cooperation of ha-ras and bcl-2 during multistep skin carcinogenesis

Cooperation of Ha-ras and Bcl-2 DuringMultistep Skin Carcinogenesis

Sangjun Lee,1,5 Nikhil S. Chari,1,5 Hyung Woo Kim,2 Xuemei Wang,2 Dennis R. Roop,4

Song H. Cho,1,5 John DiGiovanni,3 and Timothy J. McDonnell1,5*1Department of Hematopathology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas2Department of Biostatistics, The University of Texas M.D. Anderson Cancer Center, Houston, Texas3Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center, Houston, Texas4Department of Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, Houston, Texas5The University of Texas Graduate School of Biomedical Sciences at Houston, Texas

Nonmelanoma skin cancer (NMSC) is the most frequently diagnosed cancer in the United States. Deregulation of

bcl-2 and ras family members is commonly observed in NMSC. It has been previously demonstrated that simultaneousbcl-2 and Ha-ras gene expression in keratinocytes results in disordered differentiation and resistance to cell deathinduced by ultraviolet (UV) radiation. It was, therefore, interest to assess the extent of cooperation between bcl-2 and

Ha-ras during multistep skin carcinogenesis. The keratin 1 promoter was used to generate HK1.ras and HK1.bcl-2transgenic mice, which were subsequently crossed to generate HK1.ras/bcl-2 double transgenic mice. The apoptoticindex (AI) following UV-irradiation was significantly lower in HK1.bcl-2 and HKI.ras/bcl-2 epidermis compared tocontrol littermates. Interestingly, the AI of HK1.ras/bcl-2 mice was significantly lower than even HK1.bcl-2 mice

following UV-irradiation. To investigate the interaction of these oncogenes in skin tumorigenesis, a two-stagechemical carcinogenesis protocol was used to induce tumors. The individual contributions of Ha-ras and bcl-2 topapilloma latency, incidence, and growth rate in HK1.ras/bcl-2 double transgenic mice was marginally additive.

Papillomas arising in HK1.ras transgenic mice exhibited the highest rate of apoptosis whereas papillomas arising in theHK1.ras/bcl-2 double transgenic mice exhibited rates of apoptosis that were significantly lower than papillomas arisingin either control littermate or HK1.ras mice. Constitutive expression of either Ha-ras or bcl-2 exhibited similar rates of

malignant tumor progression and they were not significantly different than control littermates. Importantly, whenthese two oncoproteins were coexpressed, a significant, and synergistic, increase in malignant transformation wasobserved. � 2007 Wiley-Liss, Inc.

Key words: nonmelanoma skin cancer; oncogenes; keratinocyte tumorigenesis; proliferation

INTRODUCTION

Nonmelanoma skin cancer (NMSC) is the mostfrequently diagnosed form of cancer in the UnitedStates. The estimated incidence of these malignan-cies is approximately 1.2 million cases annuallysimilar to the aggregate incidence of all othermalignancies [1]. It is widely appreciated thatmalignant transformation is a consequence not onlyof enhanced proliferation but also resistance to celldeath. In fact, the deregulation of cell death andproliferation have been consistently demonstratedto cooperate during in vivo multistep carcinogenesis[2].

Bcl-2 is recognized as a critical regulator of celldeath and is frequently implicated in the pathogen-esis of multiple tumor types including NMSC. Theexpression of bcl-2 is precisely regulated duringkeratinocyte differentiation and is normally limitedto basal keratinocytes [3–5]. Bcl-2 enhances cellviability following various apoptotic stimuli, includ-ing ultraviolet (UV)-irradiation, the most importantcausative agent of skin carcinogenesis [3,6]. It hasalso been shown that deregulation of bcl-2 familymembers is commonly observed in NMSC [4,7,8]. A

transgenic mouse model in which bcl-2 expressionwas targeted to epidermal keratinocytes using akeratin promoter (HK1.bcl-2) demonstrated an en-hanced rate of tumor formation following a standardtwo-stage chemical carcinogenesis protocol [9].

Previous studies have shown that oncogenic ras isintegral in initiating and maintaining the malignantphenotype [10–14]. Ras signaling can regulate cellproliferation, differentiation, and apoptosis [15–18].The estimated frequency of activating ras mutations

MOLECULAR CARCINOGENESIS 46:949–957 (2007)

� 2007 WILEY-LISS, INC.

This article contains supplementary material, which may beviewed at the Molecular Carcinogenesis website at http://www.interscience.wiley.com/jpages/0899-1987/suppmat/index.html.

Abbreviations: NMSC, nonmelanoma skin cancer; UV, ultraviolet;H&E, hematoxylin and eosin; PCNA, proliferating cell nuclear anti-gen; AI, apoptotic index; DMBA, 7,12-dimethyl-benz[a]anthracene;TPA, 12-o-tetradecanoylphorbol-13-acetate; PI, proliferative index.

Sangjun Lee and Nikhil S. Chari contributed equally to this study.

*Correspondence to: Department of Hematopathology—Box89, The University of Texas M.D. Anderson Cancer Center, 1515Holcombe Blvd., Houston, TX 77030.

Received 8 November 2006; Revised 3 February 2007; Accepted12 March 2007

DOI 10.1002/mc.20334

in NMSC is approximately 20% [19]. To study thecontribution of ras deregulation in skin tumorformation, skin-specific ras transgenic mice weregenerated using cytokeratin 1, 10, or 14 promoters[20–22]. The papillomas that developed in thesemodels did not, however, undergo malignant trans-formation.

It has been previously demonstrated that simulta-neous bcl-2 and Ha-ras gene deregulation in organo-typic keratinocyte cultures results in disordereddifferentiation and resistance to cell death inducedby UV radiation. It was, therefore, of interest todetermine whether, in fact, Ha-ras and bcl-2 couldcooperate during multistep skin carcinogenesisin vivo.

MATERIALS AND METHODS

Generation and Genotyping of Transgenic Mice

HK1.bcl-2 and HK1.ras transgenic mice weregenerated as previously described [9,23]. The mousestrains used were as below; the HK1.bcl-2 foundermice were mated with C57BL/6 mice to generate F1mice, and HK1.v-Ha-ras founder mice were matewith ICR mice to generate F1 mice. To generateHK1.ras/bcl-2 double transgenic mice, the pupsfrom each genotype were mated and screened usingPCR reaction with primer sets as follows: (a) Bcl-2primer set, Bcl-2S (0-CGACGACTTCTCCCGCCGC-TACCGC-30) and Bcl-2 AS (50-CCGCATGCTGGGGCCGTACAGTTCC-30); (b) v-Ha-ras primer set, Ras S(50-GGATCCGATGACAGAATACAAGC-30) and RasAS (50-ATCGATCGAACACTTGCA-30). PCR reactionsconsisted of 30 cycles under the following condi-tions: 948C, 30 s for DNA denaturation; 608C, 1 minfor annealing; 728C, 1 min for polymerization. PCRproducts were resolved on a 1.0% agarose gel andvisualized with ethidium bromide. The HK-1.v-Ha-ras generated the predicted 611 bp band while theHK-1.bcl-2 transgene generated a 350 bp band. Theprocedures for performing animal experiments werewith approval and in accordance with guidelines laidby the University of Texas M.D. Anderson CancerCenter Institutional Review Board. Sequence of Ha-RAS used in the generation of the mice can be foundunder accession number J02207.

Morphologic and Immunohistochemical Analysis

Skin samples from neonatal transgenic mice werefixed in 10% buffered formalin for 10 h. The fixedsamples were dehydrated in an ascending series ofalcohol and xylene and then embedded in paraffin.Tissue sections, 4 mm-thick, were deparaffinized inxylene and rehydrated in a descending alcohol seriesand routinely stained with hematoxylin and eosin(H&E). For immunohistochemical studies, tissuesections used were treated with 3% H2O2 to blockendogenous peroxidase activity and with a serum-free protein block (DAKO Corp., Capinteria, CA) to

prevent artificial color reaction. To assess prolifera-tion in papillomas, mouse primary anti-human/mouse proliferating cell nuclear antigen (PCNA)antibody was used (DAKO Corp.). Sections wereincubated with primary antibody for 1 h, and thenwashed in PBS. The slides were next incubated withHPR-conjugated secondary antibody (DAKO Corp.)for 30 min and visualized using standard DABtechniques (DAKO Corp.) and counterstaining withMayer’s hematoxylin.

Assessment of Epidermal Proliferation

The incorporation of 5-bromo-20-deoxyuridine-50-triphosphate (BrdU) (Sigma, St. Louis, MO) into theepidermis was used to assess the extent and distribu-tion of proliferative keratinocytes in vivo. Pups wereinjected intraperitoneally with 500 mg of BrdU in50 mL of sterile saline. After 1 h of incubation, theepidermis was harvested from the pups and dehy-drated in a graded series of cold alcohol andprocessed in cold xylene for 24 h followed by paraffinembedment. Paraffin sections were deparaffinized incold xylene and rehydrated in a descending series ofcold alcohol. Tissue sections were treated in 2N HClfor 30 min to denature the DNA. After blocking andquenching the sections using 1% normal goat serum,30% methanol, and 0.3% H2O2, the slides wererinsed in PBS and incubated with anti-BrdU antibody(Becton Dickinson, San Jose, CA) for 1 h. The slideswere incubated with anti-mouse HRP (AmershamLife science, Piscataway, NJ) for 45 min, and thenvisualized using standard DAB techniques (DAKOCorp.). The sections were counterstained withMayer’s hematoxylin for 3 min. Proliferative indiceswere calculated as the mean percentage of basal layerkeratinocytes having BrdU incorporated nuclei. Atotal of 7–10 mice per genotype (control, HK1.bcl-2,HK1.ras, and HK1.ras/bcl-2) were used in theseexperiments. Approximately 1500 cells were count-ed for each sample.

Assessment of Keratinocyte Apoptosis inNeonatal Mouse Skin

One-day-old neonatal mice were exposed to UVusing a FS40 sun lamp (Westinghouse, Bloomfield,NJ) with 62.5% UV-B, 37.2% UV-A, and 0.3% UV-Coutput. The neonates were exposed to a dose of 2 J/cm2 of UV-B as determined by an IL 700 radiometerfitted with a WN 320 filter specific for UV-Bwavelengths and an A127 quartz diffuser (Interna-tional Light, Newburyport, MA). The sunburneddorsal skin was removed 24 h after treatment withUV radiation and fixed in 10% buffered formalin for10 h before paraffin embedding. The paraffinsections were prepared as described above. Afterdeparaffinization and rehydration, the slides werestained using H&E. The apoptotic index (AI) wascalculated as a percentage of total keratinocytesexhibiting apoptotic morphology. A total of three

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Molecular Carcinogenesis DOI 10.1002/mc

mice per genotype were used and greater than1000 cells (three randomly selected areas were usedto harvest epidermis from each mouse, i.e. total9 sections/genotype) were counted. Multiple fieldswere counted for each section. For calculatingspontaneous AI the tissues were harvested from theprimary papillomas at the end of the 1 yr observationperiod and a similar procedure as described abovewas used.

Two-Stage Chemical Carcinogenesis

Briefly, 7,12-Dimethyl-benz[a]anthracene (DMBA)and 12-o-tetradecanoylphorbol-13-acetate (TPA) wereused as the initiator and the promoter, respectively,in a previously described two-stage chemical skincarcinogenesis protocol [9]. The dorsal skin ofneonatal mice was painted once with 20 mg of DMBAdissolved in 200 mg of acetone and after 1 week micereceived an application of 2.5 mg of TPA in 100 mg ofacetone twice a week for 3 weeks. The mice were thentreated with 5 mg of TPA in acetone twice a week fora total of 17 weeks. Mice were inspected weekly, andtotal papilloma incidence, growth rate, and meanlatency of papilloma formation were monitored for1 yr.

Statistical Analysis of Chemical Carcinogenesis

The probability of chemically induced tumordevelopment was assessed using a 2�2 factorialdesign. Box plots were used to illustrate the distribu-tion of incidence and growth rate of papillomas bytreatment group. A frequency table was prepared tosummarize the number and percentage of mice withcancer development in each group. NonparametricWilcoxon rank sum tests were carried out to assessthe difference in tumor incidence or growth ratebetween transgenic mice groups. A log-logisticsurvival model was fit for the time to tumordevelopment and the difference in the median timeto tumor development was compared across treat-ment groups. We also fit a multiple logistic regres-sion model for the binary endpoint of cancerdevelopment and assessed the main treatmenteffects due to Ha-ras, Bcl-2 as well as the synergisticeffect between these two. All statistical analyses werecarried out in Splus [24].

RESULTS

Proliferation and Cell Death in HK1.Ha-ras/bcl-2

Expressing Keratinocytes

To generate HK1.ras/bcl-2 double transgenic mice,the pups from HK1.ras and HK1.bcl-2 [9,23] micewere mated and screened for genotype using PCR asdescribed in the Materials and Methods. The pheno-type of HK1.bcl-2 neonates did not differ signifi-cantly from control neonates. Neonates expressingthe v-Ha-ras transgene exhibited a distinctive phe-notype with thickened and wrinkled skin (data not

shown). Double transgenic neonates, were indistin-guishable from Ha-ras transgenic neonates. Theeffects of Ha-ras and bcl-2 on epidermal proliferationand cell death were examined. The extent anddistribution of proliferating epidermal keratinocyteswere assessed by BrdU incorporation (Figure 1).Immunohistochemical assessment of tissue sectionsshowed that essentially all of the BrdU-positive cellswere located in the basal layer of control and bcl-2transgenic epidermis (Figure 1a). However, some ofthe proliferating keratinocytes in Ha-ras and Ha-ras/bcl-2 transgenic mice were also detected in thesuprabasal layers (Figure 1a). The proliferative index(PI) was calculated as percentage of BrdU positivekeratinocytes as described in the Materials andMethods. The PI of HK1.bcl-2 (6.74�0.32%) wasmodestly, but significantly, lower than that ofcontrol mice (9.02� 0.4%, P< 0.001) (Figure 1b).Similarly, the PI of the Ha-ras/bcl-2 double trans-genic epidermis (14.71� 0.82%) was significantlylower than the PI in Ha-ras transgenic epidermis(17.7�0.79%, P<0.012) (Figure 1b).

The AI was determined by quantifying the percen-tage of apoptotic epidermal keratinocytes followingUV irradiation as described in the Materials andMethods. The AI of the bcl-2 transgenic epidermis

Figure 1. Increased epidermal proliferation in Ha-Ras and Ha-ras/bcl-2 transgenic mice by BrdU incorporation. (a) Epidermal prolifera-tion of keratinocytes in transgenic mice was estimated using theBrdU incorporation assay. (b) The percentage of BrdU positive cellswas calculated as described. Bcl-2 over expression in the Bcl-2 andHK1.ras/bcl-2 transgenic mice epidermis, results in lower levels ofproliferative basal keratinocytes compared to the control andHK1.ras transgenic mice (P<0.001 and P<0.012, respectively).

MULTISTEP SKIN CARCINOGENESIS 951


was 9.94�2.15%, compared with 20.61�3.69% forcontrol littermates (P<0.0001, Figure 2a). Theepidermis of ras transgenic mice had an AI of14.05�3.39% following UV treatment, comparedwith 6.29�1.16% for the ras/bcl-2 double transgeniclittermates (P< 0.0001, Figure 2b).

Incidence of Tumor Formation in Control and

Bitransgenic Mice

To assess the roles of Ha-ras and bcl-2 cooperationduring multistep skin carcinogenesis, we applied astandard two-step chemical carcinogenesis protocolto control, HK1.ras, HK1.bcl-2, and HK1.ras/bcl-2double transgenic mice. Skin tumors were inducedusing DMBA as an initiator and TPA as a promoter asdescribed in the Materials and Methods. The micewere inspected weekly, and the incidence, latency,and growth rate of papilloma formation weremonitored for 1 yr. To assess the probability of tumordevelopment in chemically induced skin carcino-genesis, the latency for papilloma development

was compared using a 2�2 factorial study designconsisting of control, bcl-2 alone, ras alone, or bothras and bcl-2. A log-logistic survival model was fittedto the data (Figure 3). The median time to tumordevelopment was significantly shorter in the bcl-2group, compared to the control group (P¼0.025)Similarly, the median time to tumor developmentwas significant shorter in the ras group, compared tothe control group (P<0.0001). Relative reductionsin median time to tumor development due to bcl-2and ras were 16.4% and 36.3%, respectively, whencompared to the control. However, we observedneither a synergistic nor an antagonistic relationshipbetween bcl-2 and Ha-ras in our data (P¼0.65).

Tumor Number and Size

The number of papillomas per mouse was assessedfor each of the four genotypes (Figure 4A). In the boxplots (Figures 4A and B), the white bars werecorresponding to the median value and the lowerborder and upper border of each black box represent

Figure 2. Evaluation of apoptotic sensitivity in transgenic epider-mis following UV treatment. Epidermis of neonatal transgenic micewere exposed to 3.2 kJ/m2 of UV irradiation (2 kJ/m2 of UVB), andthe apoptotic sensitivity of each transgenic epidermis was evaluatedby counting the number of apoptotic cells 24 h after UV treatment.The AI was calculated from H&E stained tissue sections using light

microscopy. Apoptotic keratinocytes had condensed/fragmentedchromatin, eosinophilic cytoplasm, and a halo surrounding the cells.(a) The bcl-2 transgenic epidermis showed a lower number ofapoptotic keratinocytes than control. (b) The keratinocytes in theras/bcl-2 double transgenic epidermis were more resistant toUV-mediated apoptosis induction than ras transgenic epidermis.

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the 25th percentile and 75th percentile, respectively.The median value for the control and HK1.bcl-2transgenics was 0 and 1.5 papillomas per mouse,respectively, and was not significantly different(P¼0.20). When bcl-2 was coexpressed with Ha-ras,the median value of 5.0 papillomas per mousehowever did not quite achieve statistical signifi-cance, greater than the median value of 3.0 papillo-mas per mouse observed in HK1.ras transgenic mice(P¼0.08). However, it was significantly greater thanthe median value of 1.5 papillomas per mouseobserved in HK1.bcl-2 transgenic mice (P¼ 0.002).

The growth rate of papillomas was estimated usingthe mean number of papillomas with a diameter of5 mm or larger (Figure 4B). No mice in the controlgroup had any papillomas meeting this criteria andthere was a single mouse in the HK1.bcl-2 mousewith a papilloma greater than 5 mm. The mediannumber of large papillomas (>5 mm) in HK1.ras/bcl-2 double transgenic mice was 1, papillomas/mouse,which was not significantly different from themedian of 1, papilloma per mouse observed inHK1.ras transgenic mice (P¼ 0.15).

Proliferation and Apoptotic Rates in Papillomas

All grossly apparent tumors visible at the end of the1 yr observation period were fixed in formalin andembedded in paraffin for light microscopic morpho-logic assessment. Histologically, papillomas fromeach genotype were indistinguishable, consisting ofwell-differentiated keratinocytes. The extent of pro-liferation and the distribution of proliferative kera-tinocytes in chemically induced papillomas in theeach genotype of mouse were examined using

immunohistochemical detection of PCNA. Theextent of PCNA staining was similar in the papillo-mas from each genotype (not shown). The sponta-neous AI was quantitated in papillomas from mice ofeach genotype by counting the number of keratino-cytes exhibiting the characteristic features of apop-tosis including loss of junctional continuity withneighboring cells, cell shrinkage, nuclear condensa-tion, and blebbing as previously described (Figure 5).The spontaneous AI was expressed as the percentageof cells exhibiting apoptotic morphology in thepapillomas (Figure 5b). The AI for HK1.bcl-2 mice of1.95�0.37% and for HK1.ras/bcl-2 double trans-genic mice of 3.15� 1.29%, was significantly lower(P< 0.001) than the AI of 7.45�3.28% observed incontrol littermate mice and the AI of 8.44�2.25%observed in HK1.ras transgenic mice, respectively.

Conversion Rates of Tumors to Cancer

A subset of the tumors examined histologicallyconsisted of invasive squamous cell carcinomas(Figure 6). These tumors were comprised of moder-ately differentiated squamous cell carcinomas thatinvaded into the underlying dermis. Morphologicevidence of tumor dissemination to metastatic siteswas not observed. Based on the fitted multiplelogistic regression model (Table 1), the incidence ofcarcinomas in HK1.ras or HK1.bcl-2 mice was notsignificantly different (P¼0.32 and 0.31, respec-tively) than that of control animals (Table 1). Incontrast to the rate of papilloma formation in whichthe combined effect of Ha-ras and bcl-2 deregulationwas additive, the rate of malignant transformationwas clearly synergistic in the HK1.ras/bcl-2 doubletransgenic mice (P¼0.01) (Table 1). The incidence ofcarcinomas in the HK1.ras/bcl-2 double transgenicmice was significantly higher (P¼ 0.05) than that ofcontrol animals indicating an increased rate ofcancer formation in the bitransgenic mice.

DISCUSSION

Activating ras mutations and upregulation of bcl-2are common events in cutaneous malignancies [25–28]. In order to assess the extent of cooperationbetween deregulated Ha-ras and bcl-2 during multi-step skin carcinogenesis, in vivo, we utilized gene-tically engineered strains of mice. Geneticallyengineered mice provide a powerful means to assessthe contributions of molecular alterations to thepathogenesis of skin cancer. The results of many skincarcinogenesis studies using murine models consis-tently demonstrate that epidermal keratinocytes areremarkably resistant to malignant transformation. Ithas previously been demonstrated using thesemodels that Ha-ras and bcl-2 deregulation are,individually, insufficient for malignant transforma-tion of epidermal keratinocytes [9,29].

Our findings demonstrate that Ha-ras is moreeffective than bcl-2 in facilitating tumor formation

Figure 3. Probability of tumor development in chemical skincarcinogenesis. Estimated survival curves by genotype. After TPAtreatments for 17 weeks, cumulative percentage of tumor-bearingmice was compared between each group. v-Ha-ras transgenic micehad higher risk of tumor development comparing to control mice(P< 0.0001). Coexpression of bcl-2 and ras in HK1.ras/bcl-2double transgenic mice showed additive effects in chemical skincarcinogenesis .The median time to tumor formation for control,HK1.bcl-2, HK1.ras, and HK1.ras/bcl-2 mice was 16.4, 13.7, 10.5,and 8.8 weeks, respectively.



following standard two-stage chemical initiationand promotion. The incidence and growth rate ofpapillomas in HK1.ras mice consistently exceededthat observed in HK1.bcl-2 mice. Further, papillomaformation occurred following a significantly longerlatency period in the HK1.bcl-2 mice compared tothose that developed in HK1.ras mice. HK1.ras/bcl-2double transgenic mice were slightly more suscep-tible to tumor-promotion than either HK1.ras orHK1.bcl-2 mice. The individual contributions of Ha-

ras and bcl-2 to papilloma latency, incidence, andgrowth rate in HK1.ras/bcl-2 double transgenic micewas marginally additive. In this context, it isnoteworthy that in organotypic keratinocyte raftcultures, bcl-2 is able to significantly reduce theproliferative effect resulting from constitutive Ha-rassignaling activity [5]. This finding is consistent withour observation that the HKI.bcl-2 mice and, sug-gests that the anti-proliferative activity of bcl-2 isinsufficient to inhibit tumor formation in HK1.ras/

Figure 4. (A) Incidence of tumor development in chemical skincarcinogenesis. Median numbers of papillomas/mouse show thatepidermal expression of bcl-2 alone cannot be a significant factor forthe tumor incidence in chemical skin carcinogenesis (P¼ 0.20).However, when bcl-2 was coexpressed with Ha-ras, it showed highlycooperative effect on the incidence of tumor development comparedto bcl-2 alone (P¼0.002). (B) Growth rate of papillomas in chemicalskin carcinogenesis. Mean numbers of large papillomas (>0.5 cm)/

mouse were calculated to estimate the papillomas growth rate.Control mice had no large papillomas and HK1.bcl-2 transgenic micehad one, which did not give any statistical significance. Whereas, thepapillomas developed in HK1.ras/bcl-2 transgenic mice have shownto grow faster than HK1.ras transgenic mice but were not statisticallysignificant (P¼0.15). The white lines in each box represent themedian with, the lower and upper borders representing the 25th and75th percentile, respectively.

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bcl-2 double transgenic mice. Alternatively, theantiproliferative activity of bcl-2 could be compen-sated for, by yet to be defined, secondary molecularalterations during tumor formation.

Ras has been implicated both in inhibiting andpromoting apoptosis, depending on the cell type andexperimental conditions. The ras-mediated activa-tion of the PI3 kinase pathway has been shown tofunction as a prosurvival signal [30,31]. Additionally,it has been shown that NF-kB-mediated apoptosis

inhibition requires Ha-ras signaling [32,33]. Incontrast, other studies have demonstrated that ras-mediated signaling can facilitate cell death [34–36].For example, constitutively activated Ha-ras cansignal apoptosis following serum withdrawal or theinhibition of PKC [37]. Ras activation has also beenimplicated in mediating cell death through theCD95 ‘‘extrinsic’’ (Fas/Apo1) cell death pathway[38,39].

Available evidence suggests that bcl-2 proteins canfacilitate Ha-ras-mediated tumorigenesis by inhibit-ing apoptosis associated with constitutive ras activa-tion [40–42]. Previously, we demonstrated inkeratinocytes grown in organotypic culture thatbcl-2 could inhibit apoptosis associated with con-stitutive Ha-ras signaling [5]. In this study, wedemonstrated that bcl-2 could also significantlyprotect against UV-induced apoptosis in the epider-mis of HK1.ras/bcl-2 double transgenic mice as

Figure 5. Morphology of chemically induced papillomas and theirspontaneous apoptosis index. (a) Chemically induced papillomaswere stained using hematoxylin and eosin. The panels represent themorphology of papillomas from control (upper left), HK1.bcl-2(upper right), HK1.ras (lower left), and HK1.ras/bcl-2 (lower right)transgenic epidermis. Inset images of representative apoptotic cells.(b) The spontaneous apoptotic index was assessed as described in themethods. The percentage of apoptotic keratinocytes in papillomas ofbcl-2 and ras/bcl-2 transgenic mice was lower than that of controland ras transgenic mice, respectively (P<0.001 in both).

Table 1. Fitted Multiple Logistic Regression Model for‘‘Cancer Development’’ (N¼ 277)

Variable Coefficient SE P-value

Intercept 2.361 0.468 <0.001Bcl2 �0.745 0.752 0.323Ras �0.878 0.858 0.307Bcl2a Ras 2.649 1.071 0.014

aSE: standard error.

Figure 6. Malignant transformation of papillomas. (a) Tissuesections of skin lesions were stained with H&E and examined bylight microscopy. The morphologic features of benign papillomas(upper panel) and invasive squamous cell carcinoma (lower panel) areshown. (b) Table summarizing the frequency of squamous cellcarcinoma at 1 yr arising in each genotype.



compared to HK1.ras skin. Together, these findingssuggest that the functional basis of Ha-ras and bcl-2cooperation during multistep skin tumorigenesis isthe ability to inhibit apoptosis associated withderegulated ras during tumor promotion withoutsignificantly altering rates of proliferation. Indeed,papillomas arising in HK1.ras transgenic miceexhibited the highest rate of spontaneous apoptoticcell death whereas papillomas arising in the HK1.ras/bcl-2 double transgenic mice exhibited rates ofapoptosis that were significantly lower than papillo-mas arising in either control littermate or HK1.rasmice.

Forced expression of either Ha-ras or bcl-2 trans-gene in the epidermis resulted in similar rates ofmalignant tumor progression furthermore, theywere not appreciably different than that observedfor control mice. These findings suggest that thebiologic impact of independently deregulating thesegenes may be in the earlier stages of multistepcarcinogenesis, but not malignant transformationper se. However, when these two oncoproteins werecoexpressed, a significant, and synergistic, increasein malignant transformation was observed. A similartransgenic mouse model targeted bcl-2 transgeneexpression to the basal layer of the epidermis using akeratin 14 promoter [21]. In contrast to our results,the coexpression of both ras and bcl-2 oncoproteinsin basal keratinocytes paradoxically delayed chemi-cally induced skin tumorigenesis. Together thesefindings may suggest that the spatial distribution ofprooncogenic proteins may be an important deter-minant in the susceptibility of keratinocytes toundergo tumor formation and malignant progres-sion.

The mechanistic basis of the synergism betweenHa-ras and bcl-2 during multistep skin carcinogen-esis is, at present, speculative. The synergistic effectof Ha-ras and bcl-2 may be entirely, or in part,attributable to facilitating the acquisition of requi-site complementary molecular alterations neededfor malignant transformation. Alternatively, thefindings could suggest the possibility of ‘‘cross-talk’’between these divergent signaling pathways. It isconceivable that such cross-talk could result in theamplification of a signal mediating a critical featureof malignant transformation, such as angiogenesis orinvasion into the underlying stroma. Such signalscould imply synergism of functions other thansimply cell survival or proliferative advantage. Inthis regard, it may be relevant that in prostate cancerxenografts, bcl-2 was associated with an augmenta-tion in VEGF production in response to hypoxia [43].The bcl-2 expressing tumor xenografts exhibited anincrease in vessel density and more aggressivegrowth compared to control tumors [43].

These findings may have more generalized sig-nificance with respect to redundant functions inthe bcl-2 gene family. The expression of the anti-

apoptotic protein, bcl-xL, within the epidermis hasbeen shown to facilitate chemically induced papillo-matogenesis [44]. Additionally, mice deficient in theproapoptotic bcl-2 family member protein, bax,exhibited a significant enhancement in tumor for-mation using a similar two-stage chemical carcino-genesis protocol [45,46]. These observations suggestthat the deregulated expression of other anti-apop-totic bcl-2 family members, or conversely thedisrupted expression of proapoptotic bcl-2 proteins,may contribute to multistep skin carcinogenesis. Theextent, and manner, in which these bcl-2 familymembers cooperate with other common molecularalterations in NMSC, including Ha-ras, remains to bedetermined. In conclusion ras and bcl-2, two well-characterized oncogenes cooperate in giving kerati-nocytes a selective advantage in their increasedproliferation potential and the reduced rates ofapoptosis in response to stress. Using in vivo murinemodels, we have been able to show that bcl-2 and rashave a synergistic effect in the malignant transfor-mation of papillomas to cancer. Further more, thebitransgenic mice shows a reduced tumor latencyand an increase in tumor frequency as compared tothe control animals.

ACKNOWLEDGMENTS

This work was supported by grants NIH UO1CA105491 and UO1 CA83701 Song H. Cho wassupported by NIH training Grant T32 CA67759.

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cooperation of ha-ras and bcl-2 during multistep skin carcinogenesis

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