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Cell Growth & Differentiation Vol. 10, 739-748, November 1999
© 1999 American Association for Cancer Research


Articles

Effect of Elevated Levels of Ornithine Decarboxylase on Cell Cycle Progression in Skin1

Susan K. Gilmour2, Mary Birchler, Mary K. Smith, Kathryn Rayca and Judith Mostochuk

Lankenau Medical Research Center, Wynnewood, Pennsylvania 19096

Abstract

By crossing TG.AC v-Ha-ras and K6/ODC transgenic mice, we found previously that an activated ras and follicular ornithine decarboxylase (ODC) overexpression cooperate to generate spontaneous tumors in the skin. Cellular proliferation was dramatically increased in the K6/ODC transgenic skin, as evidenced by elevated proliferating cell nuclear antigen and Ki67 expression compared with nontransgenic littermates. Keratinocytes isolated from transgenic skin also displayed increased clonal growth. Paradoxically, expression of the growth inhibition-associated proteins p53, p21Waf1, p27Kip1, and Bax was increased with ODC overexpression in the skin. ODC overexpression did not affect cyclin D/cyclin-dependent kinase 4 (Cdk4)-dependent phosphorylation of retinoblastoma protein but stimulated cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity, with minimal effect on the levels of these proteins. Thus, ODC/polyamine-induced activation of cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity may cooperate with the ras induction of cyclin D/Cdk4/6-associated retinoblastoma protein phosphorylation to not only stimulate proliferation but ultimately contribute to tumor development.

Introduction

Increased levels of ODC3 and polyamines have been implicated as playing an important role in epithelial tumorigenesis, largely because of the early induction of ODC by tumor promoters such as TPA (1, 2, 3, 4) , and to studies using inhibitors of ODC (5, 6, 7, 8) . Although ODC overexpression is not sufficient to transform normal diploid keratinocytes or fibroblasts, it does cooperate with genetic defects in premalignant epidermal cells to promote tumor progression (9) . Moreover, we have shown recently that ODC overexpression directed to cells in the outer root sheath of hair follicles in transgenic mice cooperates with a single oncogene, an activated ras, to generate spontaneous papillomas, many of which convert rapidly to carcinomas (10) .

The mechanism by which elevated ODC and polyamines influence epithelial tumor development is not known. Polyamines readily bind polyanionic macromolecules such as DNA, RNA, and phospholipids, resulting in far-reaching effects on DNA replication, transcription, and translation. Thus, it is not surprising that numerous studies using specific inhibitors of polyamine biosynthesis have documented the essential role played by polyamines in cell growth and differentiation (11 , 12) . However, use of these polyamine inhibitors or analogues have produced varying effects in different phases of the cell cycle (13, 14, 15, 16, 17, 18) . Because ODC is the first and rate-limiting enzyme in polyamine biosynthesis, it is significant that ODC activity is elevated in the late G1 phase of many different cell types (19 , 20) . In addition to its association with entry into the S phase of the cell cycle, ODC is transactivated by Myc protein, which is an important regulator of cell cycle progression, differentiation, and apoptosis (21, 22, 23, 24) . However, despite evidence implicating increased levels of ODC with cellular proliferation and transformation, the effect of elevated levels of ODC on the control of the cell cycle has not been addressed.

Progression through the mammalian cell cycle is regulated by a set of kinases, termed Cdks, and their regulatory subunits, the cyclins (25) . Cdks by themselves are inactive and must bind to a cyclin to be active. The cyclical synthesis and degradation of cyclins D, E, A, and B1 correspond to their activation in the G1, G1-S, S/G2, and G2-M phases of the cell cycle, respectively. Cdk activity is further regulated by phosphorylation (both inhibitory and activating) and CKIs (p15, p16, p18, p21Waf1, and p27Kip1) that bind to cyclins, Cdks, or cyclin-Cdk complexes. Mitogenic growth factors act during the G1 phase of the cell cycle. The critical point in late G1 at which a cell commits to another round of DNA replication is most likely regulated by the cyclins D1, D2, or D3 complexed to either Cdk4 or Cdk6. The D-type cyclins are induced as part of a delayed early response to mitogenic stimulation by growth factors. D-type cyclins combine with Cdk4 or Cdk6 to form active complexes that phosphorylate the product of the retinoblastoma gene (pRb), which results in the removal of the G1 phase block caused by underphosphorylated Rb. Cyclin E is induced later in G1 than D-type cyclins, and its binding to Cdk2 is essential for the initiation and progression of DNA replication. Once a cell passes the G1-S checkpoint, progression through the cell cycle is independent of mitogenic stimulation.

Because the G1-associated cyclins/Cdks factor so significantly into the commitment of the cell to proceed through the cell cycle, it is not surprising that aberrant expression of G1 cyclins and/or their Cdk partners is often found in tumor cells (25) . Although it has been demonstrated that ODC overexpression plays a causal role in epidermal tumorigenesis, it is not known whether elevated levels of ODC and polyamines affect the expression and/or activity of proteins responsible for regulating the cell cycle. We provide evidence that ODC overexpression in the skin of transgenic mice stimulates cellular proliferation and indirectly activates cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity.

Results

Dermal Elevations of ODC Stimulate Cellular Proliferation.
Previously, we have shown that follicular overexpression of ODC using a keratin 6 promoter cooperates with a mutated Ha-ras to produce spontaneous skin tumors in ODC/Ras double transgenic mice (10) . In contrast, K6/ODC transgenic mice that overexpress ODC but do not express a mutated Ha-ras do not form tumors (10) . Indeed, the skin of K6/ODC transgenic mice does not display epidermal hyperplasia as is characteristically found in skin stimulated to proliferate as a result of wounding or treatment with a tumor promoter. When ODC expression is directed to follicular outer root sheath cells, the primary skin abnormalities are degeneration of hair follicles and the formation of follicular cysts in the dermis. Interestingly, spontaneous tumors that form in ODC/Ras double transgenic mice appear to be of follicular origin, and often cells in the tumors appear to be continuous with the cells lining the follicular cysts (10) . Because tumor development is thought to arise by clonal growth of initiated cells, we examined the effect of ODC overexpression on proliferation in the targeted skin of K6/ODC transgenic mice. Immunohistochemical staining of skin from K6/ODC transgenic mice and from their normal littermates demonstrated elevated levels of ODC in the cells lining the follicular cysts in the dermis (Fig. 1)Citation . This ODC staining colocalized with staining for the proliferation marker Ki67, which is only expressed in the nucleus of cycling cells (26) . Whereas very little Ki67 nuclear staining was detected in either epidermal or dermal cells of the normal littermate skins (Fig. 1A)Citation , the majority of cells lining the follicular cysts in the dermis of transgenic skin exhibited nuclear Ki67 staining (Fig. 1B)Citation . Levels of PCNA (27) were also elevated in the K6/ODC skin compared with the skin from their normal littermates (Fig. 2)Citation . Not surprisingly, PCNA typically was found to be increased to similarly high levels in spontaneous tumors that developed in ODC/Ras double transgenic mice. As shown previously (10) , the truncated but fully active K6/ODC transgene protein product is expressed in both the epidermis and dermis of K6/ODC transgenic skin and to a greater extent in the spontaneous tumors from the ODC/Ras double transgenic mice. There was a slight induction of endogenous wild-type ODC in TPA-induced tumors from TG.AC mice not possessing the K6/ODC transgene but, as expected, no expression of the lower molecular weight K6/ODC transgene product (Fig. 2)Citation .



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Fig. 1. Immunohistochemistry of normal littermate skin (A and C) and K6/ODC transgenic mouse skin (B and D) using an anti-Ki67 antibody (A and B) or an anti-ODC antibody (C and D). B, inset, brown-stained, Ki67-positive nuclei in cells lining the follicular cysts in the dermis of the K6/ODC transgenic skin. Arrows, ODC-stained cells lining the follicular cysts in D.

 


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Fig. 2. Proliferation is increased with elevated levels of ODC in the skin. Equal amounts of protein lysate from the epidermis (E) or dermis (D) of K6/ODC transgenic mouse skin and from normal, nontransgenic littermate skin and from spontaneous tumors (1 2 3 4) in ODC/Ras double transgenic mice were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Tumor 5 was from a TPA-promoted TG.AC v-Ha-ras transgenic mouse that did not carry the K6/ODC transgene. ODC and PCNA protein were detected by immunoblotting. The locations of the endogenous ODC protein and the truncated K6/ODC transgene protein product are indicated. As a loading control, the blot was stripped and probed with an antimouse IgG antibody, and the heavy chain mouse IgG is shown in the bottom panel.

 
Clonal growth of keratinocytes is a measure of their proliferative capacity (28) . We isolated keratinocytes from the epidermis of K6/ODC transgenic mice and their normal littermates for use in determining the effect of ODC on clonal growth of keratinocytes. The isolated epidermal cells were identical in age and genetic background but differed in that the cultured transgenic epidermal cells expressed ODC to very high levels compared with the nontransgenic littermate epidermal cells (29) . Suspensions of freshly isolated single epidermal cells were plated at clonal density onto irradiated feeder cells. After 2 or 3 weeks in culture, cells were fixed and stained with rhodamine B. As illustrated in Fig. 3Citation , colonies of ODC-overexpressing epidermal cells were larger and more numerous than colonies from normal epidermal cells. Thus, elevated levels of ODC increased the clonal growth of epidermal cells.



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Fig. 3. Clonal growth assay of normal and K6/ODC transgenic keratinocytes. Keratinocytes isolated from the skin of 3-day-old K6/ODC transgenic mice and their normal littermates were seeded at clonal density on a layer of irradiated Swiss 3T3 feeder cells. After 2 weeks, colonies were fixed and stained with rhodamine B.

 
Analysis of Cell Cycle-related Proteins in ODC Transgenic Skin.
In an effort to better understand the mechanism by which high levels of ODC and polyamines stimulate cell proliferation, their effect on the expression of cell cycle-related proteins was evaluated. To avoid the complicating effects of chemical carcinogens and tumor promoters, we examined tissue lysates from non-tumor-bearing skin of K6/ODC transgenic mice and in spontaneous tumors arising in ODC/Ras double transgenic mice. Use of the transgenic model allows the analysis of: (a) the effects of only ODC overexpression in the skin of K6/ODC transgenic mice; and (b) the effect of the expression of two genetic lesions, elevated ODC levels and an activated Ha-ras, found in the spontaneous tumors of ODC/Ras double transgenic mice (10) . Tissue lysates from transgenic mice were compared with those of nontransgenic littermates with the same age and genetic background.

With the exception of cyclin B1, there were usually higher levels of cell cycle-related proteins in the ODC/Ras tumors compared with the non-tumor-bearing skins of K6/ODC transgenic mice or their normal littermates (Fig. 4)Citation . In particular, levels of cyclin D1 and cyclin E were elevated in tumors resulting from high levels of ODC activity and expression of v-Ha-ras. As reported previously by others, we detected three isoforms of cyclin E with molecular weights between Mr 50,000 and Mr 55,000, which likely represent alternatively spliced variants of this gene (30 , 31) . Although there appeared to be a cooperative effect of ODC and v-Ha-ras in causing the accumulation of the G1-related cyclins D1 and E, overexpression of ODC had only a minimal effect on levels of cyclins D1, E, B1, or the Cdks 2 or 4. Cyclin A, however, was elevated in both the epidermis and dermis of K6/ODC transgenic mice.



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Fig. 4. Western analysis of cell cycle-related proteins in transgenic skin and tumors. Equal amounts of protein in Tween 20 lysates of epidermal (E) and dermal (D) tissue from K6/ODC (Transgenic) mouse skin and from nontransgenic littermate (Normal) skin and of spontaneous tumors from ODC/Ras double transgenic mice were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Equal protein loading and transfer to the membrane was assessed by Ponceau S staining. Cyclin D1, Cdk4, cyclin E, Cdk2, cyclin A, and cyclin B1 protein were detected by immunoblotting.

 
Activation of Cyclin/Cdk Complexes with ODC Overexpression.
Because cell cycle progression is dependent on the kinase activity of cyclin/Cdk complexes, we examined the histone H1 or Rb-associated kinase activity of immunoprecipitated complexes of cyclins D, E, A, and B1 and of Cdks 4 and 2. In agreement with the immunoblot results shown in Fig. 4Citation , cyclin B1-associated kinase activity is not elevated in K6/ODC transgenic skin or in the ODC/Ras tumor lysates. Kinase activity was elevated in immunoprecipitated complexes of cyclin E, Cdk2, and cyclin A from K6/ODC skin (particularly in the dermis) and from spontaneous ODC/Ras tumors compared with that in the epidermis and dermis of nontransgenic littermates (Fig. 5)Citation . In contrast, cyclins D1-, D2-, and D3-associated Rb kinase activity was not elevated in the non-tumor-bearing skin of K6/ODC mice (Figs. 5Citation and 6)Citation . However, as reported previously, expression of a mutated Ha-ras in the tumors resulted in increased Rb phosphorylation by immunoprecipitated Cdk4 in spontaneous tumors in which ODC was overexpressed (Fig. 5)Citation . This suggests that activation of cyclin E/Cdk2 and cyclin A/Cdk2 in ODC-overexpressing skin would promote S-phase entry to allow the cell to progress through another round of the cell cycle.



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Fig. 5. Kinase assays of transgenic skin and spontaneous ODC/Ras tumors. Equal amounts of Tween 20 lysates of epidermal (E) and dermal (D) tissue from K6/ODC (Transgenic) mouse skin and from nontransgenic littermate (Normal) skin and of spontaneous tumors from ODC/Ras double transgenic mice were immunoprecipitated with either cyclin D1, Cdk4, cyclin E, Cdk2, cyclin A, or cyclin B1 antibody. The resulting immunoprecipitates were used in in vitro kinase assays with either GST-Rb or histone H1 as a substrate, as indicated. Kinase reactions were resolved by 12% SDS-PAGE, and the dried gel was subjected to autoradiography.

 


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Fig. 6. ODC stimulates cyclin E- but not cyclin D-associated kinase activity. Equal amounts of Tween 20 lysates of epidermal (E) and dermal (D) tissue from K6/ODC (Transgenic) mouse skin and from nontransgenic littermate (Normal) skin were immunoprecipitated with either cyclin D1, cyclin D2, cyclin D3, or cyclin E antibody. The resulting immunoprecipitates (ip) were used in in vitro kinase assays with GST-Rb as a substrate, except for cyclin E immunocomplexes where histone H1 was used as a substrate. Kinase reactions were resolved by SDS-PAGE, followed by autoradiography.

 
Increased p53 and Cell Cycle Inhibitory Proteins p21Waf1 and p27Kip1 in K6/ODC Skin.
Because cell cycle-related kinase inhibitors play an important role in regulating cyclin/Cdk activity, we looked at the expression of the cyclin-dependent kinase inhibitors p21Waf1, p27Kip1, and p53 in K6/ODC transgenic skin. Because ODC overexpression stimulates proliferation in the skin, we were surprised to find accumulations of p53, p21Waf1, and p27Kip1 in skin tissues that have elevated levels of ODC. Fig. 7Citation shows that levels of p53, p21Waf1, and p27Kip1 were increased in both the epidermis and dermis of K6/ODC transgenic mice as well as in the resulting spontaneous skin tumors that arise from the simultaneous expression of v-Ha-ras and ODC. Additional immunoblot analyses did not detect either p19INK4a or p57Kip2 in tumors or in skin from either the K6/ODC transgenic mice or their normal littermates (data not shown). Because wild-type p53 can transactivate several genes including p21Waf1 and Bax (32 , 33) , we investigated whether Bax protein levels were elevated in K6/ODC skin. Examination of transgenic skin for expression of Bax revealed that ODC overexpression also led to accumulations of Bax but not another proapoptotic protein, Bak, both in the epidermis and dermis of K6/ODC mice compared with their normal littermates (Fig. 7)Citation .



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Fig. 7. ODC stimulates expression of growth inhibition-associated proteins. Equal amounts of RIPA lysates from the epidermis (E) or dermis (D) of K6/ODC (Transgenic) mouse skin and from nontransgenic littermate (Normal) skin and of spontaneous tumors from ODC/Ras double transgenic mice were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed for p53, p21Waf1, p27Kip1, Bax, and Bak protein by immunoblotting.

 
We also examined the relative binding of p27Kip1 to cyclin D and cyclin E complexes because studies have shown that the ratio of p27Kip1 or p21Waf1 CKIs bound to either cyclin D/Cdk4 or to cyclin E/Cdk2 complexes may play a role in determining to what extent the kinase activity is inhibited (34, 35, 36) . Proteins associated with either cyclin E or cyclin D2 were immunoprecipitated with the respective antibodies, and the level of p27Kip1 was determined by immunoblotting (Fig. 8)Citation . There was no difference in the level of p27Kip1 bound to cyclin E complexes in transgenic and normal mouse skin. In contrast, more p27Kip1 was complexed with cyclin D in ODC overexpressing skin compared with nontransgenic littermate skin. Likewise, there was more cyclin D1 protein found in p27Kip1 immunoprecipitates from K6/ODC transgenic skin than in normal littermate skin. A similar pattern of binding to cyclin/Cdk complexes was seen with p21Waf1 (data not shown). Thus, the net effect of more inhibitory proteins p27Kip1 or p21Waf1 bound to cyclin D in K6/ODC skin compared with nontransgenic littermate skin probably contributes to the lack of increased kinase activity associated with cyclin D/Cdk4 complexes in the transgenic skin.



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Fig. 8. Elevated levels of ODC cause sequestration of p27Kip1 in cyclin D protein complexes. Equal amounts of protein lysates from the epidermis (E) or dermis (D) of K6/ODC (Transgenic) mouse skin and from nontransgenic littermate (Normal) skin were immunoprecipitated with either cyclin E (top panel), cyclin D2 (middle panel), or p27Kip1 (bottom panel) antibody. The resulting immunocomplexes were resolved using SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis using antibody to p27Kip1 (top and middle panels) or cyclin D1 (bottom panel).

 
TUNEL Staining for Apoptosis.
To address whether the ODC-induced accumulations of p53 and Bax in K6/ODC transgenic skin were indicative of apoptosis, we used a terminal deoxynucleotidyl transferase-based TUNEL assay to detect cells within the skin containing DNA fragmentation associated with apoptosis. No stained nuclei were found in the nontransgenic littermate skin (Fig. 9A)Citation . However, positively stained nuclei were found in cells lining the follicular cysts in the dermis of K6/ODC transgenic skin (Fig. 9B)Citation . All of these positively stained cells were located in the more suprabasal layer of cells lying closest to the lumen of the follicular cysts. Because differentiating cells found in the suprabasal layers of the epidermis can sometimes stain positive in this TUNEL assay because of DNA fragmentation, it is possible that these positively stained cells are terminally differentiating cells rather than apoptotic cells. However, no suprabasal cells in the epidermis of either transgenic mice or their nontransgenic littermates were positively stained.



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Fig. 9. TUNEL staining of K6/ODC transgenic and nontransgenic littermate skin. Skin from nontransgenic littermates (A) and K6/ODC transgenic mice (B) were fixed overnight with 10% formalin and paraffin embedded. Sections were stained for the TUNEL reaction, as described in "Materials and Methods." Arrows, cells staining positive for the TUNEL reaction.

 
Discussion

We have shown that sustained elevated levels of ODC and polyamines stimulate proliferation in the skin of transgenic mice. Although high levels of ODC have minimal or no effect on the levels of G1 cyclin and Cdk proteins, the coexpression of ODC and v-Ha-ras found in tumor tissue results in elevated levels of both cyclin D and cyclin E proteins. In addition, we have found that ODC overexpression does not affect cyclin D/Cdk4-dependent phosphorylation of Rb but does stimulate cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity. Our results confirm that both cyclin D levels and associated kinase activity are stimulated by a mutated Ha-ras (37 , 38) . However, in the absence of a mutated ras, elevated levels of ODC stimulate growth without increasing cyclin D expression or its associated kinase activity. Our data suggest that the ODC activation of cyclin E/Cdk2- and cyclin A/Cdk2-associated kinase activity allows DNA synthesis to proceed, thus promoting proliferation in the K6/ODC skin. Several studies have established a link between ras and the G1 Cdk/Rb/E2F pathway (37, 38, 39) . Cyclin D1 associates with Cdk 4/6 and phosphorylates and inactivates pRb during the early to mid-G1 phase. Cyclin E/Cdk2 acts after cyclin D1 and before cyclin A in regulating the G1-S phase transition. Thus, ODC/polyamine-induced activation of cyclin E/Cdk2 and cyclin A/Cdk2-associated kinase activity may cooperate with the ras stimulation of cyclin D/Cdk4/Cdk6 complexes (25) and the subsequent cyclin D/Cdk4/Cdk6-associated Rb phosphorylation to not only stimulate proliferation but to ultimately contribute to tumor development.

Although there is a good correlation between ODC-stimulated proliferation and increased cyclin/Cdk kinase activity, the mechanism by which ODC activates Cdk2 kinase activity is unclear. The kinase activity of the cyclin/Cdks is dependent on several factors including the expression levels of the cyclins and Cdks themselves, the phosphorylation status of the Cdks, and the stoichiometric ratio of complexes of proteins composed of cyclins, Cdks, and CKIs. The data presented here suggest that ODC activation of cyclin E/Cdk2 is only partially attributable to increased levels of cyclin or Cdk protein. In this respect, there are similarities between ODC and Myc protein with regard to their effect on the cell cycle. The induction of Myc in growth factor-depleted cells stimulates the activation of cyclin E/Cdk2 kinase, not cyclin D/Cdk4, without altering the level of Cdk2 or cyclin E (40) . In this case, Cdk2 is activated by dephosphorylation via the phosphatase Cdc25A, which is a transcriptional target of Myc (41) . Although we have observed no consistent increase in Cdc25A protein levels after ODC overexpression (data not shown), Cdc25A may also be activated by phosphorylation. Because ODC is transcriptionally activated by Myc (21, 22, 23, 24) , it is possible that ODC may mediate some of the biological effects of Myc.

Alternatively, it is possible that the protein inhibitors, such as p21Waf1, p27Kip1, or p16 are redistributed among various cyclin/Cdk complexes when ODC and polyamine levels increase. We were intrigued to find levels of p21Waf1 and p27Kip1 CKIs elevated in ODC-overexpressing skin and tumors, despite the increased level of proliferation. However, several reports indicate that the ratio of p21Waf1 or p27Kip1 bound to either cyclin D/Cdk4 or to cyclin E/Cdk2 complexes plays an important role in determining to what extent the kinase activity is inhibited (34, 35, 36) . In addition, it has been postulated that low stoichiometric levels of p27Kip1 or p21Waf1 may serve to stimulate the assembly of cyclin/Cdk complexes that is necessary for kinase activation (34 , 42 , 43) . Thus, our observation that K6/ODC transgenic skin has more p27Kip1 and p21Waf1 bound to cyclin D complexes, but not with cyclin E complexes, compared with nontransgenic littermate skin suggests that some of the elevated levels of these CKIs is sequestered in cyclin D complexes in K6/ODC skin. This sequestration of CKIs would presumably partially reverse the inhibitory effect on cyclin E/Cdk2 and cyclin A/Cdk2 kinase activity that would be expected with the increased levels of p21Waf1 and p27Kip1 in ODC-overexpressing skin.

It is intriguing that p53 protein levels are increased in the skin of K6/ODC transgenic mice. The tumor suppressor protein p53 has been implicated in a variety of pathways in the cell, including growth arrest (44) , apoptosis (45) , and differentiation (46 , 47) . Accumulation of p53 protein in response to DNA damage after UV irradiation of the skin leads to either cell cycle arrest or apoptosis (48, 49, 50) . This has been attributed, in part, to the transcriptional activity of p53 because p53 can stimulate transcription of several genes including p21waf1, Bax, GADD45, and MDM-2 (32 , 33 , 44 , 51 , 52) . It is possible that high levels of ODC and polyamines directed to the skin can increase genomic instability or damage, thus triggering the accumulation of p53. Furthermore, it is unclear whether the accumulation of p53 protein in K6/ODC transgenic skin is causing growth arrest or apoptosis in some cells within the skin. It has been reported that perturbations in the levels of ODC and polyamines can trigger apoptosis (53, 54, 55, 56, 57) . This is supported by our finding of increased expression of the proapoptotic gene Bax and the detection of positively stained cells using an in situ TUNEL assay in K6/ODC transgenic skin compared with nontransgenic littermates. We have also found a correlation between ODC overexpression in skin and increased levels of p27Kip1, which has been reported recently to induce apoptosis in some cells (58) . It is puzzling that although the expression of proapoptotic genes is elevated in the K6/ODC epidermis and dermis (Fig. 8)Citation , TUNEL-stained cells are found only in cells lining the follicular cysts in the dermis of K6/ODC transgenic mice (Fig. 9)Citation . In part this may be attributed to differences in the follicular epithelial cells found lining the dermal cysts and the remaining interfollicular cells in the epidermis of these hairless transgenic mice. Apoptosis is the result of altered expression of a cascade of many genes (in addition to the ones assayed in Fig. 8Citation ), and our results may suggest that the ODC-induced apoptosis detected in follicular cells in the dermis of the K6/ODC transgenic skin is due to alterations in genes besides those found in p53, p21Waf1, p27Kip1, and Bax. For instance, it is possible that there is a difference between follicular and interfollicular epidermal cells in both their expression of other essential proapoptotic genes and in their subsequent biological response (i.e., whether they undergo apoptosis) to elevated levels of ODC and polyamines.

Whereas normal proliferating keratinocytes are found only in the basal layer of the epidermis, cells that have acquired a mutated ras form squamous papillomas, in which cells continue to proliferate even in the suprabasal layers. The increased proliferation of epidermal cells that express high levels of ODC but no mutated ras may force some cells out of the basal compartment of the epidermis and into suprabasal epidermal layers, where proliferation is normally suppressed and terminal differentiation is triggered with detachment from the basement membrane (59) . Indeed, we have observed increased expression of differentiation-associated keratin 1 and loricrin in K6/ODC transgenic skin (data not shown). Because keratinocyte differentiation leads to DNA fragmentation, the suprabasal localization of the TUNEL-stained nuclei found around follicular cysts may reflect increased differentiation rather than apoptosis within these cells. Thus, our results suggest that increased ODC/polyamines not only stimulate entry into the cell cycle but also promote the commitment of more cells to differentiate or to undergo apoptosis.

It has been proposed that the association between proliferation and apoptosis is an important feature of homeostasis in the epidermis (60) . There is evidence that apoptosis requires the cell to be cycling because senescent cells fail to undergo apoptosis (61 , 62) . In fact, apoptotic "sunburn cells" are found in the proliferative basal layer of UV-irradiated skin (63) . Moreover, increased levels of p53 protein have been found in proliferating primary cultures of keratinocytes (47 , 64) . Interestingly, the transforming growth factor {alpha} gene is transcriptionally activated by p53 (65) . Thus, it has been proposed that p53 may play a dual role, one involving the repair or elimination of genetically damaged cells, and the other as part of a proliferative response necessary for replacement of damaged cells (65) . The efficient replacement of damaged cells in an epithelium such as skin is critical toward maintaining the integrity of the epithelium.

These results suggest one mechanism by which elevated levels of ODC and polyamines cooperate with a mutated Ha-ras to produce malignant epidermal tumors. ODC overexpression in the skin stimulates cell proliferation, despite the seemingly paradoxical activation of cyclin E/Cdk2-associated kinase activity, which allows S-phase entry, and the accumulation of wild-type p53, which is generally associated with growth arrest and apoptosis. Moreover, activation of cyclin E/Cdk2 activity may control the phosphorylation of the transcriptional coactivators CBP/p300, thus increasing the transcriptional coactivating function of CBP/p300 (66 , 67) . Cyclin E/Cdk2 has also been found to regulate transcriptional activation by nuclear factor-{kappa}B through interactions with p300, thus coordinating transcriptional activation with cell cycle progression (67) . Thus, it is clear that the stimulatory effects of elevated levels of ODC and polyamines on the level and activity of cell cycle-related proteins can have far-reaching effects on not only genes associated with proliferation but perhaps other genes that play an important role in the development of an invasive, malignant phenotype.

Materials and Methods

Tissue Collection.
K6/ODC transgenic mice were derived as described previously (68) . Mice hemizygous for the K6/ODC transgene were bred with TG.AC mice homozygous for the v-Ha-ras transgene (obtained from Taconic Labs, Germantown, NY). Offspring hemizygous for both the TG.AC ras transgene and the K6/ODC transgene were identified by PCR genotyping. These ODC/Ras double transgenic mice developed spontaneous skin tumors at 8–10 weeks of age and were sacrificed 4–6 weeks after the initial appearance of the tumors. All mice were fed ad libitum and maintained at 70–72°F with a 12/12-h light/dark cycle.

For analyses of tumor-free skin from K6/ODC mice, the epidermis and dermis were separated by immersing excised skin into 55°C water for 20 s, followed by scraping. Tissue collected from groups of two to three mice of either K6/ODC transgenic mice and of nontransgenic wild-type littermates was then frozen in liquid nitrogen, ground to a fine powder, and used for subsequent protein analyses. To circumvent problems arising from tumor heterogeneity, tumors from the ODC/Ras double transgenic mice were frozen in liquid nitrogen, ground, and stored at -80°C, if not used immediately after collection.

Protein Analyses.
For immunoblots, tissues were homogenized in RIPA buffer [50 mM Tris-HCl (pH 7.5), 1% NP40, 0.25% sodium deoxycholate, 0.25% SDS, 150 mM NaCl, and 1 mM EGTA] containing 2 µg/ml each of aprotinin, leupeptin, and pepstatin, 1 mM NaF, 1 mM sodium orthovanadate, and 1 mM Pefabloc by passing through a syringe needle after a 30-min incubation on ice. Tissue lysates were clarified by centrifugation, and protein content was determined by a Lowry assay. Equal amounts of protein were electrophoresed through a 7.5 or 12% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH). To assess for equal protein loading and transfer to the membranes, the protein on the membranes was stained with Ponceau S solution (Sigma-Aldrich, St. Louis, MO). Immunoblots were incubated for 1 h at room temperature in blocking solution (PBS with 10% milk and 0.05% Tween 20), followed by the primary antibody diluted in 0.1% milk blocking solution for 1–2 h. The immunoblots were developed with a horseradish peroxidase-conjugated secondary antibody, followed by detection using enhanced chemiluminescence according to the manufacturer’s directions (Pierce Corp., Rockford, IL).

Antibodies and Other Reagents.
Antibodies to Cdk2 (M2), Cdk4 (C-22), Cdk6 (C-21), cyclin A (C-19), cyclin B1 (GNS1), cyclin D1 (C-20), cyclin E (M-20), PCNA (PC10), p21Waf1 (C-19), p27Kip1 (C19), and p53 (FL-393) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal antibodies to cyclin D1 (Ab-2), cyclin D2 (Ab-3), and cyclin D3 (Ab-3) were purchased from NeoMarkers, Inc., (Fremont CA). For immunocytochemical studies, we used a rabbit antibody to Ki67 from Novacastra Laboratories (Newcastle, United Kingdom). Polyclonal antibodies to Bax and Bak were obtained from Upstate Biotechnology (Lake Placid, NY). Protein G-agarose was purchased from Life Technologies, Inc. (Grand Island, NY). Horseradish peroxidase-conjugated donkey antimouse and donkey antirabbit were obtained from Amersham (Arlington Heights, IL). [{gamma}-32P]ATP was from New England Nuclear (Boston, MA).

Immunoprecipitations and Kinase Assays.
Tissues were lysed in Tween 20 buffer [50 mM HEPES (pH 7.5) containing 150 mM NaCl, 2.5 mM EGTA, 1 mM EDTA, 0.01% Tween 20, 1 mM DTT, and 10 mM ß-glycerophosphate] containing protease and phosphatase inhibitors. The tissue lysates were frozen at -80°C and not thawed until used for the assay. Equal amounts of protein (300 µg to 1 mg) were immunoprecipitated at 4°C for 2 h with saturating amounts of antibodies. After the addition of 50 µl of protein G-agarose beads for 1 h, the immunocomplexes were washed with Tween 20 buffer three times. For Western blotting, the immunocomplexes were boiled in Laemmli sample buffer and separated by SDS-PAGE.

Immunocomplexes were washed twice with kinase buffer [50 mM HEPES (pH 7.5), 10 mM MgCl2, 10 mM ß-glycerophosphate, 2.5 mM EGTA, 20 µM ATP, 1 mM NaF, 100 µM NaVO4, and 1 mM DTT] containing protease inhibitors. For cyclin D/Cdk4 immunocomplexes, 30 µl of kinase buffer with 7.5 µCi of [{gamma}-32P]ATP and 2 µg of GST-Rb were added to each tube and incubated at 30°C for 30 min (69) . For all other cyclin/Cdk-associated kinase assays, 2 µg of histone H1 were used as a substrate instead of GST-Rb. The reaction was stopped by the addition of 35 µl of 2x sample buffer containing SDS and 10% freshly added ß-mercaptoethanol. The samples were boiled for 5 min and centrifuged, and the supernatants were separated by SDS-PAGE. Phosphorylation of GST-Rb or histone H1 was visualized by autoradiography of the dried gel. All of the immunoblot and kinase assays were repeated twice, or in some cases, three times to demonstrate reproducibility.

Histology and Immunohistochemistry.
Tissues were fixed in Fekete’s solution (60% ethanol, 3.2% formaldehyde, and 0.75 M acetic acid) overnight and embedded in paraffin. Sections of all tissues were stained with H&E for histopathological evaluation. Freshly cut tissue sections for Ki67 staining were microwaved in 10 mM sodium citrate (pH 6.0), twice for 5 min each, to expose the antigen. Goat serum was used to suppress nonspecific binding. Tissue sections were incubated with either rabbit ODC antiserum (a gift from Dr. Oili Hietala, University of Oulu, Oulu, Finland) at a 1:500 dilution or rabbit Ki67 polyclonal antibody at 1:700 dilution for 2 h at room temperature in a humidity chamber. The sections were then incubated with biotinylated antirabbit secondary antibody, followed by an incubation with an avidin horseradish peroxidase complex (Vectastain Elite ABC kit; Vector Laboratory, Burlingame, CA). Immunoreactive cells were localized by incubating the sections with a diaminobenzidine chromagen solution and then counterstained with hematoxylin. Normal serum was used as the primary antibody to serve as negative controls.

In Situ Apoptosis Detection.
Tissues were fixed in 10% formalin and paraffin embedded. Apoptosis was evaluated in freshly sectioned tissues using the terminal deoxynucleotidyl transferase-based TUNEL assay (TumorTACS; Trevigen, Gaithersburg, MD) according to the manufacturer’s protocol. Briefly, 3' ends of cleaved DNA fragments in deparaffinized tissue sections were labeled by the transferase using biotinylated nucleotides. These labeled DNA fragments were visualized by binding streptavidin-horseradish peroxidase, followed by reaction with diaminobenzidine to generate a dark brown precipitate and counterstaining with methyl green. Thus, apoptotic cells were distinguished by the dark brown nuclear staining.

Primary Cultures of Epidermal Cells.
Primary cultures of epidermal cells were isolated from 2- to 3-day-old K6/ODC transgenic and normal littermate mice by a trypsin flotation procedure (70 , 71) . Newborn mice heterozygous for the K6/ODC transgene were distinguished from their normal littermates by PCR genotyping of tail DNA using the primers described previously (68) . Single-cell suspensions of freshly isolated epidermal cells were plated at 500 viable cells/60-mm culture dish prepared previously with a feeder layer of Swiss 3T3 cells (passage 119–130). One day prior to plating the keratinocytes, subconfluent cultures of Swiss mouse 3T3 were irradiated to 5000 rads with a Shephard Mark 1 Cesium irradiator in DMEM (Life Technologies, Inc.). Four h later, the 3T3 cells were plated at a density of 1.4 x 105/cm2 on collagen/fibronectin-coated 60-mm dishes. The next day, the DMEM was removed, and the epidermal cells were plated at clonal density on this 3T3 feeder layer in William’s supplemented medium essentially as described previously (72) . The medium was changed three times weekly. Two or 3 weeks later, the dishes were fixed with 10% buffered formalin and stained with rhodamine B (Sigma Chemical Co., St. Louis, MO).

Acknowledgments

We thank Dr. Rebecca Morris for supplying medium and feeder cells and helpful advice in the clonal growth studies and Dr. Oili Hietala for the ODC antibody. We gratefully acknowledge Drs. Cheryl Hobbs, Janet Sawicki, Karen Knudsen, and Kathryn Lawson for helpful discussions and critical reading for the manuscript and Loretta Rossino and Michelle Darby for manuscript preparation.

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 NIH Grant R01-CA-70739 (to S. K. G.). Back

2 To whom requests for reprints should be addressed, at Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA, 19096. Phone: (610) 645-8429; Fax: (610) 645-2205; E-mail: gilmours{at}mlhs.org. Back

3 The abbreviations used are: ODC, ornithine decarboxylase; TPA, 12-O-tetradecanoylphorbol-13-acetate; Cdk, cyclin-dependent kinase; GST-Rb, glutathione S-transferase-retinoblastoma protein; CKI, cyclin-dependent kinase inhibitor; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine nick end-label; PCNA, proliferating cell nuclear antigen. Back

Received for publication 2/ 9/99. Revision received 9/ 7/99. Accepted for publication 9/13/99.

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