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 p73β (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 p73β levels, which is also functional as it activated the known target gene p21WAF1/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.
The p53 tumor suppressor gene encodes a transcription factor, which plays an important role in cell cycle control, angiogenesis, apoptosis and genomic stability through the transactivation of its target genes. It is one of the most frequently inactivated genes in human tumors. Inactivation of p53 not only results in the development of tumor, it also results in chemoresistance in many types of cancer. Therefore, introduction of wild-type p53 gene has been pursued as a potential therapeutic strategy against various types of cancers. This strategy relies on the ability of p53 to induce cell cycle arrest and apoptosis in cancer cells. However, p53-based gene therapy is not suitable in certain cases where functional inactivation of p53 takes place, like amplification of mdm-2, mutational inactivation of p14ARF making p53 mediated tumor suppression inoperative.1,2,3,4,5,6,7 Moreover, in clinical trials the restoration of wild type p53 gene does not always lead to tumor regression or tumor growth inhibition suggesting resistance of some tumors to exogenous p53.8,9,10
p73 is a member of p53 gene family and encodes proteins, which have significant homology with p53 both structurally and functionally. p73 can bind to p53 responsive elements and upregulate some of the p53 target genes suggesting that it has the potential for functional overlap with p53. When exogenously overexpressed, p73β, one of the p73 isoforms, can transactivate some of the p53 target genes leading to induction of growth arrest and apoptosis.11,12 Although, initially it was reported that p73 is not induced by DNA damage, subsequent studies reported that p73 is indeed induced by a wide variety of DNA-damage-inducing drugs like adriamycin, etoposide, cisplatin, etc.11,13,14,15,16,17,18 It was also reported that both p73 and p63 (another p53 family member) play a crucial role in DNA-damage-induced p53-mediated apoptosis.19 It was shown that combined loss of p63 and p73 results in failure of cells containing wild-type p53 to undergo apoptosis in response to DNA damage. A recent study has also linked chemosensitivity of cancer cells to functional p73 irrespective of p53 status.18 It was shown that abrogation of p73 function in mutant p53 background leads to chemoresistance. These results suggest that p73 is a determinant of chemosensitivity and could be used as a candidate for cancer gene therapy.
We have recently shown that p73 overexpression by infecting cells with a replication-deficient adenovirus expressing p73β specifically inhibits the growth of cancer cells expressing human papilloma virus E6 oncogene.20 Another independent study demonstrated the potential therapeutic use of p73 expressing adenovirus against colorectal cancer cells.21 In the present study, we investigated the cytotoxic potential of a replication-deficient recombinant adenovirus expressing p73β (Ad-p73) against a panel of human cancer cell lines of diverse tissue origin. We analyzed the infectivity of Ad-p73 by monitoring the cellular p73 and p21WAF1/CIP1 levels as well as the cytotoxicity against a variety of cancer cells. In addition, we tested the ability of Ad-p73 to induce cell cycle arrest and apoptosis in all cancer cells as well as to sensitize the cancer cells to chemotherapeutic drugs. Finally, we tested cytotoxic property of Ad-p73 against human normal lung fibroblasts and immortalized keratinocytes.
Materials and methods
Tumor cell lines and culture conditions
Human cancer cell lines 293 (human embryonic kidney cells transformed with adenoviral fragments), SW480 (colon carcinoma), H460 (lung carcinoma), Saos-2 (osteosarcoma), HCT116 (colon carcinoma), U2OS (osteosarcoma) and H1299 (lung carcinoma) were described previously.22,23,24 A375 (melanoma), MCF7 (breast carcinoma), PA-1 (ovarian carcinoma), HepG2 (hepatic carcinoma), U373MG (glioblastoma), SkBr3 (breast carcinoma), C33A (cervical carcinoma) and T47D (breast carcinoma) were grown in DMEM (Sigma) supplemented with 10% FBS. Human normal lung fibroblasts (HEL 299) and immortalized keratinocytes (HaCaT) were obtained from NCCS, Pune, India.
Adenovirus reagents and infections
The following adenoviruses were used: Ad-LacZ and Ad-p53 lack both E1A and E1B but carries β-galactosidase and p53 respectively.25 Ad-p73 lacks both E1A and E1B but carries simian p73β.20 Adenoviral infections were carried out as described.26
Western and immunohistochemical analysis
Western analysis and immunohistochemical staining were performed as described before22 with mouse anti-human p21WAF1/CIP1 (Ab-1; Oncogene), mouse anti-human p73 monoclonal (Ab-2; Oncogene) and Actin (Ab- 1; Oncogene). Cells were harvested or fixed at the indicated time point as described after virus infection and subjected to analysis.
MTT assay was carried out as described previously.27 A total of 1.5 × 103 cells per well were plated in a 96-well plate. After 24 hours of plating, the cells were infected with Ad-LacZ or Ad-p73 at multiplicities of infection (MOI) of 2, 10 and 20. MTT (20 μl of 5 mg/ml) was added 48 hours after infection. MTT is a tetrazolium salt that is converted by living cells into blue formazan crystals. The medium was removed from the wells 3 hours after MTT addition and 200 μl of DMSO was added to dissolve the formazan crystals and then the absorbance was measured at 550 nm in an ELISA reader.
Cell cycle analysis
Ad-p73 infection results in induced p73 and p21WAF1/CIP1 expression in human cancer cells
We have previously reported the generation of a replication-deficient adenovirus expressing p73β (Ad-p73).20 To explore the therapeutic potential of Ad-p73 against cancer cells, we tested the ability of Ad-p73 to infect and express functional p73 protein in a panel of human cancer cell lines. Different cancer cell lines (n=12) of different tissue types were infected with Ad-p73 at 10 MOI and the expression of p73 was monitored after 24 hours postinfection. In all cell lines tested, Ad-p73-infected cells showed intense nuclear p73 staining in comparison to a control adenovirus (Ad-LacZ), which expresses β-galactosidase (Fig 1a). We also monitored the induced expression of p73 and its target gene p21WAF1/CIP1 upon Ad-p73 infection by Western blotting. Ad-p73 infected cells showed several-fold higher amounts of p73 and p21WAF1/CIP1 in comparison to Ad-LacZ infected cells (Fig 1b). These results show that Ad-p73 infects all the cell lines of different tissue origins very efficiently leading to the overexpression of functionally active p73.
p73 overexpression inhibits the growth of cancer cells of varying cell types
Next we tested the cytotoxic potential of Ad-p73 by monitoring the ability of Ad-p73 to inhibit the growth of different cancer cell lines. Cells were infected with varying MOI of Ad-p73 or Ad-LacZ and the cytotoxicity was analyzed by measuring the viability (Fig 2). Ad-p73 inhibited the growth of all the cell lines very efficiently in comparison to Ad-LacZ (Fig 2). Ad-p73 reduced the viability of cells by 60–80% at 20 MOI (Table 1). These results suggest that Ad-p73 is cytotoxic to all cell lines irrespective of the p53 status and Ad-p73 is a potent inhibitor of cancer cell growth.
Ad-p73 infection induces cell cycle arrest and apoptosis
Exogenous expression of p73 has been shown to induce growth arrest and apoptosis.11,12,20,24,29 In order to study the mechanism of growth inhibition by p73 in different cell types, we analyzed the cell cycle profile of four different cell lines of different tissue types infected with Ad-p73. HCT116, H1299, HepG2 and A375 cells were infected with either Ad-p73 or Ad-LacZ virus and the cells were collected at different time points after infection and subjected to fluorescence-activated cell sorting (FACS) (Fig 3). The results were quantified and are represented graphically in Figure 4. p73 overexpression resulted in a time-dependent DNA synthesis inhibition and induction of apoptosis in all the cell lines tested (Fig 3). The proportion of cells actively replicating DNA, which represent S-phase cells, decreased to less than 50% by 24 hours in all cell lines except HCT116 cells and to very small levels (less than 5%) by 48 hours in all cell lines infected with Ad-p73 in comparison to Ad-LacZ (Fig 4b, d). Cells with less than 2n content of DNA, which represent apoptotic cells, appeared in significant levels by 24 hours, which increased to a very high proportion by 48 hours in Ad-p73-infected cells in comparison to Ad-LacZ-infected cells (Fig 4a, c). These results suggest that p73 is a potent inducer of cell cycle arrest and apoptosis in cancer cells.
Ad-p73 increases the chemosensitivity of cancer cells
p73 is an important determinant of chemosensitivity of cancer cells.18 In addition, p73 and p63 has been shown to be essential for DNA-damage-induced p53-mediated cytotoxicity.19 This raises the possibility that cells overexpressing p73 may be more sensitive to chemotherapeutic drugs. To address this point, different cancer cell lines with varying p53 status were tested for chemosensitivity to commonly used anticancer drug adriamycin after infection with Ad-p73. Two colon cancer cell lines, HCT116 (p53 wt) and SW480 (p53 mutant), two lung cancer cell lines, H460 (p53 wt) and H1299 (p53 null), a breast cancer cell line SkBr3 (p53 mutant) and a cervical cancer cell line C33A (p53 mutant) were infected with 4 MOI of either Ad-p73, Ad-p53 and Ad-LacZ and 6 hours later increasing concentrations of adriamycin was added. The chemosensitivity was determined by measuring the cell viability at 48 hours postdrug addition (Fig 5a). IC50 values of these cell lines to adriamycin at different conditions and the fold change in their IC50 values upon infection with Ad-p73 are given in Figure 5b, c. Ad-LacZ infection did not have any appreciable effect on the chemosensitivity of any of the cell lines tested. Both Ad-p73 and Ad-p53 infection increased the sensitivity of all the cell lines tested to adriamycin by 2–5-fold. However, Ad-p73 increased the chemosensitivity of cell lines harboring mutant p53 more efficiently than Ad-p53 (Fig 5c). Thus the results show that p73 overexpression by Ad-p73 infection increases the chemosensitivity of cancer cells irrespective of p53 status and Ad-p73 is more efficient than Ad-p53 in sensitizing the mutant p53 harboring cancer cells to chemotherapeutic drugs.
Effect of Ad-p73 on normal cells
To determine if the growth inhibitory actions of p73 were specific to the malignant cell lines, HEL 299 cells which are normal lung fibroblasts and HaCaT, immortalized keratinocytes. In the first experiment, HEL 299 cells were infected with varying MOI of Ad-LacZ or Ad-p73 and the cytotoxicity was analyzed by measuring the viability (Fig 6a). Ad-LacZ-infected cells showed no cytotoxicity. Ad-p73-infected cells showed some marginal cytotoxcity at higher MOI (75% viability at 20 MOI of Ad-p73 infection). Ad-p73 infection also led to increased levels of p73 protein in HEL 299 cells (Fig 6b). To investigate further, we monitored the cell cycle profile of Ad-p73-infected cells by FACS. HEL 299 cells were infected with either Ad-LacZ or Ad-p73 and at different time points cells were collected and subjected to dual parameter FACS analysis (Fig 6c). Ad-LacZ-infected cells showed no change in cell cycle profile even after 48 hours of virus infection. However, upon infection with Ad-p73, a 35% decrease in S phase (34–22%) with a concomitant increase in G1-phase cells could be seen at 24 hours, which further increased to 56% inhibition of DNA synthesis at 48 hours (34–15%). The kinetics of DNA synthesis inhibition in HEL 299 cells by p73 is much slower compared to cancer cells, where 60% reduction at 24 hours and 92% reduction in DNA synthesis at 48 hours are seen after Ad-p73 infection (Fig 4d). More importantly, no apoptosis was detected in Ad-p73-infected HEL 299 cells even after 48 hours (Fig 6c), unlike cancer cells, where 25–35% of cells become apoptotic after 48 hours of Ad-p73 infection (Fig 4c).
In the next experiment, HaCaT cells were infected with either Ad-LacZ or Ad-p73 and at different time points cells were collected and subjected to FACS analysis. Ad-p73 infection led to increased levels of p73 protein in HaCaT cells (Fig 7a). p73 overexpression did not induce apoptosis in HaCaT cells, even though it induced massive apoptosis in HCT116 cells, which are derived from colon carcinoma, in time-dependent fashion (Fig 7b). As expected, Ad-LacZ infection did not induce apoptosis in HaCaT as well as in HCT116 cells (Fig 7b). Taken together from these results, we conclude that cytotoxicity of Ad-p73 is specific to cancer cells.
We show here that Ad-p73, a replication-deficient adenovirus expressing p73β can infect several cancer cells of different tissue types very effectively and express high levels of p73β protein, which is functional as it induces the levels of p21WAF1/CIP1. Ad-p73 is also cytotoxic to all the cell lines tested irrespective of their p53 status. The cytotoxicity of Ad-p73 is due to a combination of both cell cycle arrest and apoptosis induction. Moreover, p73 overexpression sensitized the cancer cells, in particular those harboring mutant p53, to common chemotherapeutic drug adriamycin by many fold. Furthermore, Ad-p73 is found to be not cytotoxic to normal cells suggesting that Ad-p73 is a potential cytotoxic agent specifically against cancer cells.
Mutations in p53 have been observed in about 50% of most human primary tumors.30,31 Most of these mutations are missense mutations producing a faulty protein, which is highly overexpressed. Inactivation of p53 is involved not only in carcinogenesis but also confers chemoresistance. Introduction of wild-type p53 back into tumor cells can result in tumor regression or tumor growth inhibition as p53 can induce cell cycle arrest as well as apoptosis; at the same time, it can also make the tumors sensitive to chemotherapeutic drugs. For these reasons, p53-based gene therapy strategy has been pursued against several types of cancer. However, p53-based gene therapy is not responsive in certain cases where functional inactivation of p53 takes place, like amplification of mdm-2, mutational inactivation of p14ARF and presence of papilloma virus oncogene E6 making p53-mediated tumor suppression inoperative.1,2,3,4,5,6,7,32,33,34 Additionally, in clinical trials, the restoration of wild-type p53 gene does not always lead to tumor regression or tumor growth inhibition.8,9,10 Hence, there exists a need for identification of alternative candidates for effective gene therapy, in particular under conditions where p53 is ineffective.
When p73 was identified, it was reported that p73 is not induced by DNA damage,11 but later on it was found that p73 is indeed induced by a wide variety of DNA-damage-inducing drugs like adriamycin, etoposide, cisplatin, etc.13,14,15,16,17,18 A recent study has also linked chemosensitivity of cancer cells to functional p73 irrespective of p53 status.18 It was shown that specific abrogation of p73 leads to chemoresistance. Moreover, both p73 and p63 (another p53 family member) have been found to play a crucial role in DNA-damage-induced p53-mediated apoptosis.19 It was shown that combined loss of p63 and p73 results in failure of cells containing wild-type p53 to undergo apoptosis in response to DNA damage indicating that possibly p73 and p63 help p53 to transactivate its proapoptotic target genes like PERP, BAX and NOXA. Together, these results suggest that p73 plays a very important role in determining the chemosensitivty of cancer cells.
Our results demonstrate that Ad-p73 can be used to infect variety of cancer cells of different tissue type very effectively as can be seen from the fact that Ad-p73-infected cells show elevated levels of p73β and p21WAF1/CIP1. p73 overexpression is also found to be highly cytotoxic to all the cell lines tested as well as it induced cell cycle arrest and apoptosis. Very importantly, Ad-p73 is able to inhibit the growth of cancer cells regardless of their p53 status. In addition, prior infection with Ad-p73 results in increased chemosensitivity of cancer cells and it is more effective than Ad-p53 in cancer cells harboring mutated p53. This aspect of Ad-p73 has immense clinical significance due to two reasons: (1) there are reports which show that tumor-derived p53 mutants inhibit p53 more strongly as compared to p73 and (2) p53 is mutated in more than 50% of cancers.31,35,36
Another important aspect to be considered while developing a therapeutic agent is its effect on normal cells. Our results show that Ad-p73 infection in normal cells does not induce apoptosis and is only very marginally growth inhibitory to normal cells as it induces cell cycle arrest. Thus our results clearly indicate that Ad-p73 could be a candidate approach to treat human cancers.
We thank Dr Omana Joy for technical assistance in FACS experiments. This study was supported partly by grants from Life Science Research Board, DRDO, and Council of Scientific and Industrial Research (CSIR), Government of India. Funding from ICMR (Center for Advanced studies in Molecular Medicine), DBT (Program support), DST (FIST) and UGC (Special assistance) to Department of Microbiology and Cell Biology is also acknowledged. KS is a Wellcome Trust International Senior Research Fellow. SD, SN, and SA are supported by fellowships from CSIR, Government of India.
multiplicity of infection