem. Sci., 20: 187-190, 1995.[Medline]
  11. Munger K., Pieternpol J. A., Pittelkow M. R., Holt J. T., Moses H. L. Transforming growth factor B1 regulation of c-myc expression, pRb phosphorylation, and cell cycle progression in keratinocytes. Cell Growth Differ., 3: 291-298, 1992.[Abstract]
  12. Nevins J. R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science (Washington DC), 258: 424-429, 1992.[Abstract/Free Full Text]
  13. Kress S., Sutter C., Strickland P. T., Mukhtar H., Schweizer J., Schwarz M. Carcinogen-specific mutational pattern in the p53 gene in ultraviolet B radiation-induced squamous cell carcinosmas of mouse skin. Cancer Res., 52: 6400-6403, 1992.[Abstract/Free Full Text]
  14. Ruggeri B., Caamano J., Goodrow T. Alterations of the p53 tumor suppressor gene during mouse skin tumor progression. Cancer Res., 51: 6615-6621, 1991.[Abstract/Free Full Text]
  15. Robles A. I., Conti C. J. Early overexpression of cyclin D1 protein in mouse skin carcinogenesis. Carcinogenesis (Lond.), 16: 781-786, 1995.[Abstract/Free Full Text]
  16. Bianchi A. B., Fischer S. M., Robles A. I., Rinchik E. M., Conti C. J. Overexpression of cyclin D1 in mouse skin carcinogenesis. Oncogene, 8: 1127-1133, 1993.[Medline]
  17. Robles A. I., Larcher E., Whalin R. B., Murillas R., Richie E., Gimenez-Conti I. B., Jorcano J. L., Conti C. J. Expression of Cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia. Proc. Natl. Acad. Sci. USA, 93: 7634-7638, 1996.[Abstract/Free Full Text]
  18. Robles A. I., Rodriguez-Puebla M. L., Glick A. B., Trempus C., Hansen L., Sicinski P., Tennant R. W., Weinberg R. A., Yuspa S. H., Conti C. J. Reduced skin tumor development in cyclin D1-deficient mice highlights the oncogenic ras pathway in vivo. Genes Dev., 12: 2469-2474, 1998.[Abstract/Free Full Text]
  19. Rodriguez-Puebla M. L., LaCava M., Gimenez-Conti I. B., Jonhson D. G., Conti C. J. Deregulated expression of cell-cycle proteins during premalignant progression in SENCAR mouse skin. Oncogene, 17: 2251-2258, 1998.[Medline]
  20. Rodriguez-Puebla M. L., Robles A. I., Johnson D. G., LaCava M., Conti C. J. Synchronized proliferation induced by TPA treatment of mouse skin: an in vivo model for cell cycle regulation. Cell Growth Differ., 9: 31-39, 1998.[Abstract]
  21. Rodriguez-Puebla M. L., Robles A. I., Conti C. J. Ras activity and cyclin D1 expression: an essential mechanism of mouse skin tumor development. Mol. Carcinog., 24: 1-6, 1999.[Medline]
  22. Balmain A., Pragnell I. Mouse skin carcinomas induced in vivo by chemical carcinogens have a transforming Harvey-ras oncogene. Nature (Lond.), 303: 72-74, 1983.[Medline]
  23. Bizub D., Wood A., Skalka A. Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons. Proc. Natl. Acad. Sci. USA, 83: 6048-6052, 1986.[Abstract/Free Full Text]
  24. Quintanilla M., Brown K., Ramsden M., Balmain A. Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature (Lond.), 322: 78-80, 1986.[Medline]
  25. Roop D., Lowy D., Tambourin P., Strickland J., Harper J., Balaschak M., Spangler E., Yuspa S. An activated Harvey ras oncogene produces benign tumors on mouse epidermal tissue. Nature (Lond.), 323: 822-824, 1986.[Medline]
  26. Pierce A. M., Fischer S., Conti C. J., Johnson D. G. Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development. Oncogene, 16: 1267-1276, 1998.[Medline]
  27. Jiang W., Kahn S., Zhou P., Zhang Y., Cacace A., Infante A., Doi S., Santella R., Weinstein I. Overexpression of cyclin D1 in rat fibroblasts causes abnormalities in growth control, cell cycle progression and gene expression. Oncogene, 8: 3447-3457, 1993.[Medline]
  28. Imoto M., Doki Y., Jiang W., Han E. K., Weinstein I. B. Effects of cyclin D1 overexpression on G1 progression-related events. Exp. Cell Res., 236: 173-180, 1997.[Medline]
  29. Musgrove E., Lee C., Buckley M., Sutherland R. Cyclin D1 induction in breast cancer cells shortens G1 and is sufficient for cells arrested in G1 to complete the cell cycle. Proc. Natl. Acad. Sci. USA, 91: 8022-8026, 1994.[Abstract/Free Full Text]
  30. Walker D. H., Maller J. L. Role for cyclin A in the dependence of mitosis on completion of DNA replication. Nature (Lond.), 354: 314-317, 1991.[Medline]
  31. Girard F., Strausfeld U., Fernandez A., Lamb N. J. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell, 67: 1169-1179, 1991.[Medline]
  32. Desdouets C., Sobczak-Thepot J., Murphy M., Brechot C. Cyclin A: function and expression during cell proliferation. Prog. Cell Cycle Res., 1: 115-123, 1995.[Medline]
  33. Zindy F., Lamas E., Chenivesse X., Sobczak J., Wang J., Fesquet D., Henglein B., Brechot C. Cyclin A is required in S phase in normal epithelial cells. Biochem. Biophys. Res. Commun., 182: 1144-1154, 1992.[Medline]
  34. Di Cunto F., Topley G., Calautti E., Hsiao J., Ong L., Seth P. K., Dotto G. P. Inhibitory function of p21Cip1/WAF1 in differentiation of primary mouse keratinocytes independent of cell cycle control. Science (Washington DC), 280: 1069-1072, 1998.[Abstract/Free Full Text]
  35. Guo K., Wang J., Andres V., Smith R., Walsh K. MyoD-induced expression of p21 inhibits cyclin-dependent kinase activity upon myocyte terminal differentiation. Mol. Cell. Biol., 15: 3823-3829, 1995.[Abstract/Free Full Text]
  36. Jiang H., Lin J., Su Z., Collar F. R., Huberman E., Fisher P. B. Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21/WAF1/CIP1, expression in the absence of p53. Oncogene, 9: 3397-3406, 1994.[Medline]
  37. Parker S. B., Eichele G., Zhang P., Rawls A., Sands A. T., Bradley A., Olson E. N., Harper J. W., Elledge S. J. p53-independent expression of p21Cip1 in muscle and other terminally differentiating cells. Science (Washington DC), 267: 1024-1027, 1995.[Abstract/Free Full Text]
  38. Labaer J., Garret M. D., Stevenson L. F., Slingerland J. M., Sandhu C., Chou H. S., Fattaey A., Harlow E. New functional activities for the p21 family of CDK inhibitors. Genes Dev., 11: 847-862, 1997.[Abstract/Free Full Text]
  39. Xiong Y., Hannon G., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature (Lond.), 366: 701-704, 1993.[Medline]
  40. Xiong Y. Why are there so many CDK inhibitors?. Biochim. Biophys. Acta, 1288: 1-5, 1996.
  41. Nevins J., Chellappan S., Mudryj M., Hiebert S., Devoto S., Horowitz J., Hunter T., Pines J. E2F transcription factor is a target for the RB protein and the cyclin A protein. Cold Spring Harb. Symp. Quant. Biol., 56: 157-162, 1991.[Abstract/Free Full Text]
  42. Jiang W., Zhang Y-J., Khan S. M., Hollstein M. C., Santella R. M., Lu S. H., Harris C. C., Montesano R., Weinstein I. B. Altered expression of the cyclin D1 and retinoblastome genes in human esophageal cancer. Proc. Natl. Acad. Sci. USA, 90: 9026-9030, 1993.[Abstract/Free Full Text]
  43. Bartkova J., Lukas J., Strauss M., Bartek J. The PRAD1/cyclin D1 oncogene product accumulate aberrantly in a subset of colorectal carcinomas. Int. J. Cancer, 58: 568-573, 1994.[Medline]
  44. Schauer I. E., Siriwardana S., Langan T. A., Sclafani R. A. Cyclin D1 overexpression versus retinoblastoma inactivation: implications for growth control evasion in non-small cell and small cell lung cancer. Proc. Natl. Acad. Sci. USA, 91: 7827-7831, 1994.[Abstract/Free Full Text]



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