Adenovirus-mediated p53 gene therapy in nasopharyngeal cancer

EBV-associated nasopharyngeal cancer (NPC) occurs with high frequency in China and is a major cause of morbidity and mortality. To explore the potential use of adenovirus-mediated tumor suppressor p53 gene therapy In NPC, we first examined the in vitro effects of p53 introduced into the NPC cell lines RPMI 2650, Fadu and Detroit 562. p21(WAF1/CIP1) induction by chemotherapy was used as a functional assay which revealed that RPMI 2650 expresses wild-type p53 whereas Fadu and Detroit 562 encode mutant p53. Infection with p53-expressing adenovirus (Ad-p53) induced apoptosis and inhibited cell growth in all three NPC cell lines, regardless of the endogenous p53 status. Adenovirus infectivity was greatest in RPMI 2650 cells, with 100% of the cells expressing beta-galactosidase following Ad-LacZ infection using an MOI of 100, as compared to 20-30% infectivity with the other NPC lines. Using RPMI 2650 cells injected into nude mice, we developed an animal model for nasopharyngeal cancer. Established tumors (0.6-0.8 cm) were injected with 5x10(9) PFU Ad-LacZ, Ad-p53 or PBS in a 100 mu l volume. We found evidence for in vivo expression of beta-galactosidase or p53 and p21 up to two weeks following Ad-LacZ or Ad-p53 virus injection respectively. Objective regression of tumor size was observed at two weeks in 4/6 Ad-p53-treated tumors, but not in Ad-LacZ or PBS-treated tumors. The results provide an animal model for human nasopharyngeal cancer, and indicate a potential use of p53 in its therapy in vivo.     

Acute overexpression of wt p53 facilitates anticancer drug-induced death of cancer and normal cells

The relationship between chemosensitivity and p53 is currently considered from two mutually exclusive points of view: (1) wt p53 increases chemosensitivity due to apoptosis and (2) wt p53 decreases chemosensitivity due to growth arrest and DNA repair. We used p53-expressing adenovirus (Ad-p53) to directly evaluate effect of p53 on sensitivity to anticancer drugs. When p53 was expressed at sublethal levels, it sensitized cells to the DNA-damaging drugs Adriamycin, mitomycin C, actinomycin D, etoposide (VP16), cisplatin and CPT11. This sensitization was observed in cancer cell lines (N=10) regardless of endogenous p53 status and also in normal human lung and skin fibroblasts. The degree of sensitization appeared to be greater in cancer cells with mutant p53. Normal fibroblasts required significantly higher doses of Ad-p53 to affect a drug's sensitivity partly because of their lower infectivity by adenovirus. Wt p53 not only decreased IC₅₀ but also accelerated cell death induced by DNA-damaging drugs. In contrast, sensitization to microtubule-active drugs by p53 was shown only in a few cell lines. We conclude that exogenous wt p53 accelerates cell death induced by DNA damaging agents in both normal and cancer cells and offers no protection from anticancer drugs. Int. J. Cancer75:933–940, 1998. © 1998 Wiley-Liss, Inc.     

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.     

Efficient growth inhibition of HPV 16 E6-expressing cells by an adenovirus-expressing p53 homologue p73beta

Tumor suppressor p53 functions are downregulated in most cervical cancers, because the product of human papilloma virus (HPV) oncogene E6 binds to and inactivates p53 by promoting its degradation. p73, a p53 homologue, is similar to p53 in structure and function but yet not degraded by HPV E6 gene product. In this study, we have developed a replication-deficient recombinant adenovirus, which expresses p73beta (Ad-p73). Infection of human cancer cells with Ad-p73 results in several fold increase of p73beta levels as well as its known target genes like p21(WAF1/CIP1). Ad-p73-infected cells showed reduced cellular DNA synthesis, arrest in G1 phase of cell cycle and induction of apoptosis. Ad-p73 inhibited the growth of cancer cells of different types. More importantly, Ad-p73 inhibited the growth of cell lines carrying HPV E6 gene, which was introduced by stable integration, more efficiently in comparison to an Ad-p53. Furthermore, Ad-p73 also inhibited the growth of HeLa cells, a cell line derived from cervical cancer, very efficiently. The ability of Ad-p73 to inhibit the growth of HPV E6-expressing cells and HeLa cells correlated with the stable expression of functional p73 in the presence of E6. These results suggest that Ad-p73 could be used as a potential gene therapy agent against cervical cancer.     

Overexpression of p21WAF1/CIP1 induces cell differentiation and growth inhibition in a human glioma cell line

p21^WAF1/CIP1 is a downstream mediator of p53 and mediates growth arrest by inhibiting the action of G₁ cyclin-dependent kinases. Since cellular differentiation is frequently characterized by G₁ arrest, we examined whether p21^WAF1/CIP1 overexpression would induce growth suppression and differentiation in p53-defective human glioma cells. Overexpression of p21^WAF1/CIP1 resulted in an accumulation of cells in G₁, altered morphology, growth arrest and cell differentiation. The extent of cell differentiation correlated with the level of p21^WAF1/CIP1 as well as of proliferating cell nuclear antigen, cyclin E, and cdk 2, which associates with p21^WAF1/CIP1. Our data suggest that gene transfer of p21^WAF1/CIP1 may arrest glioma cell growth in vivo by committing malignant glioma cells to a pathway of terminal differentiation. Int. J. Cancer 75:643–648, 1998. © 1998 Wiley-Liss, Inc.     

Late resistance to adenoviral p53-mediated apoptosis caused by decreased expression of Coxsackie-adenovirus receptors in human lung cancer cells

Adenovirus-mediated wild-type p53 gene transfer induces apoptosis in a variety of human cancer cells. Although clinical trials have demonstrated that a replication-deficient recombinant adenovirus expressing the wild-type p53 gene (Ad-p53) is effective in suppressing growth of non-small cell lung cancer (NSCLC), we often experienced late resistance to this treatment. To elucidate the mechanism of late resistance to Ad-p53 in human lung cancer cells, we generated 5 different resistant variants from p53-susceptible H1299 NSCLC cells by repeated infections with Ad-p53. We first examined the transduction efficiency of adenoviral vector by Ad-LacZ transduction followed by X-gal staining in parental and 5 resistant H1299 cell lines. Their sensitivity to viral infection decreased in correlation with the magnitude of resistance, and Ad-p53-mediated tumor suppression could be restored by dose escalation of Ad-p53 in the resistant variants. The expression of Coxsackie and adenovirus receptor (CAR) and alphaV integrins, which are cellular receptors for attachment and internalization of the virus, respectively, was next investigated in these cell lines. Flow cytometry revealed that alphaVbeta3 and alphaVbeta5 integrin expression was consistent, while p53-resistant cell lines showed that diminished CAR expression correlated with the magnitude of the resistance. Our results demonstrated that decreased CAR expression could be one of the mechanisms of late resistance to Ad-p53, which may have a significant impact on the outcome of adenovirus-based cancer gene therapy.     

The mitogen-activated protein kinase pathway mediates growth arrest or E1A-dependent apoptosis in SKBR3 human breast cancer cells

Previously, we have shown that phorbol ester (PMA) induces p21^WAF1/CIP1-dependent growth arrest in SKBr3 breast cancer and LNCaP prostate cancer cells. Here, I demonstrate that inhibition of Raf-1 kinase by dominant-negative Raf-1 or pharmacological depletion of Raf-1 prevented PMA-mediated induction of p21^WAF1/CIP1. Similarly, PD98059, a specific inhibitor of MEK, abolished p21^WAF1/CIP1induction and PMA-induced growth arrest. Like PMA, the H-ras oncogene, another activator of the Raf-1/MEK/MAPK pathway, transactivated p21^WAF1/CIP1in SKBr3 cells. I further investigated PMA-induced growth arrest following infection of SKBr3 cells with 12S E1A-expressing adenovirus. Although high levels of E1A oncoprotein prevented both PMA-induced p21^WAF1/CIP1and growth arrest, smaller amounts of E1A abrogated growth arrest without down-regulation of p21^WAF1/CIP1. Therefore, E1A can stimulate proliferation downstream of p21^WAF1/CIP1. Albeit less effective than full activity, either Rb- or p300-binding activity of E1A was sufficient for the abrogation of PMA-mediated growth arrest. E1A-driven proliferation of PMA-treated SKBr3 cells was accompanied by apoptosis. New therapeutic approaches can be envisioned that would utilize stimulation of the Raf-1/MEK/MAPK pathway to inhibit growth of PMA-sensitive cancer cells. Int. J. Cancer 78:511–517, 1998. © 1998 Wiley-Liss, Inc.     

Mitomycin C and doxorubicin elicit conflicting signals by causing accumulation of cyclin E prior to p21WAF1/CIP1 elevation in human hepatocellular carcinoma cells

Proteins involved in the G1 phase of the cell cycle are aberrantly expressed, sometimes in mutated forms, in human cancers including human hepatocellular carcinoma. Upon attack by a DNA-damaging anticancer drug, a cell arrests at the G1 phase; this is a safety feature prohibiting entry of DNA-damaged cells into S-phase. p21WAF1/CIP1 prevents damaged cells from progressing to the next cell cycle. Here, we show that, in response to mitomycin C and doxorubicin, human hepatocellular carcinoma cells generate conflicting signals, mediated by cyclin E and p21WAF1/CIP1, which respectively accelerates and represses cell cycle transition. Exposure to these anticancer drugs led to rapid accumulation of cyclin E in both p53-proficient HepG2 and p53-deficient Hep3B cells. Such anticancer drug-induced cyclin?E accumulation influenced the G1-S-phase transition, but not DNA fragmentation-mediated death. In p53-proficient HepG2 cells, accumulation of cyclin E was followed by an increase in the level of p53-dependent p21WAF1/CIP1, thereby inhibiting further the G1-S-phase transition. Sublethal drug concentrations also induced rapid accumulation of cyclin E, but p21WAF1/CIP1 accumulation was delayed, further facilitating the G1-S-phase transition. Eventually, most cells arrested in G2/M. Thus, mitomycin C- or doxorubicin-induced conflicting signals, mediated by cyclin E and p21WAF1/CIP1, are in play in human hepatocellular carcinoma cells. Damaged G1 cells either immediately enter S-phase, or do not do so at all, depending on the extent of DNA damage.     

The administration schedule of cyclin-dependent kinase inhibitor gene therapy and etoposide chemotherapy is a major determinant of cytotoxicity.

Cyclin-dependent kinase inhibitors are potent suppressors of cell growth and have been proposed as targets for gene replacement therapy in cancer. Expression of either p16INK4a or p21WAF1 protected cells from the cytotoxic effects of the topoisomerase II inhibitor, etoposide. A lower level of p53 was induced in CDK inhibitor-expressing etoposide-exposed cells suggesting that protection may be due to lower levels of DNA damage in the growth arrested cells. Exposure of human osteosarcoma cells to either p16INK4a or p21WAF1 prior to and during etoposide therapy protected cells against etoposide-induced cell death. Infection of the cells by Ad-p16INK4a or Ad-p21WAF1 following exposure to etoposide resulted in loss of the protective effect with evidence of enhanced growth inhibition. The results suggest that the schedule of administration of DNA damaging etoposide chemotherapy and cell cycle inhibitory therapy is a major determinant of the resulting cytotoxicity.     

Synergistic growth inhibition by combination of adenovirus mediated p53 transfer and cisplatin in ovarian cancer cell lines

Objective

This study was to investigate the synergistic growth inhibitory effect by combination of adenovirus mediated p53 gene transfer and cisplatin in ovarian cancer cell lines with different p53 gene mutation patterns.

Methods

Three ovarian cancer cell lines, p53 deleted SKOV3, p53 mutated OVCAR-3, and PA-1 with wild-type p53 were transduced with human adenovirus vectors carrying p53 gene (Ad-p53) and treated with a sublethal concentration of cisplatin before and after Ad-p53. The cell number was counted daily for 5 days after Ad-p53 transduction. Western blotting was used to identify p53 and p21 protein expressions, and flow cytometric analysis was performed to investigate any change of DNA ploidy after Ad-p53 transfer.

Results

Ad-p53 transduced cells successfully expressed p53 and p21 proteins after 48 hours of Ad-p53 transduction. Synergistic growth inhibition by combination of Ad-p53 and cisplatin was detected only in SKOV3 and OVCAR-3 cells, but not in PA-1 cells. In p53 deleted SKOV3 cells, cisplatin treatment after Ad-p53 showed higher growth inhibition than the treatment before Ad-p53 transduction, and reverse relationship was observed in p53 mutated OVCAR-3 cells. In SKOV3 cells, the fraction of cells at G2/M phase increased after cisplatin treatment, however, it decreased dramatically with Ad-p53 transduction.

Conclusion

The synergistic growth inhibition by combination of Ad-p53 and cisplatin may depend on the p53 status and the temporal sequence of cisplatin treatment, suggesting judicious selective application of this strategy in clinical trials.     

Therapeutic potential of recombinant p53 overexpression in breast cancer cells expressing endogenous wild-type p53

Reconstitution of the p53-dependent apoptotic pathway by gene transfer of a recombinant wild-type p53 minigene leads to rapid apoptotic cell death in breast and other cancer cell types expressing null or mutant p53. Tumour cells expressing wild-type p53 have been reported to be more resistant to this treatment strategy, presumably as a result of mutations in downstream regulators of p53-dependent apoptotic signalling. The MCF-7 breast cancer cell line is representative of this class of tumour cell. Our recent observation of a p53-dependent apoptotic response following adenovirus-mediated HSV thymidine kinase gene transfer and gancyclovir treatment led us to reexamine recombinant p53 cytotoxicity in MCF-7 cells. Infection with a recombinant adenovirus expressing wild-type p53 resulted in a dramatic increase in p53 protein levels and was accompanied by an increase in p21^{WAF 1/CIP1} protein levels and G1 arrest within 24 hours post-infection. A significant decrease in MCF-7 cell viability was first observed at 5 days post-infection and coincided with the appearance of morphological and biochemical changes consistent with apoptotic cell death. By day 7 post-treatment, cell viability decreased to 45% and clonogenic survival was reduced to 12% of controls. The results demonstrate that persistent, high level expression of recombinant p53 can induce programmed cell death in MCF-7 cells. While the mechanism by which p53 overexpression overcomes the defect in downstream apoptotic signalling is not clear, our data suggests that this treatment strategy may be beneficial for the class of tumour cells represented by the MCF-7 cell line.     

人乳腺癌细胞株WAF1／GIP1／p21基因的表达及其与细胞生物学特性的关系

OBJECTIVE: To study the WAF1/CIP1 gene DNA status, mRNA and protein expressions in human breast cancer cell line and its significance. METHODS: Using cell culture, molecular biological techniques such as Southern blot, Northern blot and immunocytochemical methods, the WAF1/CIP1 gene DNA status, mRNA and protein expression levels in MCF-7 expressing wild type p53(wtp53) and MDA-MB-231 expressing mutant p53 (mtp53) human breast cancer cell lines were detected respectively. The p53 and mdm-2 protein expression levels and cytobiological features of the 2 cell lines were compared and correlated to their WAF1/CIP1 gene expression levels. RESULTS: (1) There was no difference in WAF1/CIP1 gene DNA status in the two breast cancer cell lines. Neither of them showed gene amplificatian or deletion. However, the WAF1/CIP1 mRNA and p21WAF1/CIP1 protein expression levels of MCF-7 cells were higer than those of MDA-MB-231 cells (P < 0.05). (2) The character and cellular distribution of p53 protein in the two cell lines were clearly different. The expression level of mdm-2 proteion was significantly higher in MCE-7 than in MDA-MB-231 cells (P < 0.05). (3) Compered to the other breast cancer cell line, MCF-7 cells were better differentiated, grew more slowly and adhered more closely with each other. CONCLUSION: The WAF1/CIP1 gene expression at mRNA and protein levels is associated with p53 phenotype and some cytobiological features of human breast cancer.     

Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition.

Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>ALLN>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition.     

International cancer research grants

Ahderrant cyclin expression has been implicated in oncogenesis in a number of human cancers. Since alterehd function of regulators of cyclin-dependent kinase (CDK) activity other than cyclins, in particular CDK inhibitors, might play a similar role in oncogenesis, we examined the expression and regulation of the CDK inhibitors p16^INk4 p15^INK4B and p21^WAF1/CIP1 in human breast cancer cell lines. Both the INK4 and INK4B genes were homozygously deleted in 3 cell lines, while INK4 alone was deleted in 2 cell lines. A further 2 cell lines displayed loss of an allele at this locus, and in 1 of these the remaining allele contained a mis-sense mutation within the coding region of the p16^INK4 protein. The majority of cell lines examined, including 2 normal mammary epithelial cell strains, expressed low levels of INK4 mRNA and low or undetectable levels of INK4B mRNA. However, INK4 mRNA was expressed at high levels in 5 cell lines, and this was associated with deletion or inactivation of the retinoblastoma susceptibility gene product pRB but not with mutation of TP53. No deletions of the WAF1/CIP1 gene were observed, but WAF1/CIP1 mRNA levels were reduced in cell lines with TP53 mutation. Transfection of a p16^INK4 expression vector into MDA-MB-231 cells lacking the INK4 gene failed to produce any p16^INK4-expressing cell lines, suggesting that such cells were selected against in continuous culture. Despite the frequent deletion of INK4 in breast cancer cell lines, no evidence was obtained for INK4 deletions in DNA from 45 primary breast carcinomas. Thus, homozygous deletion of the INK4 gene appears to be a rare event in primary breast cancer.