The proapoptotic Bcl-2 family member Bim plays a central role during the development of virus-induced hepatitis

The proapoptotic Bcl-2 homolog Bim was shown to control the apoptosis of both T cells and hepatocytes. This dual role of Bim might be particularly relevant for the development of viral hepatitis, in which both the sensitivity of hepatocytes to apoptosis stimuli and the persistence of cytotoxic T cells are essential factors for the outcome of the disease. The relevance of Bim in regulating survival of cytotoxic T cells or induction of hepatocyte death has only been investigated in separate systems, and their relative contributions to the pathogenesis of T cell-mediated hepatitis remain unclear. Using the highly dynamic model system of lymphocytic choriomeningitis virus-mediated hepatitis and bone marrow chimeras, we found that Bim has a dual role in the development of lymphocytic choriomeningitis virus-induced, T cell-mediated hepatitis. Although the absence of Bim in parenchymal cells led to markedly attenuated liver damage, loss of Bim in the lymphoid compartment moderately enhanced hepatitis. However, when both effects were combined in Bim(-/-) mice, the effect of Bim deficiency in the lymphoid compartment was overcompensated for by the reduced sensitivity of Bim(-/-) hepatocytes to T cell-induced apoptosis, resulting in the protection of Bim(-/-) mice from hepatitis.

BCL-2 family member BOK is widely expressed but its loss has only minimal impact in mice

BOK/MTD was discovered as a protein that binds to the anti-apoptotic Bcl-2 family member MCL-1 and shares extensive amino-acid sequence similarity to BAX and BAK, which are essential for the effector phase of apoptosis. Therefore, and on the basis of its reported expression pattern, BOK is thought to function in a BAX/BAK-like pro-apoptotic manner in female reproductive tissues. In order to determine the function of BOK, we examined its expression in diverse tissues and investigated the consequences of its loss in Bok(-/-) mice. We confirmed that Bok mRNA is prominently expressed in the ovaries and uterus, but also observed that it is present at readily detectable levels in several other tissues such as the brain and myeloid cells. Bok(-/-) mice were produced at the expected Mendelian ratio, appeared outwardly normal and proved fertile. Histological examination revealed that major organs in Bok(-/-) mice displayed no morphological aberrations. Although several human cancers have somatically acquired copy number loss of the Bok gene and BOK is expressed in B lymphoid cells, we found that its deficiency did not accelerate lymphoma development in Eμ-Myc transgenic mice. Collectively, these results indicate that Bok may have a role that largely overlaps with that of other members of the Bcl-2 family, or may have a function restricted to specific stress stimuli and/or tissues.

Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

Induction of apoptosis and enhancement of chemosensitivity in human prostate cancer LNCaP cells using bispecific antisense oligonucleotide targeting Bcl-2 and Bcl-xL genes

OBJECTIVE: To determine whether a specifically designed bispecific (Bcl-2/Bcl-xL) antisense oligonucleotide (ASO) induces apoptosis and enhances chemosensitivity in human prostate cancer LNCaP cells, as Bcl-2 and Bcl-xL are both anti-apoptotic genes associated with treatment resistance and tumour progression in many malignancies, including prostate cancer. MATERIALS AND METHODS: Inhibition of Bcl-2 and Bcl-xL expression by the bispecific ASO was evaluated using real-time reverse transcription-polymerase chain reaction and Western blotting, while growth inhibition and induction of apoptosis were analysed by a crystal violet assay, flow cytometry and Western blotting of apoptosis-relevant proteins. The effect of combined treatment with bispecific ASO and chemotherapy or small-interference RNA (siRNA) targeting the clusterin gene was also investigated. RESULTS: Bispecific ASO reduced Bcl-2 and Bcl-xL expression in LNCaP cells in a dose-dependent manner. There was cell growth inhibition, increases in the sub-G0-G1 fraction, and cleavage of caspase-3 and poly(ADP-Ribose) polymerase proteins in LNCaP cells after bispecific ASO treatment. Interestingly, Bcl-2/Bcl-xL bispecific ASO treatment also resulted in the down-regulation of Mcl-1 and up-regulation of Bax. The sensitivity of LNCaP cells to mitoxantrone, docetaxel or paclitaxel was significantly increased, reducing the 50% inhibitory concentration by 45%, 80% or 90%, respectively. Furthermore, the apoptotic induction by Bcl-2/Bcl-xL bispecific ASO was synergistically enhanced by siRNA-mediated inhibition of clusterin, a cytoprotective chaperone that interacts with and inhibits activated Bax. CONCLUSIONS: These findings support the concept of the targeted suppression of Bcl-2 anti-apoptotic family members using multitarget inhibition strategies for prostate cancer, through the effective induction of apoptosis.

AKT/mTOR pathway activation and BCL-2 family proteins modulate the sensitivity of human small cell lung cancer cells to RAD001

PURPOSE: The Akt/mammalian target of rapamycin (mTOR) pathway is frequently activated in human cancers and plays an important role in small cell lung cancer (SCLC) biology. We investigated the potential of targeting mTOR signaling as a novel antitumor approach in SCLC. EXPERIMENTAL DESIGN: The expression of mTOR in patient specimens and in a panel of SCLC cell lines was analyzed. The effects on SCLC cell survival and downstream signaling were determined following mTOR inhibition by the rapamycin derivative RAD001 (Everolimus) or down-regulation by small interfering RNA. RESULTS: We found elevated expression of mTOR in patient specimens and SCLC cell lines, compared with normal lung tissue and normal lung epithelial cells. RAD001 treatment impaired basal and growth factor-stimulated cell growth in a panel of SCLC cell lines. Cells with increased Akt pathway activation were more sensitive to RAD001. Accordingly, a constitutive activation of the Akt/mTOR pathway was sufficient to sensitize resistant SCLC cells to the cytotoxic effect of RAD001. In the sensitive cells, RAD001 showed a strong additive effect to the proapoptotic action of the chemotherapeutic agent etoposide. Intriguingly, we observed low Bcl-2 family proteins levels in the SCLC cells with a constitutive Akt pathway activation, whereas an increased expression was detected in the RAD001-resistant SCLC cells. An antisense construct targeting Bcl-2 or a Bcl-2-specific inhibitor was able to sensitize resistant SCLC cells to RAD001. Moreover, SCLC tumor growth in vivo was significantly inhibited by RAD001. CONCLUSION: Together, our data show that inhibiting mTOR signaling with RAD001 potently disrupts growth and survival signaling in human SCLC cells.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP3Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP3R1 and IP3R2, and barely to IP3R3, and essentially all cellular Bok is IP3R bound in cells that express substantial amounts of IP3Rs. Binding to IP3Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP3Rs (amino acids 1895–1903 of IP3R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP3R binding, the following was observed: (i) Bok does not appear to control the ability of IP3Rs to release ER calcium stores, (ii) Bok regulates IP3R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP3R degradation, and (iv) Bok protects IP3Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP3Rs and that the most significant consequence of this binding appears to be protection of IP3Rs from proteolysis. Thus, Bok may govern IP3R cleavage and activity during apoptosis.

The role of BCL-2 in regulation of apoptosis susceptibility by modulation of glutathione

The role of oxidative stress and apoptosis has recently been recognized as an important determinant in the development of a variety of diseases known to man. The oncogene BCL-2 is known to regulate sensitivity to induction of apoptosis and appears to function in an antioxidant pathway by regulating glutathione. We have investigated various steps in the oxidative stress cascade to determine possible sites of action for BCL-2. The fluorescent probes H2DCFDA, dihydroethidium and cis-parinaric acid were used to quantitate generation of peroxides, superoxide and lipid peroxidation, respectively. While each of these agents was able to detect substantial increases in oxidative stress following exposure of cells to ionizing radiation, there was no significant difference between cells expressing high or low levels of BCL-2. Investigation of mitochondrial dysfunction during apoptosis revealed a possible site of bcl-2 intervention, but, analysis of kinetic events occurring during apoptosis suggested that the observed effect is not in the direct apoptotic effector pathway. When glutathione was studied, localization to the nucleus was observed in cells overexpressing BCL-2 that did not occur in cells lacking BCL-2. Additionally, nuclear accumulation of glutathione was sufficient to block granzyme b-mediated nuclear DNA fragmentation, poly (ADP-ribose) polymerase cleavage and caspase activity suggesting that nuclear accumulation of glutathione via a bcl-2 dependent process is functionally relevant to suppression of apoptosis. Thus, a model system emerges where BCL-2 is able to regulate a cell's ability to prevent apoptosis by modifying the cell's antioxidant systems at the organelle level to compensate for oxidative stresses placed upon it. ^

Alphaviruses induce apoptosis in Bcl-2-overexpressing cells: evidence for a caspase-mediated, proteolytic inactivation of Bcl-2

Bcl-2 oncogene expression plays a role in the establishment of persistent viral infection by blocking virus-induced apoptosis. This might be achieved by preventing virus-induced activation of caspase-3, an IL-1beta-converting enzyme (ICE)-like cysteine protease that has been implicated in the death effector phase of apoptosis. Contrary to this model, we show that three cell types highly overexpressing functional Bcl-2 displayed caspase-3 activation and underwent apoptosis in response to infection with alphaviruses Semliki Forest and Sindbis as efficiently as vector control counterparts. In all three cell types, overexpressed 26 kDa Bcl-2 was cleaved into a 23 kDa protein. Antibody epitope mapping revealed that cleavage occurred at one or two target sites for caspases within the amino acid region YEWD31 (downward arrow) AGD34 (downward arrow) A, removing the N-terminal BH4 region known to be essential for the death-protective activity of Bcl-2. Preincubation of cells with the caspase inhibitor Z-VAD prevented Bcl-2 cleavage and partially restored the protective activity of Bcl-2 against virus-induced apoptosis. Moreover, a murine Bcl-2 mutant having Asp31, Asp34 and Asp36 substituted by Glu was resistant to proteolytic cleavage and abrogated apoptosis following virus infection. These findings indicate that alphaviruses can trigger a caspase-mediated inactivation of Bcl-2 in order to evade the death protection imposed by this survival factor.

Bcl-2 predicts response to neoadjuvant chemotherapy and is overexpressed in lymph node metastases of urothelial cancer of the bladder

PURPOSE To assess whether Bcl-2, an inhibitor of the apoptotic cascade, can predict response to neoadjuvant chemotherapy in patients with urothelial cancer of the bladder (UCB). METHODS Bcl-2 expression was analyzed in 2 different tissue microarrays (TMAs). One TMA was constructed of primary tumors and their corresponding lymph node (LN) metastases from 152 patients with chemotherapy-naive UCB treated by cystectomy and pelvic lymphadenectomy (chemotherapy-naive TMA cohort). The other TMA was constructed of tumor samples obtained from 55 patients with UCB before neoadjuvant chemotherapy (transurethral resection of the bladder cancer) and after cystectomy with pelvic lymphadenectomy (residual primary tumor [ypT+], n = 38); residual LN metastases [ypN+], n = 24) (prechemotherapy/postchemotherapy TMA cohort). Bcl-2 overexpression was defined as 10% or more cancer cells showing cytoplasmic immunoreactivity. RESULTS In both TMA cohorts, Bcl-2 overexpression was significantly (P<0.05) more frequent in LN metastases than in primary tumors (chemotherapy-naive TMA group: 18/148 [12%] in primary tumors vs. 39/143 [27%] in metastases; postchemotherapy TMA: ypT+7/35 [20%] vs. ypN+11/19 [58%]). In the neoadjuvant setting, patients with Bcl-2 overexpression in transurethral resection of the bladder cancer specimens showed significantly (P = 0.04) higher ypT stages and less regression in their cystectomy specimens than did the control group, and only one-eighth (13%) had complete tumor regression (ypT0 ypN0). In survival analyses, only histopathological parameters added significant prognostic information. CONCLUSIONS Bcl-2 overexpression in chemotherapy-naive primary bladder cancer is related to poor chemotherapy response and might help to select likely nonresponders.

Apoptosis and the molecular complementation of bcl-2, myc and p53 in multistep lymphomagenesis

Follicular lymphoma is the most common lymphoid malignancy in humans. The bcl-2 transgenic mice, which mimic the human follicular lymphoma, initially exhibit a polyclonal hyperplasia due to the overriding of apoptosis by deregulated bcl-2. After a latency period of 15 month 20% of the animals developed clonal lymphomas. Approximately, 50% of these high grade lymphomas presented chromosomal translocations involving c-myc, suggesting that deregulation of this gene is important in the complementation with bcl-2. E$\mu$-myc x bcl-2 double transgenic mice were generated to assess the ability of this two genes to complement in an in vivo system. The double transgenic mice presented a shortened latency (3-4 weeks) and higher incidence of tumor development. Quantification of the extent of programmed cell death indicated that bcl-2 can abrogate the high rate of apoptotic cell death that results from myc deregulation. Bcl-2-Ig, E$\mu$-myc, and bcl-2/E$\mu$-myc lymphomas were examined using PCR-SSCP to detect the presence of p53 mutations in exons 5-9. A high incidence of p53 mutations in E$\mu$-myc lymphomas suggested that inactivating lesions of p53 may represent an important step in the genetic complementation of c-myc in lymphomagenesis. Surprisingly, p53 mutations were quite uncommon in bcl-2 lymphomas suggesting that inactivating mutations of p53 and overexpression of bcl-2 may not cooperate in lymphoma progression. To assess this question, we generated mice that contained a deregulated bcl-2 gene and were nullizygous for p53 (p53KO). No reduction in the tumor latency was observed in the p53KO/bcl-2-Ig hybrid mice when compared with p53 KO mice. Using splenic mononuclear cells isolated from p53KO mice and bcl-2 transgenic mice we demonstrated that bcl-2 suppresses p53 mediated apoptosis in response to DNA damage initiated by $\gamma$-radiation even though p53 protein is induced normally in the bcl-2 overexpressing cells. Western analysis of the expression of p53 target proteins after $\gamma$-radiation showed a correlation between the p53-dependent induction of bax protein after radiation and the ability of p53 to mediate apoptosis. ^

Potential roles for bcl-2 in the pathogenesis, progression, and treatment of prostate cancer

The most common molecular alterations observed in prostate cancer are increased bcl-2 protein expression and mutations in p53. Understanding the molecular alterations associated with prostate cancer are critical for successful treatment and designing new therapeutic interventions. Hormone-ablation therapy remains the most effective nonsurgical treatment; however, most patients will relapse with hormone-independent, refractory disease. This study addresses how hormone-ablation therapy may increase bcl-2, develops a transgenic model to elucidate the role of bcl-2 multistep prostate carcinogenesis, and assesses how bcl-2 may confer resistance to cell death induction using adenoviral wild-type p53 gene therapy. ^ Two potential androgen response elements were identified in the bcl-2 promoter. Bcl-2 promoter luciferase constructs were transfected into the hormone- sensitive LNCaP prostate cell line. In the presence of dihydrotestosterone, the activity of one bcl-2 promoter luciferase construct was repressed 40% compared to control cells grown in charcoal-stripped serum. Additionally, it was demonstrated that both bcl-2 mRNA and protein were downregulated in the LNCaP cells grown in the presence DHT. This suggests that DHT represses bcl-2 expression through possible direct and indirect mechanisms and that hormone-ablation therapy may actually increases bcl-2 protein. ^ To determine the role of bcl-2 in prostate cancer progression in vivo, probasin-bcl-2 mice were generated where human bcl-2 was targeted to the prostate. Increased bcl-2 expression rendered the ventral prostate more resistant to apoptosis induction following castration. When the probasin-bcl-2 mice were crossed with TRAMP mice, the latency to tumor formation was decreased. The expression of bcl-2 in the double transgenic mice did not affect the incidence of metastases. The double transgenic model will facilitate the study of in vivo effects of specific genetic lesions during the pathogenesis of prostate cancer. ^ The effects of increased bcl-2 protein on wild-type adenoviral p53-mediated cell death were determined in prostatic cell lines. Increased bcl-2 protected PC3 and DU145 cell lines, which possess mutant p53, from p53-mediated cell death and reductions in cell viability. Bcl-2 did not provide the same protective effect in LNCaP cell line, which expresses wild-type p53. This suggests that the ability of bcl-2 to protect against p53-mediated cell death is dependent upon the endogenous status of p53. ^

Participation of bcl -2 and bax in epidermal homeostasis and non-melanoma skin cancer

Non-melanoma skin cancer (NMSC) is the most frequently diagnosed form of cancer in United States. As in many other cancers, this slow growing malignancy manifests deregulated expression of apoptosis regulating proteins including bcl-2 family member proteins. To understand the role of apoptosis regulating protein in epidermal homeostasis and progression of NMSC, we investigated keratinocyte proliferation, differentiation and tumorigenesis in bcl-2 and bax null mice. The rate and the pattern of proliferation and spontaneous cell death were the same between the null and the control mice. Both bcl-2 and bax null epidermis showed decreased levels of cytokeratin 14 expression compared to the control littermates. Also, the gene knock out mice showed higher expression of cytokeratin 1 and loricrin in epidermis compared to the control mice. The apoptotic response to genotoxic agent, UV radiation (UVR), was assessed by counting sunburn cells. The bax null keratinocytes showed a resistance to apoptosis while bcl-2 null mice showed an increased susceptibility to cell death compared to the control mice. Moreover, we demonstrated an increase in tumor incidence in bax null mice compared to control littermates in the in vivo chemical carcinogenesis study. Next, we examined the tumor suppressor role of bax protein in NMSC by studying its participation in repair of UVR-mediated DNA lesions. In UVR treated primary keratinocytes from bax deficient mice, the level of CPD remaining was twice that of control cells at 48 hours. Similar results were obtained using embryonic fibroblasts from bax null and bax +/+ embryos, and also with a bax deficient prostate cancer cell line in which bax expression had been restored. However, the repair rate of 6-4 PP was unaffected by the absence of bax protein in all three of above mentioned cell types. In conclusion, bax protein may have a dual function in its role as tumor suppressor in NMSC. Bax may directly or indirectly facilitate DNA repair, or programmed cell death if DNA damage is too severe, thus, in either function, preserving genomic integrity following a genotoxic event. ^

Regulation of Bcl-2 expression by the product of the prostate apoptosis response gene -4, Par-4

Elevated expression levels of the bcl-2 proto-oncogene have been correlated with the appearance of androgen independence in prostate cancer. Although bcl-2 was first cloned as the t (14:18) translocation breakpoint from human follicular B cell lymphoma, the mechanism of overexpression of bcl-2 is largely undefined for advanced prostate cancer, there being no gross alterations in the gene structure. We investigated the role of the product of the prostate apoptosis response gene-4 (Par-4) and the product of the Wilms' tumor 1 gene (WT1) in the regulation of Bcl-2 expression in prostate cancer cell lines. We observed growth arrest and apoptosis, upon decreasing Bcl-2 protein and transcript in the high Bcl-2 expressing, androgen-independent prostate cancer cell lines, by all trans-retinoic acid treatment but this did not occur in the androgen-dependent cell lines expressing low levels of Bcl-2. Changes in localization of Par-4, and an induction in the expression of WT1 protein accompanied the decrease in the Bcl-2 protein and transcript following all trans-retinoic acid treatment, in the androgen-independent prostate cancer cell line. In stable clones expressing ectopic Par-4 we observed decreased Bcl-2 protein and transcript. This was accompanied by an induction in WT1 expression. Finally, we detected Par-4 and WT1 proteins binding to a previously identified WT1 binding site on the bcl-2 promoter both in vitro and in vivo leading to a decrease in transcription from the bcl-2 promoter. We conclude that Par-4 regulates Bcl-2 through a WT1 binding site on the bcl-2 promoter. ^

Functional studies of *Ras and Bcl-2 oncoproteins in keratinocyte homeostasis and multistep skin carcinogenesis

To assess the effect of deregulated Ha-ras and bcl-2, individually and in combination on epidermal keratinocyte homeostasis and during multistep skin carcinogenesis, we generated skin-specific transgenic mice and keratinocyte transfectants constitutively expressing oncogenic Ha-ras and bcl-2 proteins. The deregulated Ha-ras and bcl-2 expression contributing to homeostatic imbalances in the skin had an additive effect on the probability of tumor development. They were also cooperative in incidence, growth, and latency of tumor formation, and they exhibited synergistic cooperation in malignant transformation of benign papillomas. To explain the homeostatic imbalances by Ha-ras and bcl-2 overexpression in the skin, we investigated the three major cellular processes of proliferation, cell death, and differentiation. Epidermal expression of Bcl-2 retarded keratinocyte proliferation in the epidermis of neonatal mice compared with results for control littermates. Constitutive expression of Ha-ras increased keratinocyte proliferation, and co-expression of bcl-2 modestly suppressed the ras-mediated abnormal proliferation of neonatal keratinocytes. Bcl-2 proteins in keratinocytes protected UV-treated cells from apoptotic cell death regardless of oncogenic ras expression in both non-neoplastic neonatal epidermis and human keratinocyte cell lines. The spontaneous apoptotic index (AI) was also lower in papillomas constitutively expressing bcl-2 compared with the ones that developed in control mice. Ras-overexpressing epidermis, including that in ras/bcl-2 double transgenic mice, had abnormal differentiation patterns compared with controls. The oncogenic ras protein had alterations in both epidermal distribution and the extent of cytokeratin 14 and involucrin expression. Abnormal expression of the hyperproliferation marker cytokeratin 6 and modest down regulation of cytokeratin 1 were also detected. Late appearance of filaggrin was another abnormal phenotype of the ras-expressing epidermis. Overexpression of bcl-2 had no effect on epidermal differentiation. Together, these findings suggest that constitutive expression of oncogenic Ha-ras and bcl-2 are important determinants of epidermal proliferation, viability and differentiation. In summary, our results demonstrated that the disruption of epidermal homeostasis by overexpressed ras and bcl-2 predisposes to hyperplastic growth of the epidermis and to papilloma development and that these proteins with distinct mechanisms for oncogenesis are functionally synergistic for malignant transformation of chemically induced skin carcinogenesis. ^