Intratumor injection of small interfering RNA-targeting human papillomavirus 18 E6 and E7 successfully inhibits the growth of cervical cancer

Human papillomavirus (HPV) 18 is related not only to squamous cell carcinoma of the cervix, but also to adenocarcinoma and small cell carcinoma of the cervix, in which prognosis is known to be poor. Small interfering RNA (siRNA) that targets HPV18 E6 and E7 was tested in HPV18-positive cell lines to investigate its effect and investigate its mechanism of action. Nude mice were also tested in a combination of siRNA and atelocollagen to determine whether it might be useful as a new molecule-targeting therapy for cervical cancer. siRNAs targeting HPV18 E6 and E7 were transfected into cervical cancer cells in vitro and they were investigated for cell growth inhibition, expression of E6 and E7 mRNA, expression of retinoblastoma protein, and senescence-associated beta-galactosidase staining. Sequence-specific siRNA inhibited cell growth. Decreased expression of E6 and E7 mRNA followed with E7 protein was observed in the transfected cells, but the expression of retinoblastoma protein and the beta-galactosidase staining increased, suggesting cell growth inhibitory effect through senescence. Treatment of xenografts established from SKG-II cells with siRNA specific for E6 and E7 obviously suppressed tumor growth in vivo. These results indicate that atelocollagen-mediated delivery of siRNA HPV18 E6 and E7 can be used as a novel therapeutic approach for cervical cancer.     

Down-regulation of HPV18 E6, E7, or VEGF expression attenuates malignant biological behavior of human cervical cancer cells

To investigate the effect of down-regulation of VEGF (vascular endothelial growth factor) and HPV18 E6/E7 by hairpin RNA (shRNA) on cell proliferation, apoptosis, migration, invasion, and adhesion abilities of cervical carcinoma cells, recombinant plasmids including pS-E6 shRNA, pS-E7 shRNA, and pS-VEGF shRNA were constructed and transfected into HeLa cells. The levels of E6 mRNA, E7 mRNA, or VEGF mRNA were significantly reduced after transfection of pS-E6 shRNA (76.0%), pS-E7 shRNA (74.4%), and pS-VEGF shRNA (46.7%). VEGF expression was down-regulated by pS-E6 shRNA (55.1%) and pS-E7 shRNA (46.6%). The apoptosis of HeLa cells was increased, and the proliferation, invasion, and adhesion abilities were decreased significantly. For in vivo study, cancer cells that stably expressed the plasmids were cultured. Cells were transplanted subcutaneously into nude mice to establish xenograft tumor model. Finally, expression of E6 shRNA, E7 shRNA, and VEGF shRNA in cancer cells led to inhibition of the growth of xenograft. Hence, RNA interference could effectively suppress the expression of HPV18 E6/E7 and VEGF in human cervical cancer cells. This suppression attenuates malignant biological behavior of human cervical cancer cells. RNA interference of HPV E6/E7 or VEGF expression implies an effective anti-cervical cancer strategy.     

Therapeutic potential of an adenovirus expressing p73 beta, a p53 homologue, against human papilloma virus positive cervical cancer in vitro and in vivo

Human papilloma virus (HPV) infection is the most important risk factor for cervical cancer development. p53 based gene therapy is not suitable for cervical cancer because HPV oncoprotein E6 inactivates p53 protein by targeting it for ubiquitin mediated degradation. Here we evaluated the efficiency of Ad-p73, a replication deficient adenovirus expressing p73beta a p53 homologue, to inhibit the growth of HPV positive cervical cancer cells in vitro using tissue culture system and in vivo using human xenografts in nude mice. Ad-p73, but not Ad-p53 (p53 adenovirus), inhibited the growth in vitro of three different HPV positive cervical cancer cell lines, HeLa, ME180, and SiHa, efficiently, which correlated with stable expression of functional p73 protein. However, the growth of a HPV negative cervical cancer cell line, C33A, was inhibited equally by both Ad-p73 and Ad-p53. In addition, we show that Ad-p73 preinfected HeLa cells and HCT116 E6 cells, an E6 stable cell line, failed to form tumors in nude mice unlike Ad-p53 or Ad-LacZ preinfected cells. Moreover, Ad-p73, but not Ad-p53, inhibited completely the growth of already established tumors of HeLa or HCT116 E6 cells. Furthermore, the ability of p73 to inhibit the growth of these tumors correlated with the stable expression of p73 protein with the concomitant induction of its target gene p21(WAF1/CIP1) and induction of apoptosis in tumor cells. These results suggest that Ad-p73 inhibits efficiently the growth in vitro and tumorigenicity and tumor growth in vivo of HPV positive cervical cancer cells and that p73-based approach should be explored as a potential therapeutic model for the treatment of cervical cancer.     

Highly potent and specific siRNAs against E6 or E7 genes of HPV16- or HPV18-infected cervical cancers

Infection with high-risk types (type 16 or type 18) of human papillomaviruses (HPVs) increases a patient's risk of cervical cancer. Given the importance of the cervix and the severe side effects resulting from traditional cancer therapies, this study aimed to achieve targeted inhibition of viral oncogenes in tumor cells using small interfering RNAs (siRNA). To accomplish this, we developed nine siRNAs against either the E6 or E7 genes of HPV-16 or HPV-18 in several combinations, yielding siRNAs targeting 16E6, 16E7, 18E6 and 18E7. We measured the effectiveness of the siRNAs by examining E6 or E7 mRNA expression after transfection of the siRNAs into HPV-positive CaSki (HPV-16) or HeLa (HPV-18) cell lines. We found that the HPV-siRNAs significantly reduced cell growth and colony formation in both cell lines. Flow cytometry analysis revealed a significant increase in apoptosis. The siRNAs had no effect on cell growth, colony formation or apoptosis in HPV-negative C33A cells, demonstrating a lack of off-target effects. In addition, an in vivo xenograft study showed that intra-tumoral injection of the siRNAs reduced tumor growth in BALB/c nude mice. In conclusion, we have developed highly specific and potent HPV-siRNAs that successfully suppress tumor growth and induce apoptosis in HPV-positive cervical cancer cells. siRNA treatment has potential for further development as an adjuvant therapy for cervical cancer.     

Differential suppression of human cervical cancer cell growth by adenovirus delivery of p53 in vitro: arrest phase of cell cycle is dependent on cell line.

It has been reported that overexpression of wild-type p53 protein induces suppression of tumor cell growth in vivo and in vitro. In this study, we further evaluated the differential effects of p53 delivered in an adenovirus vector on the cell growth, apoptosis and cell cycle progression in cervical cancer cell lines. We constructed a recombinant adenovirus expressing p53 and then delivered this into cervical carcinoma cell lines (CaSki, SiHa, and HeLa, HeLaS3) along with adenovirus expressing beta-galactosidase as a negative control. Adenovirus-delivered p53 overexpression resulted in a more significant suppression of cell growth in HPV 18-infected cells (HeLa and HeLaS3) and a lesser suppression in HPV 16-infected cells (CaSki and SiHa). However, no suppression was observed in cells infected with a negative control virus. p53 overexpression also induced apoptosis and cell cycle arrest, as determined by annexin V and propidium iodide staining. In particular, the cell cycle was arrested in the G(2)/M phase in CaSki cells. In contrast, cell cycles were arrested in the G(1) phase in HeLa cells, suggesting that the arrest phase is dependent upon the cervical cancer cell line. Taken together, these data support the idea that overexpressed p53 protein plays a differential role in suppressing cervical cancer cell growth through apoptosis and cell cycle arrest in either G(1) or G(2)/M phase, depending on the cancer cell line.     

The synergistic therapeutic effect of cisplatin with Human papillomavirus E6/E7 short interfering RNA on cervical cancer cell lines in vitro and in vivo

Human papillomavirus (HPV) types 16 and 18 are the major etiologic factors in the development of cervical epithelial neoplasia. Our study was designed to validate antiviral short interfering RNA (siRNA) targeting the E6 and E7 oncogenes as a potential chemosensitizer of cisplatin (cis-diaminedichloroplatinum II; CDDP) in cervical carcinoma. Specifically, the therapeutic efficacy of combination of CDDP and E6/E7-specific siRNA was assessed in an in vivo cervical cancer xenograft models. The combination of CDDP and E6/E7-specific siRNA had greater efficacy than the combination of CDDP and E6-specific siRNA especially in terms of inducing cellular senescence. Through in vitro and in vivo experiments, the mechanism of synergy between these two treatments was revealed, demonstrating that the combination of E6/E7-specific siRNA and CDDP therapy was significantly superior to either modality alone. In vitro, long-term exposure of HeLa cells to the combination of CDDP and E6/E7-specific siRNA induced apoptosis and cellular senescence. In vivo, E6/E7-specific siRNA potentiated the antitumor efficacy of CDDP via induction of apoptosis, senescence and antiangiogenesis. Our results suggest that E6/E7-specific siRNA may be an effective sensitizer of CDDP chemotherapy in cervical cancer.     

Antitumor effect of vaccinia virus in glioma model.

The ability of certain viruses to lyse cancer cells suggests that they may have potential as oncolytic agents. We investigated the effect of vaccinia virus (VV) and its recombinant derivatives (recVV2, rVV-p53) on growth of C6 rat glioma cells that form fast growing tumors in athymic nude mice. VV effectively infected C6 cells in vitro, inducing high level of foreign gene expression. Most of C6 cells infected in vitro with rVV-p53 expressing the tumor suppressor p53 protein showed apoptosis specific morphological changes in DAPI-stained nuclei and DNA fragmentation pattern on gel electrophoresis; infection with VV induced low level of cell apoptosis. In an ex vivo experiment, VV-infected C6 cells were implanted s.c. in athymic nude mice and tumor development was monitored. In contrast to the control PBS group, most of mice implanted with infected cells remained tumor free until the end of the observation period. In an in vivo experiment, injection of VV or rVV-p53 after the C6 cells had been implanted in nude mice induced effective inhibition of tumor growth in comparison with control PBS groups. The oncolytic effect was greater with rVV-p53, apparently due to overexpressed p53 and p53-mediated cell apoptosis. In study of virus virulence we did not observe disease symptoms in athymic mice infected with a high dose of VV. Experimental results indicate that vaccinia virus itself and vaccinia-mediated delivery of therapeutic genes represent novel potential strategies for tumor therapy.     

Differential Suppression of Human Cervical Cancer Cell Growth by Adenovirus Delivery of p53 in vitro: Arrest Phase of Cell Cycle Is Dependent on Cell Line

It has been reported that overexpression of wild-type p53 protein induces suppression of tumor cell growth in vivo and in vitro. In this study, we further evaluated the differential effects of p53 delivered in an adenovirus vector on the cell growth, apoptosis and cell cycle progression in cervical cancer cell lines. We constructed a recombinant adenovirus expressing p53 and then delivered this into cervical carcinoma cell lines (CaSki, SiHa, and HeLa, HeLaS3) along with adenovirus expressing β-galactosidase as a negative control. Adenovirus-delivered p53 overexpression resulted in a more significant suppression of cell growth in HPV 18-infected cells (HeLa and HeLaS3) and a lesser suppression in HPV 16-infected cells (CaSki and SiHa). However, no suppression was observed in cells infected with a negative control virus. p53 overexpression also induced apoptosis and cell cycle arrest, as determined by annexin V and propidium iodide staining. In particular, the cell cycle was arrested in the G₂/M phase in CaSki cells. In contrast, cell cycles were arrested in the G₁ phase in HeLa cells, suggesting that the arrest phase is dependent upon the cervical cancer cell line. Taken together, these data support the idea that overexpressed p53 protein plays a differential role in suppressing cervical cancer cell growth through apoptosis and cell cycle arrest in either G₁ or G₂/M phase, depending on the cancer cell line.     

Combined E7-dendritic cell-based immunotherapy and human sodium/iodide symporter radioiodine gene therapy with monitoring of antitumor effects by bioluminescent imaging in a mouse model of uterine cervical cancer

Using a uterine cervical cancer cell line expressing human papillomavirus (HPV) 16 E7 antigen and bioluminescent imaging (BLI), we evaluated the therapeutic potential of combined immunotherapy using transfected dendritic cells (DC-E7) and human sodium/iodide symporter (hNIS) radioiodine gene therapy in a xenograft animal cancer model. Dendritic cells expressing either E7 antigen (DC-E7) or no-insert (DC-no insert) were made for immunization materials, and murine uterine cervical cancer cell line coexpressing E7, firefly luciferase, hNIS, and EGFP genes (TC-1/FNG) were prepared for the animal tumor model. C57BL/6 mice were divided into five therapy groups (phosphate-buffered saline [PBS], DC-no insert, DC-E7, I-131, and DC-E7+I-131 groups). Single therapy with either DC-E7 or I-131 induced greater retardation in tumor growth compared with PBS or DC-no insert groups, and it resulted in some tumor-free mice (DC-E7 and I-131 groups, 40% and 20%, respectively). Combination therapy with DC-E7 and I-131 dramatically inhibited tumor growth, thus causing complete disappearance of tumors in all mice, and these effects were further confirmed by BLI in vivo. In conclusion, complete disappearance of the tumor was achieved with combined DC-E7 vaccination and hNIS radioiodine gene therapy in a mouse model with E7-expressing uterine cervical cancer, and serial BLIs successfully demonstrated antitumor effects in vivo.     

Adenovirus lacking the 19-kDa and 55-kDa E1B genes exerts a marked cytotoxic effect in human malignant cells

The adenovirus (Ad) E1A gene exerts an antitumor effect and can induce sensitivity to treatment with DNA-damaging agents. In contrast, the Ad 19-kDa E1B protein inhibits E1A-mediated apoptosis and the 55-kDa E1B inactivates the p53 protein. In this paper, we study the in vitro and in vivo effects of a 19-kDa and 55-kDa E1B-defective Ad in several malignant human tumor cell lines. MATERIALS AND METHODS: Nontumorigenic human fibroblasts (CCD-45SK and Hs67), peripheral blood lymphocytes, and several human tumor cell lines derived from cervix, colon, and breast carcinomas, epidermoid carcinoma, and osteosarcoma (HeLa, HT29, MCF7, Saos-2, and A431 cell lines) were studied. Wild-type (wt) Ad type 5 and H5 dL118 Ad, a mutant with the deleted E1B region, were employed. The cells were infected at 20 plaque-forming units, and cell viability was evaluated by the crystal violet method. In the in vivo experiments, 2 x 10(6) cells from the carcinoma cell lines HeLa, A431 and HT29 were injected into nude mice. The tumorigenicity of previously infected cells and after an intratumoral injection of Ad was analyzed. The mice received whole-body gamma-irradiation. RESULTS: The H5 dL118 mutant produced a marked cytopathic effect in all of the malignant cells, surpassing that of the wt Ad; viability at 72 hours ranged from 11% to 20% for H5 dL118 Ad and from 70% to 93% for the wt Ad with respect to uninfected controls. In the in vivo experiments, a total inhibition of tumorigenicity was detected when cells were infected prior to injection and a partial and transitory decrease in tumorigenicity was detected when the mutant H5 dL118 was injected intratumorally. gamma-irradiation enhanced the in vivo antitumor effects. CONCLUSIONS: These results indicate that infection with completely E1B-deficient Ads induced a marked cytopathic effect on malignant cells that was higher than that seen for wt Ads; in addition, infection with such Ads exerts a tumor suppressor effect in vivo.     

Preclinical study on gene therapy of cervical carcinoma using adeno-associated virus vectors

Approximately 90% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for future tumor therapy. Using recombinant adeno-associated virus type 2 (AAV-2) vectors, two types of therapeutic genes were expressed in cervical carcinoma cells with the aim of suppressing the E6/E7 oncogenes: (a) antisense E6/E7 and ribozyme genes and (b) the monocyte chemoattractant protein-1 (MCP-1) gene encoding MCP-1. Previous studies have shown that the MCP-1 protein is able to indirectly repress E6/E7 gene expression and is consistently absent in tumorigenic HPV-positive cervical carcinoma cell lines. Here, the effect of these therapeutic genes on tumor formation is analyzed in nude mice after ex vivo gene transfer into a HPV16- or HPV18-positive cervical carcinoma cell line (HeLa or SiHa, respectively). Whereas AAV-2 vector-mediated transfer of antisense or even ribozyme genes did not significantly influence tumor formation from implanted SiHa cells, the transfer and expression of human MCP-1 strongly inhibited the development of tumors derived from either HeLa or SiHa cells. Similar results were also obtained after in vivo delivery of these genes into SiHa-derived tumors. This suggests that transfer of therapeutic genes mediating a systemic effect via recombinant AAV-2 vectors offers a promising approach for the development of gene therapies directed against papillomavirus-induced human cancers.     

Critical role for D-type cyclins in cellular transformation induced by E6/E7 of human papillomavirus type 16 and E6/E7/ErbB-2 cooperation

Recently, we reported that E6/E7 of human papillomavirus (HPV) type 16 cooperates with the ErbB-2 receptor to induce cellular transformation of human normal oral epithelial (NOE) and mouse normal embryonic fibroblast (NEF) cells. Furthermore, we demonstrated that cyclin D1 is essential for this transformation induced by E6/E7 and E6/E7/ErbB-2 cooperation using cyclin D1 antisense and knockout (D1(-/-)) cells. To determine the role of all D-type cyclins (D1, D2 and D3) in E6/E7/ErbB-2 cooperation, we examined the effects of E6/E7, ErbB-2 alone and E6/E7/ErbB-2 together in NEF, NEF-D1(-/-), NEF-D2(-/-) and NEF-D3(-/-) cells. We confirm that NEF-E6/E7 and NEF-E6/E7/ErbB-2, but not NEF-ErbB-2 cells, induce colony formation in soft agar and tumor formation in nude mice. We report that E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of D1(-/-) and D2(-/-) cells in vitro and in vivo. In contrast, E6/E7/ErbB-2 together but neither E6/E7 nor ErbB-2 alone provoke cellular transformation of D3(-/-) cells. Nevertheless, D3(-/-)E6/E7/ErbB-2 cells resulted in up to a 60 and 50% decrease in colony and tumor formation in soft agar and nude mice, respectively, compared with NEF-E6/E7/ErbB-2 cells. Furthermore, using cyclin D2 small interfering RNA we inhibited tumor and colony formation of the human NOE-E6/E7-ErbB-2-transformed cell line; in contrast, cyclin D3 small interfering RNA repressed approximately 50% of colony and 40% of tumor formation of E6/E7/ErbB-2 cooperation in this cell line. These data suggest that cyclins D1, D2 and D3 (to a lesser extent) are important downstream mediators of the cellular transformation induced by E6/E7 and E6/E7/ErbB-2 cooperation in normal cells. Our data imply that anti-D-type cyclin therapies are important in the treatment of human cancers expressing high-risk HPV or HPV/ErbB-2.     

肿瘤特异单链抗体库的构建及肿瘤血管特异性结合抗体的体内筛选

目的:研究化疗药物依托泊苷对腺病毒载体介导的外源基因在肿瘤细胞内表达水平的影响。方法:携带外源基因增强型绿色荧光蛋白(EGFP)的复制缺陷型腺病毒Ad5-CMV-EGFP(MOI为1或10)单独或联合终质量浓度为0.2、2、20、40、80、100和200μg/ml的依托泊苷感染体外培养的肿瘤细胞NCI-H446(人非小细胞肺癌细胞株)、A549(人肺腺癌细胞株)、SMMC-7721(人肝癌细胞株)、SGC7901(人胃癌细胞株)、SKBR-3(人乳腺癌细胞株)和BTT(小鼠膀胱移行上皮癌细胞株)后不同时间,流式细胞仪分析肿瘤细胞EGFP阳性率和平均荧光强度,Western blotting检测EGFP蛋白表达,RT-PCR和实时荧光定量PCR检测肿瘤细胞内EGFP的mRNA表达量和DNA拷贝数。结果:不同剂量的依托泊苷可不同程度地提高Ad5-CMV-EGFP在7种肿瘤细胞内的表达水平,但对EGFP阳性率无明显提高。10MOI的Ad5-CMV-EGFP联合40μg/ml依托泊苷分别感染肿瘤细胞NCI-H446、NCI-H460、A549、SMMC-7721、SGC7901、SKBR-3和BTT24h后,细胞内EGFP的荧光强度分别是单独感染的3.3、3.5、3.1、6.2、7.0、5.4和3.4倍。Ad5-CMV-EGFP联合应用依托泊苷后肿瘤细胞内EGFP蛋白表达增加2~5倍,EGFP mRNA表达量提高,但DNA拷贝数未见明显改变。结论:依托泊苷可提高腺病毒载体介导的外源基因在肿瘤细胞内的表达水平,该作用可能是在转录水平上发挥作用的。     

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.     

Immunotherapeutic Effects of Dendritic Cells Pulsed with a Coden-optimized HPV 16 E6 and E7 Fusion Gene in Vivo and in Vitro

Background: : Cervical cancer is the second most common cause of cancer related death of women. PersistentHPV infection, especially with high-risk types such as HPV16 and HPV18, has been identified to be the primarycause of cervical cancer. E6 and E7 are the major oncoproteins of high-risk HPVs, which are expressed exclusivelyin HPV infected tissues, and thereby represent ideal therapeutic targets for immunotherapy of cervical cancer.Materials and Methods: In this work, we used recombinant adenovirus expressing coden-optimized HPV16 E6and E7 fusion protein (Ad-ofE6E7) to prime dendritic cells (DC-ofE6E7), to investigate the ability of primed DCvaccine in eliciting antitumor immunity in vitro and vivo. Results: Our results indicated that DC-ofE6E7 vaccineco-culturing with splenocytes could strongly induce a tumor-specific cytotoxic T lymphocyte (CTL) responseand kill the TC-1 cells effectively in vitro. Moreover, DC-ofE6E7 vaccine induced protective immunity againstthe challenge of TC-1 cancer cells in vivo. Conclusions: The results suggested that the HPV16 ofE6E7 primedDC vaccine has potential application for cervical cancer immunotherapy.     

Cyclin D1 is essential for neoplastic transformation induced by both E6/E7 and E6/E7/ErbB-2 cooperation in normal cells

More than 25% of head and neck squamous cell carcinomas (HNSCC) and 99% of cervical cancers (CxCa) are positive for high-risk human papillomaviruses (HPVs). Furthermore, the type I tyrosine kinase receptor ErbB-2 is overexpressed in at least 30% of HNSCC and CxCa. Recently, we demonstrated that E6/E7 of HPV type 16 cooperate with ErbB-2 to induce cell transformation of human normal oral epithelial (NOE) cells. This is accompanied by overexpression of cyclin D1 in NOE cells. To determine the role of cyclin D1 in E6/E7/ErbB-2 cooperation, we examined the independent effects of E6/E7 and ErbB-2, and the combined effect of E6/E7 and ErbB-2 in mouse normal embryonic fibroblast (NEF), wild type (wt), and knockout cyclin D1 (D1(-/-)) cells. We report that NEF-wt cells transduced with E6/E7 alone and E6/E7/ErbB-2 together form small and large tumors in nude mice, respectively, as well as different sized colonies in soft agar; whereas ErbB-2 alone elicits neither tumor formation in vivo nor colony formation in soft agar. More importantly, E6/E7, ErbB-2 and E6/E7/ErbB-2 together all fail to induce neoplastic transformation of cyclin D1(-/-) cells in vivo and in vitro. Furthermore, using antisense cyclin D1 we completely inhibited tumor and colony formation of NEF-wt-E6/E7 and wt-E6/E7-ErbB-2 as well as human NOE-E6/E7-ErbB-2-transformed cells. These analyses reveal that cyclin D1 is the downstream target of the neoplastic transformation induced by E6/E7 or E6/E7/ErbB-2 cooperation in normal cells. Our data suggest that anti-cyclin D1 therapy may be highly specific in the treatment of all human cancers expressing high-risk HPVs or HPVs/ErbB-2.