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.     

p73 beta-expressing recombinant adenovirus: a potential anticancer agent

Tumor suppressor p53-based gene therapy strategy is ineffective in certain conditions. p73, a p53 homologue, could be a potential alternative gene therapy agent as it has been found to be an important determinant of chemosensitivity in cancer cells. Previously, we have reported the generation of a replication-deficient adenovirus expressing p73 beta (Ad-p73). In this study, we evaluated the therapeutic potential of Ad-p73 against a panel of cancer cells (n=12) of different tissue origin. Ad-p73 infected all the cell lines tested very efficiently resulting in several-fold increase in p73 beta levels, which is also functional as it activated the known target gene p21(WAF1/CIP1). Infection with Ad-p73 resulted in potent cytotoxicity in all the cell lines tested. The mechanism of p73-induced cytotoxicity in these cell lines is found to be due to a combination of cell cycle arrest and induction of apoptosis. In addition, exogenous overexpression of p73 by Ad-p73 infection increased the chemosensitivity of cancer cells by many fold to commonly used drug adriamycin. Moreover, Ad-p73 is more efficient than Ad-p53 in enhancing the chemosensitivity of mutant p53 harboring cells. Furthermore, Ad-p73 infection did not induce apoptosis in human normal lung fibroblasts (HEL 299) and human immortalized keratinocytes (HaCaT). These results suggest that Ad-p73 is a potent cytotoxic agent specifically against cancer cells and could be developed as a cancer gene therapy agent either alone or in combination with chemotherapeutic agents.     

Enhanced specificity of the p53 family proteins-based adenoviral gene therapy in uterine cervical cancer cells with E2F1-responsive promoters

p63 and p73, the p53 family proteins, are similar to p53 in many aspects: structural homology, transactivation of p53-downstream genes, and induction of apoptosis. Interestingly, they also differ from p53; in particular, they are not inhibited by viral oncoproteins such as HPV E6. This feature would be an advantage over p53 in HPV-associated cancers and therefore, we evaluated the therapeutic potentials of various p53 family proteins (p73alpha, p73beta, p63alpha and p63gamma) against HPV-infected cervical cancers. In clonogenic assay, exogenous expression of p73alpha, p73beta and p63gamma inhibited the colony formation of HPV-positive cervical cancer cells under G418- selection while p53 could not. Recombinant adenoviruses Ad/CMVp73alpha, Ad/CMVp73beta and Ad/CMVp63gamma induced potent apoptosis in all infected cervical cancers (CasKi, SiHa, HeLa, C33A, SNU682, SNU17, SNU1005, SNU703), irrespective of their HPV-infection status. Unfortunately however, Ad/CMVp73alpha, Ad/CMVp73beta, and Ad/CMVp63gamma inhibited also normal cells such as endothelial cells, fibroblasts, and keratinocytes thus, tumorspecific promoter was indispensable to the p53 family proteins-based therapy. Here we report a stringent tumor killing by p73beta in combination with ESM6, a synthetic promoter targeting the DNA tumor virusassociated cancers. Recombinant adenoviruses encoding p73beta by ESM6 (Ad/ESM6p73beta and Ad/ESM6p73bENH) expressed p73beta and induced apoptosis only in the cancer cells but not in normal cells. Collectively, we suggest that the p53 family proteins are potent therapeutic agents for HPV-associated uterine cervical cancers and ESM6 mediated expression of the p53 family proteins would be a safe and strong tumor targeting strategy.     

Chemoradiation of cervical cancer cells: targeting human papillomavirus E6 and p53 leads to either augmented or attenuated apoptosis depending on the platinum carrier ligand

Recent clinical trials comparing concurrent chemotherapy and radiation with radiation alone in cervical cancer have shown that chemoradiation reduces the risk of death by 30-50%. Despite the clinical success, treatment responses at the cellular level are still inadequately explored. A key event in cervical carcinogenesis is the disruption of p53 tumor suppressor pathway by human papillomavirus (HPV) E6 oncogene. We found that regardless of the HPV type in SiHa (HPV 16+) CaSki (HPV 16+), HeLa (HPV 18+), and UT-DEC-1 (HPV 33+) cell lines, cisplatin, carboplatin, and a novel platinum compound, oxaliplatin, activated a p53 reporter and reduced the HPV E6 mRNA. Carboplatin and oxaliplatin treatment led also to stabilization of p53, whereas none of the platinums changed p73 levels. After irradiation (IR) alone, a decrease in HPV E6 mRNA levels and an activation of the p53-reporter were detected in SiHa, CaSki, and HeLa cells, but not in UT-DEC-1 cells. Concomitant platinum treatment and IR led to poly(ADP-ribose) polymerase cleavage as a sign of caspase-3 activation and apoptosis. Clonogenic survival was enhanced by expressing a dominant negative p53 or ectopic HPV16 E6 in SiHa and HeLa cells treated with IR, carboplatin, or oxaliplatin or with a combination of IR + carboplatin or oxaliplatin. In contrast, dominant negative p53 or ectopic HPV 16 E6 sensitized the cells to cisplatin. Pt chemotherapeutics and radiation had a synergistic cytotoxic effect as determined by Bliss independence criterion. Taken together, p53 has a significant role in the cellular response to chemoradiation treatment in cervical cancer cell lines, but p53 activity may have a dramatically different effect on cell survival depending on the platinum carrier ligand.     

Leptomycin B enhances CDDP-sensitivity via nuclear accumulation of p53 protein in HPV-positive cells

Cervical cancer, which commonly contains a wild-type p53 gene, is highly correlated with human papilloma virus (HPV) infection. Because the oncoprotein E6, derived from HPV, inhibits the function of p53 protein, the inhibition of apoptosis via the p53 pathway by HPV may be related to cisplatin (CDDP)-sensitivity in cervical cancer. We conducted the present study to determine whether and how HPV is related to CDDP-sensitivity in HPV-positive cervical cancer cells. We used cervical carcinoma cell lines HeLa with integrated HPV 18 and SiHa with integrated HPV 16. An HPV-negative cell line, Yumoto, with wild-type p53 gene was used as a control. Leptomycin B (LMB) enhanced sensitivity to CDDP and CDDP-induced apoptosis in HeLa and SiHa cells, but not in Yumoto cells. After exposure to LMB or CDDP alone, we observed weak p53 staining in HeLa, SiHa and Yumoto cells. Nuclear p53 staining was significantly increased by combined treatment with CDDP and LMB in HeLa and SiHa cells, but not in Yumoto cells. The expression of p53 and Bax protein increased with exposure to CDDP and was enhanced by LMB in HeLa and SiHa cells. The present study demonstrated that LMB enhanced CDDP-sensitivity via nuclear accumulation of p53 protein in HPV-positive cells.     

榄香烯对HeLa细胞端粒酶催化亚单位基因表达的作用

目的：探讨HeLa细胞凋亡过程中,人乳头瘤病毒18型E6（HPV18-E6）基因、p53基因、端粒酶催化亚单位（hTERT)基因表达的变化,为临床诊断、治疗宫颈癌提供新思路,方法：用流式细胞仪、电镜证实细胞凋亡;用PCR和RT-PCR方法研究HPV18-E6基因、p53基因、hTERT基因表达在细胞凋亡过程中的变化及相互关系。结果：在榄香烯作用下,HPV18-E6基因、p53基因表达没有变化,但hTERT基因表达受到抑制。结论：榄香烯诱导HeLa细胞凋亡过程中的变化及相互关系。结果：在榄香烯作用下,HPV18-E6基因、p53基因表达没有变化,但hTERT基因表达受到抑制。结论：榄香烯诱导HeLa细胞凋亡过程中,hTERT基因表达受到明显抑制,对于耐药性肿瘤,端粒酶活的抑制可能代表一种新的化疗策略。     

A novel apoptotic mechanism of genetically engineered adenovirus-mediated tumour-specific p53 overexpression through E1A-dependent p21 and MDM2 suppression

Oncolytic viruses engineered to replicate in tumour cells but not in normal cells could be used as tumour-specific vectors carrying the therapeutic genes. We previously developed a telomerase-specific oncolytic adenovirus, OBP-301, that causes cell death in human cancer cells with telomerase activities. Here, we further modified OBP-301 to express the wild-type p53 tumour suppressor gene (OBP-702), and investigated whether OBP-702 induces stronger antitumour activity than OBP-301. The antitumour effect of OBP-702 was compared to that of OBP-301 on OBP-301-sensitive (H358 and H460) and OBP-301-resistant (T.Tn and HSC4) human cancer cells. OBP-702 suppressed the viability of both OBP-301-sensitive and OBP-301-resistant cancer cells more efficiently than OBP-301. OBP-702 caused increased apoptosis compared to OBP-301 or a replication-deficient adenovirus expressing the p53 gene (Ad-p53) in H358 and T.Tn cells. Adenovirus E1A-mediated p21 and MDM2 downregulation was involved in the apoptosis caused by OBP-702. Moreover, OBP-702 significantly suppressed tumour growth in subcutaneous tumour xenograft models compared to monotherapy with OBP-301 or Ad-p53. Our data demonstrated that OBP-702 infection expressed adenovirus E1A and then inhibited p21 and MDM2 expression, which in turn efficiently induced apoptotic cell death. This novel apoptotic mechanism suggests that the p53-expressing OBP-702 is a promising antitumour reagent for human cancer and could improve the clinical outcome.     

Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells

The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor β, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower β-tubulin levels and comparatively higher p53/β-tubulin or CAR /β-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.     

Upregulation of p18Ink4c expression by oncogenic HPV E6 via p53-miR-34a pathway

Binding of p53 to miR-34a promoter activates the expression of tumor-suppressive miR-34a. Oncogenic human papillomavirus (HPV) infection downregulates miR-34a expression through viral E6 degradation of p53. In our report, we found that miR-34a specifically targets p18Ink4c, a CDK4 and CDK6 inhibitor induced by E2F transactivation. HPV18(+) HeLa cells with ectopic miR-34a expression or by E6 siRNA knockdown-induced expression of endogenous miR-34a exhibited a substantial reduction of p18Ink4c in a dose-dependent manner, but had no effect on p16Ink4a, another member of CDK4/6 inhibitor family. In contrast, de novo infection by oncogenic HPVs of human keratinocyte-derived raft tissues increased p18Ink4c expression. Suppression of endogenous miR-34a in cell lines with a miR-34a inhibitor also increased p18Ink4c. We found that miR-34a suppresses the expression of p18Ink4c by binding to a specific seed match in the 5' UTR of p18Ink4c. Further investigation found remarkable increase of p18Ink4c in cervical precancer lesions and cervical cancer. Immunohistochemical staining of cervical tissue arrays showed increased expression of p18Ink4c in 68% of cervical cancer, 8.3% of chronic cervical inflammation and 4.8% of normal cervix. Although p18Ink4c inhibits cell proliferation in general and regulates E2F1 expression in HCT116 cells, it appears not to function as a tumor suppressor in cervical cancer cells lacking an intact G1 checkpoint because of viral E7 degradation of pRB. In summary, our study demonstrates an intimate connection among oncogenic HPV E6, p53, miR-34a and p18Ink4c and identifies p18Ink4c as a possible biomarker 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.     

Interference of proteins involved in the cytoplasmic sequestration of p53 with human papillomavirus E6-mediated degradation

The oncogenic human papillomaviruses (HPVs) are able to efficiently target p53 for degradation by the ubiquitin pathway. We previously demonstrated inefficient HPV E6-mediated degradation and resulting high steady-state levels of p53 in cell hybrids between a peripheral neuroepithelioma cell line and a cervical carcinoma cell line (HeLa). We now show that the p53 protein in these cell hybrids was cytoplasmically sequestered and exhibited sporadic punctate staining, which is characteristic of the p53 expression pattern observed in neuroblastic neuroblastoma (NB) cell lines, in which p53 is also sequestered. We hypothesized that the cytoplasmic sequestration of p53 in the cell hybrids might correlate with its inability to be rapidly degraded by HPV E6. Using NB cell lines as a model system to test this hypothesis, we demonstrated that the introduction of HPV E6 into two NB cell lines resulted in p53 insensitivity to HPV E6-mediated degradation. This was assessed by both pulse-chase analysis of p53 in metabolically labeled NB cells and western blotting. The enhanced stability of p53 was not due to a lack of HPV E6 expression or to a mutant conformation of the p53 protein. Our results therefore suggest that proteins involved in the cytoplasmic sequestration of p53 may also interfere with the ability of HPV E6 to target p53 for degradation.     

Proteasome inhibition mediates p53 reactivation and anti-cancer activity of 6-Gingerol in cervical cancer cells

Human papilloma virus (HPV) expressing E6 and E7 oncoproteins, is known to inactivate the tumor suppressor p53 through proteasomal degradation in cervical cancers. Therefore, use of small molecules for inhibition of proteasome function and induction of p53 reactivation is a promising strategy for induction of apoptosis in cervical cancer cells. The polyphenolic alkanone, 6-Gingerol (6G), present in the pungent extracts of ginger (Zingiber officinale Roscoe) has shown potent anti-tumorigenic and pro-apoptotic activities against a variety of cancers. In this study we explored the molecular mechanism of action of 6G in human cervical cancer cells in vitro and in vivo. 6G potently inhibited proliferation of the HPV positive cervical cancer cells. 6G was found to: (i) inhibit the chymotrypsin activity of proteasomes, (ii) induce reactivation of p53, (iii) increase levels of p21, (iv) induce DNA damage and G2/M cell cycle arrest, (v) alter expression levels of p53-associated apoptotic markers like, cleaved caspase-3 and PARP, and (vi) potentiate the cytotoxicity of cisplatin. 6G treatment induced significant reduction of tumor volume, tumor weight, proteasome inhibition and p53 accumulation in HeLa xenograft tumor cells in vivo. The 6G treatment was devoid of toxic effects as it did not affect body weights, hematological and osteogenic parameters. Taken together, our data underscores the therapeutic and chemosensitizing effects of 6G in the management and treatment of cervical cancer.     

Induction of the p53-target gene GADD45 in HPV-positive cancer cells.

The E6 oncoprotein of human papillomaviruses (HPVs) has the potential to functionally antagonize p53. In several experimental model systems, ectopic expression of E6 can block the genotoxic induction of the growth inhibitory p53 target gene gadd45, suggesting that the inactivation of this pathway may play a major role for HPV-associated cell transformation. Here, we investigated whether this reflects the regulation of gadd45 expression in carcinoma-derived HPV-positive cells. We found that the gadd45 gene is efficiently induced by mitomycin C, cisplatin, and UV irradiation in a series of HPV-positive cervical cancer cell lines. Moreover, clear induction of gadd45 gene expression was also observed following treatment with gamma-irradiation, a pathway that is strictly dependent on functional p53. This contrasted with findings in human foreskin keratinocytes experimentally immortalized by expressing the HPV16 E6, E7, or E6/E7 oncogenes from the heterologous CMV promoter, where expression of the E6 gene was linked to a lack of gadd45 induction following gamma-irradiation. These results indicate (1) that the tumorigenic phenotype of HPV-positive cancer cells is not linked to an inability to induce the gadd45 gene following DNA damage, (2) that experimental model systems in which the E6 gene is expressed ectopically and/or in a different cellular context do not necessarily reflect the regulation of p53-associated pathways in HPV-positive cancer cells and (3) that a pathway strictly depending on functional p53 is inducible in HPV-positive cancer cells, providing direct evidence that the endogenous p53 protein in these cells is competent to activate a cellular target gene, despite coexpression of the viral E6 oncogene.     

顺铂诱导宫颈癌Hela细胞凋亡及其作用机制的研究

目的:研究顺铂在体外诱导宫颈癌Hela细胞凋亡及其作用机制。方法:采用MTT法测定顺铂对Hela细胞增殖的影响;流式细胞仪和Hochest33258检测药物作用前后的细胞凋亡情况;RT-PCR检测HPVE6的mRNA水平表达;WesternBlot测定HPVE6、p53、p21、Bax、Bcl-2蛋白水平的表达。结果:顺铂抑制Hela细胞生长呈时效和量效关系;经10μg/ml顺铂分别在12、24、36、48小时作用Hela细胞,亚G1峰与对照组有显著性差异;RT-PCR提示顺铂作用Hela细胞后HPVE6的mRNA水平表达逐渐降低;WesternBlot提示顺铂作用Hela细胞后HPVE6的蛋白水平表达逐渐降低,p53、p21和Bax蛋白水平表达逐步升高,Bcl-2的表达无变化。结论:顺铂通过抑制HPVE6的表达,恢复p53的功能,引起细胞凋亡,起到杀伤肿瘤的作用。     

Functional inactivation of p73, a homolog of p53 tumor suppressor protein, by human papillomavirus E6 proteins

Human papillomavirus (HPV) is strongly implicated as a causative agent in the etiology of cervical cancer. Of its gene products, E6 binds to and inactivates p53 tumor suppressor protein by ubiquitin/proteasome-dependent degradation. Recently, p73, a novel family of p53, has been identified and demonstrated, like p53, to activate p21(WAF1). Here we show that p73 is also inactivated by HPV-E6, but ubiquitin-mediated proteolysis is not responsive. Yeast two-hybrid and GST pull-down assays indicate a physical interaction between p73 and either HPV-16 or HPV-11 E6 proteins in vivo and in vitro, respectively. The transactivation domain (amino acid residues 1 to 49) is found to be absolutely required for the interaction. Transient co-expression of E6 significantly inhibits the p73-mdiated activation of p21(WAF1) promoter in a p53-defective C33A cell line. Using Gal4-p73 fusion protein, we demonstrate that E6 inhibition of p73 transactivation function is independent of sequence-specific DNA binding, which is confirmed by a direct electrophoretic mobility shift assay. Moreover, E6 inhibits p73 function by interfering with the activity of the amino-terminal activation domain. Co-transfection of E6 mutants reveals that the same portion of E6 appears to be responsible for the inactivation of p53 and p73 function. However, the inactivation mechanism of p73 is clearly different from that of p53, because p73, unlike p53, is inactivated by both high- and low-risk E6s and is not susceptible to E6-dependent proteolysis. These overall results, consequently, suggest that in addition to the inactivation of p53, the functional interference of p73 by HPV-E6 may, at least in part, contribute to E6-mediated transformation and hyperproliferation of cervical cells.