Gene Therapy (2005) 12, 417–426 r 2005 Nature Publishing Group All rights reserved 0929-1903/05 $30.00 www.nature.com/cgt p73b-expressing recombinant adenovirus: a potential anticancer agent Sanjeev Das,1 Srikanth Nama,1 Sini Antony,1 and Kumaravel Somasundaram1 1Department of Microbiology and Cell Biology, Indian Institute of Science, Sir CV Raman Road, 560012, India.

Tumor suppressor -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 p73b (Ad-p73). In this study, we evaluated the therapeutic potential of Ad-p73 against a panel of cancer cells (n ¼ 12) of different tissue origin. Ad-p73 infected all the cell lines tested very efficiently resulting in several-fold increase in p73b 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. Cancer Gene Therapy (2005) 12, 417–426. doi:10.1038/sj.cgt.7700803 Published online 28 January 2005

Keywords: p73; adenovirus; cell cycle arrest; apoptosis; chemosensitivity

he p53 encodes a transcription structurally and functionally. p73 can bind to p53 Tfactor, which plays an important role in cell cycle control, responsive elements and upregulate some of the p53 target angiogenesis, apoptosis and genomic stability through the genes suggesting that it has the potential for functional transactivation of its target genes. It is one of the most overlap with p53. When exogenously overexpressed, p73b, frequently inactivated genes in human tumors. Inactivation of one of the p73 isoforms, can transactivate some of the p53 p53 not only results in the development of tumor, it also target genes leading to induction of growth arrest and results in chemoresistance in many types of cancer. Therefore, apoptosis.11,12 Although, initially it was reported that p73 introduction of wild-type p53 gene has been pursued as a is not induced by DNA damage, subsequent studies potential therapeutic strategy against various types of reported that p73 is indeed induced by a wide variety of . This strategy relies on the ability of p53 to induce DNA-damage-inducing drugs like adriamycin, etoposide, cell cycle arrest and apoptosis in cancer cells. However, p53- cisplatin, etc.11,13–18 It was also reported that both p73 and based gene therapy is not suitable in certain cases where p63 (another p53 family member) play a crucial role in functional inactivation of p53 takes place, like amplification DNA-damage-induced p53-mediated apoptosis.19 It was of mdm-2, mutational inactivation of p14ARF making p53 shown that combined loss of p63 and p73 results in failure mediated tumor suppression inoperative.1–7 Moreover, in of cells containing wild-type p53 to undergo apoptosis in clinical trials the restoration of wild type p53 gene does not response to DNA damage. A recent study has also linked always lead to tumor regression or tumor growth inhibition chemosensitivity of cancer cells to functional p73 irrespec- suggesting resistance of some tumors to exogenous p53.8–10 tive of p53 status.18 It was shown that abrogation of p73 p73 is a member of p53 gene family and encodes function in mutant p53 background leads to chemoresis- proteins, which have significant homology with p53 both tance. These results suggest that p73 is a determinant of chemosensitivity and could be used as a candidate for cancer gene therapy. Received May 31, 2004. Address correspondence and reprint requests to: Dr Kumaravel We have recently shown that p73 overexpression by Somasundaram, Department of Microbiology and Cell Biology, infecting cells with a replication-deficient adenovirus Indian Institute of Science, Sir CV Raman Road, Bangalore 560012, expressing p73b specifically inhibits the growth of cancer 20 India. cells expressing human papilloma virus E6 oncogene. E-mail: [email protected] Another independent study demonstrated the potential p73 adenovirus: an anticancer agent S Das et al 418 therapeutic use of p73 expressing adenovirus against addition and 200 ml of DMSO was added to dissolve the colorectal cancer cells.21 In the present study, we formazan crystals and then the absorbance was measured investigated the cytotoxic potential of a replication- at 550 nm in an ELISA reader. deficient recombinant adenovirus expressing p73b (Ad- p73) against a panel of human cancer cell lines of diverse Cell cycle analysis tissue origin. We analyzed the infectivity of Ad-p73 by monitoring the cellular p73 and p21WAF1/CIP1 levels as The single parameter FACS analysis was carried out as described before.28 The dual parameter FACS analysis well as the cytotoxicity against a variety of cancer cells. In 24 addition, we tested the ability of Ad-p73 to induce cell was done as described previously. 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 Results human normal lung fibroblasts and immortalized Ad-p73 infection results in induced p73 and keratinocytes. WAF1/CIP1 p21 expression in human cancer cells We have previously reported the generation of a replica- tion-deficient adenovirus expressing p73b (Ad-p73).20 To Materials and methods explore the therapeutic potential of Ad-p73 against cancer Tumor cell lines and culture conditions cells, we tested the ability of Ad-p73 to infect and express Human cancer cell lines 293 (human embryonic kidney functional p73 protein in a panel of human cancer cell cells transformed with adenoviral fragments), SW480 lines. Different cancer cell lines (n ¼ 12) of different tissue (colon carcinoma), H460 (lung carcinoma), Saos-2 types were infected with Ad-p73 at 10 MOI and the (osteosarcoma), HCT116 (colon carcinoma), U2OS (osteo- expression of p73 was monitored after 24 hours post- sarcoma) and H1299 (lung carcinoma) were described infection. In all cell lines tested, Ad-p73-infected cells previously.22–24 A375 (melanoma), MCF7 (breast carci- showed intense nuclear p73 staining in comparison to a noma), PA-1 (ovarian carcinoma), HepG2(hepatic control adenovirus (Ad-LacZ), which expresses b-galac- tosidase (Fig 1a). We also monitored the induced carcinoma), U373MG (), SkBr3 (breast WAF1/CIP1 carcinoma), C33A (cervical carcinoma) and T47D (breast expression of p73 and its target gene p21 upon carcinoma) were grown in DMEM (Sigma) supplemented Ad-p73 infection by Western blotting. Ad-p73 infected cells showed several-fold higher amounts of p73 and with 10% FBS. Human normal lung fibroblasts (HEL WAF1/CIP1 299) and immortalized keratinocytes (HaCaT) were p21 in comparison to Ad-LacZ infected cells obtained from NCCS, Pune, India. (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. Adenovirus reagents and infections The following adenoviruses were used: Ad-LacZ and p73 overexpression inhibits the growth of cancer cells Ad-p53 lack both E1A and E1B but carries b-galactosi- of varying cell types dase and p53 respectively.25 Ad-p73 lacks both E1A and E1B but carries simian p73b.20 Adenoviral infections were Next we tested the cytotoxic potential of Ad-p73 by carried out as described.26 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 Western and immunohistochemical analysis analyzed by measuring the viability (Fig 2). Ad-p73 Western analysis and immunohistochemical staining were inhibited the growth of all the cell lines very efficiently in performed as described before22 with mouse anti-human comparison to Ad-LacZ (Fig 2). Ad-p73 reduced the p21WAF1/CIP1 (Ab-1; Oncogene), mouse anti-human p73 viability of cells by 60–80% at 20 MOI (Table 1). These monoclonal (Ab-2; Oncogene) and Actin (Ab- 1; Onco- results suggest that Ad-p73 is cytotoxic to all cell lines gene). Cells were harvested or fixed at the indicated time irrespective of the p53 status and Ad-p73 is a potent point as described after virus infection and subjected to inhibitor of cancer cell growth. analysis. Ad-p73 infection induces cell cycle arrest and apoptosis MTT assay Exogenous expression of p73 has been shown to induce MTT assay was carried out as described previously.27 A growth arrest and apoptosis.11,12,20,24,29 In order to study total of 1.5 Â 103 cells per well were plated in a 96-well the mechanism of growth inhibition by p73 in different plate. After 24 hours of plating, the cells were infected cell types, we analyzed the cell cycle profile of four with Ad-LacZ or Ad-p73 at multiplicities of infection different cell lines of different tissue types infected with (MOI) of 2, 10 and 20. MTT (20 ml of 5 mg/ml) was added Ad-p73. HCT116, H1299, HepG2 and A375 cells were 48 hours after infection. MTT is a tetrazolium salt that is infected with either Ad-p73 or Ad-LacZ virus and the converted by living cells into blue formazan crystals. The cells were collected at different time points after infection medium was removed from the wells 3 hours after MTT and subjected to fluorescence-activated cell sorting

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 419 a H1299H460 SW480HCT116Saos2 U2OS PA-1 T-47D MCF7 U373MG HepG2 A375

Ad-LacZ

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b H1299H460 HCT116 SW480Saos2 U2OS PA-1 T-47D MCF7 U373MG HepG2 A375 Ad-LacZ + - + - + - + - + - + - + - + - + - +- + - + - Ad-p73 - + - + - + - + - + - + - + - + - + - + - + - +

p73

p21

Actin

Figure 1 Ad-p73-infected cells express functional p73. The indicated cell lines were infected with either Ad-LacZ or Ad-p73 at an MOI of 10. (a) The cells were fixed after 24 hours and analyzed by immunohistochemical staining for p73. (b) Total cell extract was made after 24 hours and analyzed by Western blotting for p73, p21WAF1/CIP1 and actin proteins.

Table 1 %Viability upon Ad-p73 infection and induction of apoptosis in all the cell lines tested (Fig 3). The proportion of cells actively replicating DNA, Origin and cell line p53 status % Viability at 20 MOI which represent S-phase cells, decreased to less than 50% Colon by 24 hours in all cell lines except HCT116 cells and to HCT116 wt 38 very small levels (less than 5%) by 48 hours in all cell lines SW480 mut 40 infected with Ad-p73 in comparison to Ad-LacZ (Fig 4b, d). Cells with less than 2n content of DNA, which Breast represent apoptotic cells, appeared in significant levels by MCF-7 wt 41 24 hours, which increased to a very high proportion by 48 T-47D mut 22 hours in Ad-p73-infected cells in comparison to Ad-LacZ- Lung infected cells (Fig 4a, c). These results suggest that p73 is a H460 wt 28 potent inducer of cell cycle arrest and apoptosis in cancer H1299 null 26 cells.

Bone Saos2 null 38 Ad-p73 increases the chemosensitivity of cancer cells U2OS wt 25 p73 is an important determinant of chemosensitivity of Brain cancer cells.18 In addition, p73 and p63 has been shown to U-373MG wt 21 be essential for DNA-damage-induced p53-mediated cytotoxicity.19 This raises the possibility that cells over- Melanoma A375 wt 27 expressing p73 may be more sensitive to chemotherapeu- tic drugs. To address this point, different cancer cell lines Liver with varying p53 status were tested for chemosensitivity HepG2 wt 19 to commonly used anticancer drug adriamycin after infection with Ad-p73. Two colon cancer cell lines, Ovarian HCT116 (p53 wt) and SW480 (p53 mutant), two lung PA-1 wt 23 cancer cell lines, H460 (p53 wt) and H1299 (p53 null), a breast cancer cell line SkBr3 (p53 mutant) and a cell line C33A (p53 mutant) were infected with (FACS) (Fig 3). The results were quantified and are 4 MOI of either Ad-p73, Ad-p53 and Ad-LacZ and 6 represented graphically in Figure 4. p73 overexpression hours later increasing concentrations of adriamycin resulted in a time-dependent DNA synthesis inhibition was added. The chemosensitivity was determined by

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 420 H460H1299 A375 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 % Viability % Viability % Viability 20 20 20 0 0 0 0 10 20 02010 02010 MOI MOI MOI U2OS Saos-2 U373MG 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 % Viability % Viability % Viability 20 20 20 0 0 0 02010 02010 02010 MOI MOI MOI MCF7 T-47D HepG2 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 % Viability % Viability % Viability 20 20 20 0 0 0 02010 02010 02010 MOI MOI MOI HCT116 SW480 PA-1 120 120 120 100 100 100 80 80 80 60 60 60 40 40 40 % Viability % Viability % Viability 20 20 20 0 0 0 02010 02010 02010 MOI MOI MOI Figure 2 Ad-p73 inhibits the growth of cancer cells. The indicated cell lines were infected with either Ad-LacZ (filled triangle) or Ad-p73 (filled circle) virus at 2, 10 or 20 MOI. After 48 hours of virus infection, proportion of live cells was quantified by MTT assay as described in Materials and methods. The absorbance of control uninfected cells was considered as 100%.

measuring the cell viability at 48 hours postdrug addition tivity of cancer cells irrespective of p53 status and Ad-p73 is (Fig 5a). IC50 values of these cell lines to adriamycin at more efficient than Ad-p53 in sensitizing the mutant p53 different conditions and the fold change in their IC50 harboring cancer cells to chemotherapeutic drugs. 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 Effect of Ad-p73 on normal cells tested. Both Ad-p73 and Ad-p53 infection increased the To determine if the growth inhibitory actions of p73 were sensitivity of all the cell lines tested to adriamycin by 2–5- specific to the malignant cell lines, HEL 299 cells which fold. However, Ad-p73 increased the chemosensitivity of are normal lung fibroblasts and HaCaT, immortalized cell lines harboring mutant p53 more efficiently than keratinocytes. In the first experiment, HEL 299 cells were Ad-p53 (Fig 5c). Thus the results show that p73 over- infected with varying MOI of Ad-LacZ or Ad-p73 and expression by Ad-p73 infection increases the chemosensi- the cytotoxicity was analyzed by measuring the viability

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 421 a 0 hr 24 hr 48 hr 500 320 320 400 300 240 240 160 160

Number 200 Ad-LacZ Number 100 Number 80 80 0 0 0 0 40 80 120 160 200 04080120 160 200 04080120 160 200 Channels Channels Channels 500 500 400 400 400 300 300 300 200 Ad-p73

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Number 200 Number 100 100 100 0 0 0 04080120 160 200 04080120 160 200 04080120 160 200 Channels Channels Channels c 600 500 280 400 400 210 300 Number Ad-LacZ 140 200 Number 200 100 Number 70 0 0 0 04080120 160 200 04080120 160 200 04080120 160 200 Channels Channels Channels 600 500 280 400 400 210 300

Ad-p73 140 Number

200 Number 200 100 Number 70 0 0 0 04080120 160 200 04080120 160 200 04080120 160 200 Channels Channels Channels

d 500 500 600 400 400 400 300 300 Number Ad-LacZ 200 200 Number 200 Number 100 100 0 0 0 04080120 160 200 04080120 160 200 04080120 160 200 Channels Channels Channels 400 600 360 320

400 270 240

Ad-p73 180 Number 160 200 Number 90 80 0 0 0 04080120 160 200 0 40 80 120 160 200 0 40 80 120 160 200 Channels Channels Channels Figure 3 Induction of cell cycle arrest and apoptosis in Ad-p73-infected cells: (a–d) HCT116, H1299, HepG2 and A375 cells were infected with either Ad-LacZ or Ad-p73 at 10 MOI. The cells were harvested at 0, 24 and 48 hours after infection and subjected to flow cytometry analysis as mentioned in the Materials and methods.

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 422 a 40 infected cells by FACS. HEL 299 cells were infected with either Ad-LacZ or Ad-p73 and at different time points 30 cells were collected and subjected to dual parameter FACS analysis (Fig 6c). Ad-LacZ-infected cells showed 20 no change in cell cycle profile even after 48 hours of virus infection. However, upon infection with Ad-p73, a 35%

% Apoptosis 10 decrease in S phase (34–22%) with a concomitant increase in G1-phase cells could be seen at 24 hours, which further 0 increased to 56% inhibition of DNA synthesis at 48 hours 02448 (34–15%). The kinetics of DNA synthesis inhibition in Time (hrs) HEL 299 cells by p73 is much slower compared to cancer b 40 cells, where 60% reduction at 24 hours and 92% reduction in DNA synthesis at 48 hours are seen after 30 Ad-p73 infection (Fig 4d). More importantly, no apop- tosis was detected in Ad-p73-infected HEL 299 cells even 20 after 48 hours (Fig 6c), unlike cancer cells, where 25–35% of cells become apoptotic after 48 hours of Ad-p73 10

% S phase cells infection (Fig 4c). In the next experiment, HaCaT cells were infected with 0 either Ad-LacZ or Ad-p73 and at different time points 02448 cells were collected and subjected to FACS analysis. Ad- Time (hrs) p73 infection led to increased levels of p73 protein in c 40 HaCaT cells (Fig 7a). p73 overexpression did not induce apoptosis in HaCaT cells, even though it induced massive 30 apoptosis in HCT116 cells, which are derived from colon carcinoma, in time-dependent fashion (Fig 7b). As 20 expected, Ad-LacZ infection did not induce apoptosis in HaCaT as well as in HCT116 cells (Fig 7b). Taken 10 together from these results, we conclude that cytotoxicity of Ad-p73 is specific to cancer cells. 0 02448 Time (hrs) d 40 Discussion

30 We show here that Ad-p73, a replication-deficient adenovirus expressing p73b can infect several cancer cells 20 of different tissue types very effectively and express high levels of p73b protein, which is functional as it induces the 10 levels of p21WAF1/CIP1. Ad-p73 is also cytotoxic to all the % S phase cellsApoptosis % cell lines tested irrespective of their p53 status. The 0 cytotoxicity of Ad-p73 is due to a combination of both 02448 cell cycle arrest and apoptosis induction. Moreover, p73 Time (hrs) overexpression sensitized the cancer cells, in particular those harboring mutant p53, to common chemothera- HCT116 HepG2 peutic drug adriamycin by many fold. Furthermore, Ad- H1299 A375 p73 is found to be not cytotoxic to normal cells suggesting that Ad-p73 is a potential cytotoxic agent specifically Figure 4 Graphical representation of FACS data presented in against cancer cells. Figure 3. %Apoptosis and %S phase cells for Ad-LacZ infection Mutations in p53 have been observed in about 50% of (a and b) and Ad-p73 infection (c and d) after 0, 24 and 48 hours of most human primary tumors.30,31 Most of these mutations infection are shown. are missense mutations producing a faulty protein, which is highly overexpressed. Inactivation of p53 is involved not only in carcinogenesis but also confers chemoresistance. (Fig 6a). Ad-LacZ-infected cells showed no cytotoxicity. Introduction of wild-type p53 back into tumor cells can Ad-p73-infected cells showed some marginal cytotoxcity result in tumor regression or tumor growth inhibition as at higher MOI (75% viability at 20 MOI of Ad-p73 p53 can induce cell cycle arrest as well as apoptosis; at the infection). Ad-p73 infection also led to increased levels of same time, it can also make the tumors sensitive to p73 protein in HEL 299 cells (Fig 6b). To investigate chemotherapeutic drugs. For these reasons, p53-based further, we monitored the cell cycle profile of Ad-p73- gene therapy strategy has been pursued against several

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 423 a 120 120 120

100 HCT116 100 H460 100 H1299 80 80 80 60 60 60

% Viability 40

% Viability 40

% Viability 40 20 20 20 0 0 0 0 0.07 0.14 0.21 0.28 0.35 0 0.05 0.1 0.15 0.2 0 0.07 0.14 0.21 0.28 0.35 Conc. (µg/ml) Conc. (µg/ml) Conc. (µg/ml) 120 120 120 SkBr3 C33A 100 SW480 100 100 80 80 80 60 60 60

% Viability 40

% Viability 40

% Viability 40 20 20 20 0 0 0 0 0.05 0.10 0.15 0.20 0 0.03 0.06 0.09 0.12 0.15 0 0.05 0.10 0.15 0.20 0.25 Conc. (µg/ml) Conc. (µg/ml) Conc. (µg/ml)

Fold change in IC b µ c 50 IC50 ( g/ml) of adriamycin in the presence of of adriamycin p53 Cell Line Cell line status -Ad-LacZAd-p73Ad-p53 Ad-p73 Ad-p53 HCT116 wt 0.23 0.20 0.06 0.04 HCT116 3.3 5.0 H460 wt 0.16 0.14 0.05 0.04 H460 2.8 3.5 H1299 null 0.27 0.25 0.11 0.08 H1299 2.3 3.1 SW480 mut 0.17 0.16 0.06 0.09 SW480 2.7 1.8 SkBr3 mut 0.09 0.09 0.03 0.05 SkBr3 3.0 1.8 C33A mut 0.14 0.12 0.04 0.07 C33A 3.0 1.7 Figure 5 Ad-p73 increases the chemosensitivity of cancer cells. (a) The indicated cell lines were mock infected (filled diamond) or infected with either Ad-LacZ (filled square) or Ad-p53 (filled triangle) or Ad-p73 (filled circle) virus at 4 MOI. Different concentrations of adriamycin was added6 hours postinfection and 48 hours postdrug addition MTT assay was carried out. The different concentrations of adriamycin used were as follows — HCT116: 0.05, 0.15, 0.25 and 0.35 mg/ml, SW480: 0.05, 0.1, 0.15 and 0.2 mg/ml, H460: 0.05, 0.1, 0.15 and 0.2 mg/ml, H1299: 0.05, 0.15, 0.25, and 0.35 mg/ml, SkBr3: 0.025, 0.05, 0.10 and 0.15 mg/ml and C33A: 0.05, 0.1, 0.15 and 0.25 mg/ml. (b)IC50 values for adriamycin under different condition tested above for all the cell lines are given. (c) Fold change in IC50 values for adriamycin in presence Ad-p73 or Ad-p53 are shown.

types of cancer. However, p53-based gene therapy is not was shown that combined loss of p63 and p73 results in responsive in certain cases where functional inactivation of failure of cells containing wild-type p53 to undergo p53 takes place, like amplification of mdm-2, mutational apoptosis in response to DNA damage indicating that inactivation of p14ARF and presence of papilloma virus possibly p73 and p63 help p53 to transactivate its oncogene E6 making p53-mediated tumor suppression proapoptotic target genes like PERP, BAX and NOXA. inoperative.1–7,32–34 Additionally, in clinical trials, the Together, these results suggest that p73 plays a very restoration of wild-type p53 gene does not always lead important role in determining the chemosensitivty of to tumor regression or tumor growth inhibition.8–10 cancer cells. Hence, there exists a need for identification of alternative Our results demonstrate that Ad-p73 can be used candidates for effective gene therapy, in particular under to infect variety of cancer cells of different tissue type conditions where p53 is ineffective. very effectively as can be seen from the fact that When p73 was identified, it was reported that p73 Ad-p73-infected cells show elevated levels of p73b and is not induced by DNA damage,11 but later on it p21WAF1/CIP1. p73 overexpression is also found to be was found that p73 is indeed induced by a wide variety highly cytotoxic to all the cell lines tested as well as it of DNA-damage-inducing drugs like adriamycin, etopo- induced cell cycle arrest and apoptosis. Very importantly, side, cisplatin, etc.13–18 A recent study has also Ad-p73 is able to inhibit the growth of cancer cells linked chemosensitivity of cancer cells to functional regardless of their p53 status. In addition, prior infection p73 irrespective of p53 status.18 It was shown that with Ad-p73 results in increased chemosensitivity of specific abrogation of p73 leads to chemoresistance. cancer cells and it is more effective than Ad-p53 in cancer Moreover, both p73 and p63 (another p53 family cells harboring mutated p53. This aspect of Ad-p73 has member) have been found to play a crucial role immense clinical significance due to two reasons: (1) there in DNA-damage-induced p53-mediated apoptosis.19 It are reports which show that tumor-derived p53 mutants

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 424

Figure 6 Effect of Ad-p73 on HEL 299 cells: (a) HEL 299 cells were infected with either Ad-LacZ (filled triangle) or Ad-p73 (filled circle) virus at 2, 10 or 20 MOI. After 48 hours of virus infection, proportion of live cells was quantified by MTT assay as described in Materials and methods. The absorbance of control-uninfected cells was considered as 100%. (b) HEL 299 cells were infected with either Ad-LacZ or Ad-p73 at 10 MOI. The cells were fixed after 24 hours and analyzed by immunohistochemical staining for p73. (c) HEL 299 cells were infected with either Ad-LacZ or Ad-p73 at 10 MOI. The cells were allowed incorporate BrdU during the last fours of the indicated time points. After BrdU incorporation, the cells harvested at 0, 24 and 48 hours of virus infection and subjected to dual parameter flow cytometry analysis as mentioned in the Materials and methods. The quantified values for cells in G1 phase (%G1), S phase (%S) and G2 phase (%G2) are given at the bottom of the figure.

Cancer Gene Therapy p73 adenovirus: an anticancer agent S Das et al 425 a Ad-LacZ Ad-p73 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.

References b 70

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