The tumor suppressor activity induced by adenovirus - Nature

Gene Therapy (2006) 13, 235–244 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt

ORIGINAL ARTICLE

The tumor suppressor activity induced by adenovirusmediated BRCA1 overexpression is not restricted to breast cancers D Marot1,2, P Opolon1, S Brailly-Tabard2, N Elie3, V Randrianarison4, E Connault1, N Foray5, J Feunteun4 and M Perricaudet1 UMR 8121 CNRS, Vectorologie et Transfert de ge`nes, Institut Gustave Roussy, Villejuif Cedex, France; 2Laboratoire d’Hormonologie et Biologie molećulaire, CHU de Biceˆtre, Le Kremlin- Biceˆtre, France; 3Laboratoire d’Imagerie du Petit Animal, Institut Gustave Roussy, Villejuif Cedex, France; 4UMR 8125, Geńe´tique Oncologique, Institut Gustave Roussy, Villejuif Cedex, France and 5U647 INSERM, Radiation Synchrotron et recherche me´dicale, European Synchrotron Radiation Facility, Grenoble, France 1

The BRCA1 (breast cancer 1) breast cancer susceptibility gene is recognized as responsible for most familial breast and ovarian cancers and is suggested to be a tissue-specific tumor suppressor gene. In this report, we investigated the tissue specificity of tumor inhibitory activities induced by a recombinant adenovirus coding for wild-type BRCA1 (wtAdBRCA1). We demonstrated a pronounced in vitro antiproliferative effect on H1299 lung and HT29 colon cells upon infection with AdBRCA1. We describe a prolonged G1 cell cycle arrest associated with a decrease in the hyperphosphorylated form of Rb, suggesting that the Rb/E2F pathway is implicated in BRCA1-induced cell growth arrest. We also

observed a significant antitumor effect in these pre-established subcutaneous tumors after in situ delivery of AdBRCA1, although these two tumors do not express wt p53, and also estrogen a and b, progesterone and androgen receptors. Moreover, BRCA1 can induce a strong prolonged cell cycle arrest and apoptotic cell death but no significant antiangiogenic effect in H1299 tumors. Finally, our data indicate that intratumor administration of wtAdBRCA1 significantly inhibits growth of lung and colon steroid hormoneindependent tumors. Gene Therapy (2006) 13, 235–244. doi:10.1038/sj.gt.3302637; published online 6 October 2005

Keywords: BRCA1; ERa; AR; angiogenesis; adenovirus

Introduction BRCA1 (breast cancer 1) mutations are responsible for most hereditary breast cancers1–3 and for almost all the hereditary breast and ovarian cancer syndromes,4 suggesting that BRCA1 gene is a tissue-specific tumor suppressor gene. BRCA1 is known to be involved in a variety of biological functions including transcription regulation, X chromosome inactivation, ubiquitination and maintenance of genome integrity.5,6 The role of BRCA1 in DNA repair is also consensually recognized, notably as a scaffold protein via its interaction with Rad51,7 Rad50/MRE11/NBS1,8 ATM,9 and BLM.10 It is also noteworthy that BRCA1 is a substrate for several DNA damage-induced kinases that are activated by a wide spectrum of genotoxic agents. In particular, the phosphorylation of BRCA1 is a sensor of DNA damage in response to ionizing radiation, UV exposure and treatment with alkylating agents.9,11–13

Correspondence: D Marot or M Perricaudet, UMR 8121 CNRS, Vectorologie et Transfert de ge`nes, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France. E-mail: [email protected] Received 9 February 2005; revised 26 July 2005; accepted 1 August 2005; published online 6 October 2005

Besides its involvement in stress signaling pathways, a number of authors have reported that BRCA1 is a transcription regulator dependent on p5314,15 and, more recently, upon the estrogen receptor a (ERa).16,17 BRCA1 is also involved in the G1/S checkpoints in a p21WAF1-18,19 and/or a Rb/E2F-dependent manner.20–22 Furthermore, wild-type (wt) BRCA1 overexpression increases cell death during cellular stress, notably that induced by serum deprivation or genotoxic stress.23,24 However, despite a considerable amount of data, the molecular mechanisms of tumor growth inhibition induced by BRCA1 remain to be investigated further. The antitumor activity of BRCA1 was demonstrated only in breast, ovarian and prostate tumors transplanted in nude mice,25–29 raising therefore the question whether the ubiquitinously expressed BRCA1 protein may regulate the proliferation of cancer cells from other tissues. Here, we have investigated the potential direct or indirect antiproliferative effect of an adenovirus (Ad)mediated expression of wtBRCA1 in lung and colon tumor models. Our observations showed that a decrease in the cell viability and clonogenic cell survival follows an in vitro infection with a recombinant Ad expressing the wtBRCA1 gene (wtAdBRCA1). Moreover, the Admediated BRCA1 delivery was seen to be associated with a strong G1 arrest dependent on the Rb/E2F pathway but independent of the expression of ERa and b (ERb),

Tumor suppressor activity induced by adenovirus-mediated BRCA1 D Marot et al

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dures described in our previous reports (Figure 1a). Two independent tests were thereafter applied to H1299 and HT29 cells to examine whether Ad-mediated wtBRCA1 expression influences the growth of lung and colon cells in vitro: the cell viability test and the clonogenic survival assay. Since about 90% of the reported BRCA1 germline mutations in the hereditary breast and ovarian cancers result in truncated proteins, we endeavored to focus here, as a first step, on the effect of C-terminal-truncated BRCA1.30 First, 25 000 H1299 and HT29 cells were infected with several Ad at a multiplicity of infection (MOI) of 600 plaque-forming units (PFU)/cell and the yield of viable cells was counted by an exclusion dye test (MTT (3-4,5dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide)) 24, 48 and 72 h postinfection (Figure 1c). A significant

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progesterone (PR) and androgen receptors (AR). We also demonstrated that intratumor administration of wtAdBRCA1 significantly inhibits growth of two lung and colon tumors pre-established in nude mice. Interestingly, we observed that wtAdBRCA1 overexpression did not induce any significant antiangiogenic effect in these steroid hormone-independent tumors.

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Figure 1 wtBRCA1 overexpression mediated by recombinant Ad inhibits the proliferation of ER- and AR-negative tumor cells. (a) Determination of optimal infection in H1299 and HT29 cells. In all, 200 000 H1299 and HT29 cells were infected with Ad LacZ at an MOI of 50, 100, 200, 400, 600 and 1000 PFU/cell and the optimal infection (MOI) was determined by staining for b-galactosidase activity 24 h postinfection. (b) Expression of different forms of BRCA1 after infection with recombinant Ad. At 48 h postinfection of H1299 and HT29 cells, 100 mg of nuclear extract were immunoblotted, as described by Randrianarison et al.29: 1. untreated cells; 2. AdCO1; 3. wtAdBRCA1; and 4. truncated AdBRCA1. (c) Effect on proliferation in response to infection with wtAdBRCA1. In all, 25 000 H1299 and HT29 cells were infected with different Ad at an MOI of 600 PFU/cell and the number of cells was measured by a colorimetric test (MTT) 24, 48 and 72 h postinfection. (d) Expression of BRCA1 and steroid hormone receptors in different cell lines used. Nuclear extracts (100 mg) from H1299 and HT29 cell lines were immunoblotted for BRCA1, as described by Randrianarison et al.,29 and for PR, AR, ERa and ERb. We used as positive controls for AR (MDA-Pca-2b cells), for ERa, ERb and PR (MCF7 cells), and for BRCA1 (MCF7 and IGROV1, a BRCA1-heterozygous cell line60), respectively. (e) Expression of ER, PR and AR postinfection with wtAdBRCA1. H1299 and HT29 cell lines were infected with Ad at an MOI of 600 PFU/ cell 2 days earlier. Nuclear cell extracts (100 mg) were separated by 4–12% Tris-Glycine gel electrophoresis, transferred onto a nitrocellulose filter and immunoblotted for PR, AR, ERa and ERb . We used, respectively, T47D cells for ERa, ERb and PR, and MDA-Pca-2b cells for AR as positive controls. Gene Therapy


Overexpression of BRCA1 induces a G1 cell cycle arrest in HT29 and H1299 tumor cells We examined thereafter the effect of Ad-mediated BRCA1 overexpression on cell cycle phase distribution. HT29 and H1299 cells were infected with Adp21WAF1, wtAdBRCA1 or truncated AdBRCA1 at an MOI of 600 PFU/cell and subsequently investigated cellular content by cytometry analysis (Figure 3a). The percent of G1-arrested cells infected with wtAdBRCA1 was 78 and 70% in HT29 and H1299 cells, respectively. The cell cycle profile of wtAdBRCA1-infected cells showed a clear increase in the G1 phase compared to that of AdCO1-infected and uninfected cells. Ad-mediated BRCA1 expression leads to an accumulation of G1arrested cells in both cell types. This effect was more pronounced in HT29 cells. Since Rb phosphorylation was shown to be a sensor of cell cycle modification, we examined the different forms of Rb protein following the wtAdBRCA1 infection.32,33 In the HT29 and H1299 control cultures, the Rb protein was present both as phosphorylated and unphosphorylated forms. After infection with wtAdBRCA1 at an MOI of 600 PFU/cell, we observed a significant increase of Rb

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decrease in proliferation was observed in the two tumor lines infected with wtAdBRCA1 compared to cells infected with truncated AdBRCA1 (Ad1025) and with the Ad control (AdCO1). It is noteworthy that the BRCA1-induced antiproliferative activity appeared to be nearly as efficient as that obtained with Ad-mediated delivery of the well-characterized antiproliferative agents, p53 and p21WAF1 (Figure 1c). The expression levels of wt or truncated forms of BRCA1 were detected by Western blot 48 h postinfection (Figure 1b). Unlike MCF7 breast tumor cells, the two cell lines (H1299, HT29) expressed neither the ERa and ERb nor the PR (Figure 1d). Furthermore, H1299 and HT29 cells did not display any significant expression of AR as compared to the MDA-Pca-2b prostate cell line31 (Figure 1d). It is also noteworthy that the expression of ERa and ERb, PR and AR was not activated following the expression of BRCA1 in HT29 and H1299 cells (Figure 1e). Secondly, the clonogenic survival of these cancer cell lines was examined after infection with wtAdBRCA1. H1299 and HT29 cells, plated at a density of 2 103 cells per six-well plates, were infected with recombinant Ad at an MOI of 1000 PFU/cell. After 10 or 14 days in culture, colonies were scored. Figure 2a showed six-well plates of two cell lines infected with various Ad after fixation and crystal-violet staining. We observed almost complete growth arrest after overexpression of wtBRCA1, 10 days postinfection in H1299 and 14 days in HT29 cells (Figure 2b). We noted a 90% decrease in clonogenic survival for H1299 cells. Despite the very large number of small colonies observed in HT29 cells, six-well plates also showed strong growth inhibition of wtAdBRCA1infected cells (Figure 2a). No significant effect was induced in cells infected with the truncated BRCA1expressing Ad vector. Again, such inhibition was similar to that obtained upon infection with Adp21WAF1 (Figure 2b). Altogether, our data indicate that Ad-mediated BRCA1 overexpression may lead to a loss of clonogenic capacities of lung and colon tumor cells, independently of the sex steroid hormone pathways.

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Figure 2 wtBRCA1 overexpression mediated by recombinant Ad inhibits clonal survival of ER- and AR-negative tumor cells. For the colony formation assay, 2 103 (H1299, HT29) cells were seeded in six-well culture plates and were infected with different Ad at 1000 PFU/cell: 1. untreated cells; 2. AdCO1; 3. wtAdBRCA1; 4. truncated AdBRCA1; 5. Adp21WAF1; and 6. Adp53. At 10–14 days postinfection, the cells were fixed and stained with 0.2% crystal violet for 20 min. The decrease in clonogenic survival after wtAdBRCA1 infection was observed in six-well culture plates in photos (a). The H1299 colonies in panel a were scored and plotted as a percentage of the number of colonies in the mock-infected well. Quantification of colony outgrowth was represented by histograms (b). This experiment was repeated twice.

overall. Such increase concerned essentially the unphosphorylated form of Rb (Figure 3b).

Overexpression of BRCA1 downregulates human telomerase reverse transcriptase (hTERT) expression in H1299 and HT29 tumor cells A number of reports showed that telomerase, an enzyme that maintains telomere length, plays major roles in cell immortalization and cancer progression. The large majority of cancer cell lines exhibit high telomerase activity that may help a continuous cell proliferation.34,35 Recently, Xiong et al.36 described a BRCA1-dependent inhibition of the telomerase activity in breast and prostate tumor cells and suggested that this phenomenon may contribute to the BRCA1 tumor suppressor activity. Human tumors express considerable heterogeneity in Gene Therapy


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Rb (105kDa) p21WAF1 (21kDa) Actin (43kDa) Figure 3 wtBRCA1 overexpression induces G1 cell cycle arrest in lung and colon tumor cells. (a) Cell cycle distribution of HT29 and H1299 cells after infection with wtAdBRCA1. HT29 and H1299 cells were infected with different Ad (AdCO1, Adp21WAF1, wtAdBRCA1, truncated AdBRCA1) at 600 PFU/cell 2 days earlier. Cells were treated, as indicated in Materials and methods, for flow cytometry analysis. The effects of Ad-mediated delivery of wtBRCA1 on the cell cycle distribution of HT29 and H1299 cells were illustrated by 10 histograms and the percent of cell cycle phases was reported in the table. (b) Expression of Rb protein in response to infection with wtAdBRCA1. The Rb protein level in HT29 and H1299 cells after infection with wtAdBRCA1 was determined by Western blot. Nuclear protein extracts from cells infected with Ad (AdCO1, wtAdBRCA1, truncated AdBRCA1) at an MOI of 400 PFU/cell were separated by 4% Tris-Glycine gel electrophoresis and immunoblotted for BRCA1 and Rb.

telomere lengths,37 whereas human telomerase reverse transcriptase (hTERT) is known to be a rate-limiting factor for assembly of a functional telomerase complex and its expression is primarily regulated at transcriptional level (see reviews in Dong et al.38). Moreover, the hTERT expression is correlated with telomerase activity in H1299 and HT29 cells.39,40 Hence, in order to know whether wtBRCA1 overexpression impacts upon telomere-specific regulation, Gene Therapy

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we have deliberately chosen to assess the mRNA and protein expression of the TERT (catalytic subunit of mammalian telomerase holoenzyme) after Ad infection since this end point is more stable and represents a common feature of tumor tissues. The mRNA and protein expression of TERT were evaluated using RT-PCR and Western blotting, respectively. In contrast with HT29 and H1299 cells infected by Ad control (AdCO1) or truncated AdBRCA1, the wtAdBRCA1-infected cells showed decreased mRNA and protein levels of hTERT (Figure 4a and b). In the same conditions, p53 delivered by the same vector induced a decrease of hTERT protein (Figure 4b). Furthermore, note also that both H1299 and HT29 tumor cells lines are p53 mutated as indicated in Materials and methods, and Ad-mediated BRCA1 expression did not impact upon the p53 yields (Figure 4b).

Overexpression of BRCA1 inhibits lung and colon tumor growth in vivo Highly proliferative HT29 colon tumors (75 mm3) established in nude mice were injected in three consecutive doses of Ad every 4 days. We observed a shrinkage in tumor growth (about 40%, 21 days after injection of wtAdBRCA1 (Figure 5a). This effect was not found following the infection with the Ad control (AdCO1) nor


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with truncated AdBRCA1 (Ad1025) (Figure 5a). The antiproliferative effect of the Ad-mediated BRCA1 expression was found to be similar to that obtained with Ad expressing the wt p53 protein (Figure 5a). Calibrated H1299 lung tumors (100 mm3) were injected under the same conditions as described above (Figure 5b). We observed a decrease in the tumor growth rate after the injection of wtAdBRCA1. At 21 days postinfection, the ratio of tumor sizes between wtAdBRCA1-treated tumors and AdCO1 control-treated tumors was 62% on average. The administration of Ad producing a C-terminal-truncated BRCA1 protein (Ad535) had no effect on tumor growth (Figure 5b). The antitumor effect mediated by wtAdBRCA1 in HT29 and H1299 tumors was found to be statistically different from that of truncated AdBRCA1 and AdCO1 treatment (Po0.05, test ANOVA). Figure 5d showed the representative histopathological slides of H1299 and HT29 tumor samples over a period of approximately 3 weeks. The growth rate of tumors injected with wtAdBRCA1 was consistently

reduced compared to that of tumors injected with truncated AdBRCA1 or control vectors. A strong antitumor growth effect was also obtained after infection with Ad vectors expressing wt p53 (Figure 5d). There was no evidence of any significant enhancement of necrosis to account for the tumor shrinkage due to the BRCA1-induced antiproliferative effect. In H1299 tumors, the proliferation and the activation of apoptosis were investigated by the Ki-67 expression (Figure 6a–d) and by the percentage of apoptotic cells (Figure 6e–h), respectively. The proliferation (Ki-67 immunostaining) and the apoptotic index (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling method (TUNEL) analysis) were scored as described previously41,42 and reported in the table in Figure 6B. The fact that Ki-67 is absent from resting cells (G0), but is present during all active phases of the cell cycle (G1, S, G2 and mitosis), makes it an excellent marker for determining the so-called growth fraction of a given cell population.43 We have detected a Gene Therapy


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and an increased apoptotic cell number (2.5 and 8%) in wtAdBRCA1- and Adp53-infected cells, respectively. Hence, a decreased cell proliferation accompanied by an increased apoptosis was observed in H1299 wtAdBRCA1-treated tumors (Figure 6), whereas it was not the case in HT29 tumors (data not shown). Finally, these data were in agreement with the rapid doubling time of HT29 cells observed in our in vitro experiments and may explain the more modest effect on HT29 tumors (size attaining 2500 mm3) compared to H1299 tumors (size attaining 1200 mm3) (Figure 5a and b). One recent report showed ERa-dependent downregulation of VEGF-A transcription by BRCA1 overexpression in breast cancer cells suggests that BRCA1 may be involved in hormone-dependent cancer vascularization.17 Hence, we tried to determine whether wtAdBRCA1 expression has an impact on intratumor vascularization in a hormone-independent context. No significant difference in the number of capillaries and the VEGF-A level was observed between the H1299 and HT29 wtAdBRCA1-treated tumors and the controls (data not shown). Altogether, these findings suggest that no significant antiangiogenic effect was induced by Admediated BRCA1 expression in steroid hormoneindependent tumors.

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Figure 6 Ki-67 expression and apoptotic marker in H1299 tumors. (A) Representative images of HT29 tumors were shown for Ki-67 expression immunostaining (a–d) and TUNEL analysis (e–h). The tumor sections were treated as described in Materials and methods. Control tumors showed high-level Ki-67 expression (a and b) and low apoptotic cells (e and f). In contrast, the tumors treated by wtAdBRCA1 and Adp53 showed low-level Ki-67 expression (c and d) and apoptotic cells (g and h), respectively. (B) Quantification of proliferation and apoptosis index. These two indexes were represented by percentage of stained area in cancer cell compartment and by score of positive cells (TUNEL analysis) per 1000 consecutive cells ( 200) as described in Materials and methods, respectively.

high-level Ki-67 expression (36–98%) and a low number of apoptotic cells (0.1%) in H1299 control tumors and trAdBRCA1-treated tumors. In contrast, we have determined a low-level Ki-67 expression (7–13% and 5–10%) Gene Therapy

That BRCA1 is a tumor suppressor gene with a specific role in breast and ovary tumor development is supported by a whole body of evidence.44,45 The BRCA1 protein is involved in mammary gland formation46,47 and also requires estrogen-dependent tissue differentiation.48 The fact that BRCA1 plays an inhibitory role in estrogensignaling pathways17,49 and that BRCA1-deficient tumors are mostly ERa negative50,51 could have suggested a strong tissue specificity of the antiproliferative capacity of BRCA1. Hence, since BRCA1 protein is expressed ubiquitously, it was worth examining whether the Admediated BRCA1 overexpression inhibits the growth of tumors different from breast, ovary and prostate cancers. For the sake of independence toward tissue specificity and steroid hormone pathways, we have deliberately chosen colon (HT29) and lung (H1299) tumors. These tumors do not express ERa and ERb, PR nor AR, and BRCA1 is not involved in colorectal and lung tumorigenesis.52,53 We observed a significant growth arrest upon expression of wtAdBRCA1 in lung and colon cell lines and this growth-inhibitory effect is similar to that obtained with the expression of Adp53 or Adp21WAF1, two wellcharacterized antiproliferative agents (Figures 1 and 2). These findings are in agreement with our previous data obtained in two breast tumor cell lines, MCF7 and HCC1937.29 It is noteworthy that the H1299 lung and HT29 colon tumor lines lack expression of p53 protein (homozygous deletion; Mitsudomi et al.54) and express a mutant p53 protein (R273H; Cottu et al.55), respectively. Consequently, the p53-dependent pathways may not impact directly upon the BRCA1-induced antiproliferative effects observed here. Although such BRCA1-induced growth arrest has been already reported in in vitro experiments including in our own report,29 such biological effect remained to be investigated further. BRCA1 is shown to play a role in


the G1/S transition via the p21WAF1 18,19 or via Rb/E2F pathway.20–22 Here, we described a strong G1 arrest of HT29 and H1299 cells 2 days postinfection with wtAdBRCA1 (Figure 3). However, in accordance with our previous data,29 and in a p53-mutated context, we have not observed any significant change in the p21WAF1 protein level upon expression of BRCA1 (Figure 3c). By contrast, the cells infected by wtAdBRCA1 did show a significant decrease in the hyperphosphorylated form of Rb. Hence, our results strongly suggest that the Ad-mediated wt BRCA1 expression is able to arrest cycle in the G1 phase via the Rb/E2F pathway in lung and colon cells, like in ovarian cancer cell lines as reported in our previous data.29 Furthermore, our findings show that estrogen- or androgen-signaling pathways are not necessarily implicated in these antiproliferative activities of BRCA1. We also explored the capacity of wtBRCA1 to interfere with the tumorigenicity of these pre-established steroid hormone receptor-negative H1299 and HT29 tumors after intratumor delivery of the AdBRCA1 vector. Unlike that seen with control Ad and truncated AdBRCA1, a significant growth arrest was observed in colon and lung tumors treated with wtAdBRCA1. The ratio of H1299 and HT29 tumor size between wtAdBRCA1-treated tumors and Ad control-treated tumors were 62 and 40% on average, respectively. Such conclusions are similar to those obtained in our previous report performed with two breast and ovarian tumors.29 Interestingly, the wtAdBRCA1 treatment of colon and lung tumors is as effective as Adp53 treatment (Figure 5a). Despite the description of various activities associated with BRCA1 in vitro, it is noteworthy that its antitumor activity is only demonstrated in breast, ovarian and prostate tumors transplanted in nude mice.25–29 In this report, however, our data show that the BRCA1 antitumor activity is not tissue specific and is not necessarily dependent of functional estrogen and androgen pathways. We also showed that multiple intratumor administrations of wtAdBRCA1 induce a G0 quiescence in a large fraction of H1299 lung cells as revealed by the Ki-67 immunostaining. In parallel, we observed that Admediated wtBRCA1 overexpression inhibits growth and formation of colonies 10–14 days postinfection (Figure 2), and also induces a decrease of hTERT expression in lung and colon tumor cells 24–72 h postinfection (Figure 4). Interestingly, recent reports showed that BRCA1 strongly inhibits telomerase enzymatic activity and induces telomere shortening in human prostate and breast cancer cell lines.36,56 It is also noteworthy that HT29 and H1299 cells do not express p16INK4A protein and wt p53 protein involved in senescence pathways.54,55,57,58 Such G0 arrest may therefore contribute to the BRCA1 tumor suppressor activity without necessarily triggering senescence. Altogether, our findings suggest therefore that high level of BRCA1 protein induced by adenoviral infection may contribute to strong prolonged cell cycle arrests independently of the steroid hormone status and the tissue origin. Moreover, we investigated whether wtBRCA1 overexpression has an impact on intratumor vascularization in a hormone-independent context. No significant difference in the VEGF-A level and the number of capillaries is observed in H1299 and HT29 tumor

sections (data not shown) despite published data showing that ERa-dependent downregulation of VEGFA transcription by BRCA1 in breast cancer cells.17 Such findings demonstrate therefore that BRCA1 overexpression in steroid hormone-independent tumors induces cell cycle arrest, senescence and/or apoptosis but has no significant antiangiogenic effect. The Ad-mediated BRCA1 antitumor activity is not restricted to breast cancers and may be applied independently of the steroid hormone status and the tissue origin. A logical extension of this study would be to explore the efficiency of combination of Ad-mediated BRCA1 gene transfer with a chemotherapeutic agent such as paclitaxel in relevant xenografted tumor protocols

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Materials and methods Cell cultures and recombinant Ad H1299, HT29, MCF7, T47D and IGROV-1 cells were maintained in red phenol-free DMEM medium (Life Technologies Inc.). PC3 and MDA-Pca-2b prostate tumor cells were grown in F12 medium (Life Technologies Inc.) and BRFF-HPC1 medium (Biological Research Faculty and Facility Inc.), respectively. All media were supplemented with 2 mM L-glutamine, and 10% fetal calf serum, 100 IU/ml penicillin and 100 mg/ml streptomycin (Gibco, Paisley, UK). Cells were cultured in phenol-free medium containing 6% charcoal dextran-stripped serum for the in vitro tests. The status of H1299 and HT29 is BRCA1wt/wt, p53mutant/mutant, and these two cell lines do not express p16INK4A protein.54,55,57,58 All the Ad vectors used throughout this study were nonreplicative E1/E3-defective recombinant Ad. AdCO1 and AdRSVbgal were control viruses carrying, respectively, no insert or the Escherichia coli LacZ transgene (bgal). Adp53, Adp21WAF1 wtAdBRCA1 and two Ad expressing a C-terminal-truncated BRCA1 gene (Ad535 and Ad1025) producing, respectively, a protein deleted for 542 and 464 C-terminal amino-acid residues were described previously.29 The optimal infection (MOI) was determined by staining for b-galactosidase activity after infection with Ad LacZ. For HT29 and H1299 cells, 600 PFU/cell was determined as an optimal compromise between a maximal efficiency of infection and a minimal virus-induced cytopathic effect. Antibodies and Western blot analysis Nuclear protein extract (100 mg) was analyzed for BRCA1 and steroid hormone receptor status by 4 or 4–12% TrisGlycine gel electrophoresis followed by Western blot analysis. Primary antibodies against BRCA1 (OP92), Rb (OP136), hTERT (K-370), AR (PC167) and ERb (GR40) were purchased from Oncogene Research Products (Cambridge, UK), antibodies against ERa (HC-20) and actin (I-19) from Santa Cruz Biotechnology (California, USA) and the monoclonal antibody against PR (Let126) was provided by JF Savouret, PhD.59 The secondary peroxidase-conjugated goat anti-mouse IgG was from Jackson Immunoresearch Laboratories (West grove, PA, USA), the donkey anti-rabbit IgG (Na934) was from Amersham Life Sciences (BuckinGene Therapy


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ghamshire, UK) and the donkey anti-goat IgG was from Santa Cruz Biotechnology (California, USA).

RT-PCR analysis Total RNAs were isolated by TRIZOL (Invitrogens), quantified by UV 260/280 nm absorption ratio and analyzed by RT-PCR. Total RNA (1 mg) was reverse transcribed by utilizing MuLvRT (Perkin-Elmer) with conditions recommended by the manufacturer. The PCR amplification conditions used were one cycle for 2 min at 941C and 25 cycles at 941C for 30 s, 551C for 30 s, 721C for 30 s and one cycle for 10 min at 721C, and 100 ng of cDNA was used. The human TERT primer sequences used were described previously.36 The primers for the control gene human b-actin were as follows: sense, 50 -GAAGCATTTGCGGTGGACCAT-30 and antisense, 50 -TCCTGTGGCATCCACCAAACT-30 (257 bp). PCR products were analyzed by electrophoresis through 2% agarose gels containing ethidium bromide, and gels were photographed under UV light. Cell viability The viral infection was carried out in triplicate in 500 ml of red phenol-free medium containing 6% charcoal dextran-stripped serum for 4 h at an MOI of 600 PFU/ cell. After infection of 25 000 H1299 and HT29 cells with different Ad, cell survival was measured by the quantitative colorimetric MTT test. After infection, the number of viable cells was determined after 24, 48 and 72 h. The cells were incubated for 4 h at 371C with 500 ml of PBS and 50 ml of a 5 mg/ml MTT solution. The cells were lysed overnight with 500 ml of lysis buffer (10 g of SDS in 50 ml DMF/H2O 1:1, pH 4.7). A measure of 200 ml of the soluble fraction was transferred into 96-well microplate and the 570-nm optical density was measured. Clonogenic assay for cell survival after infection Cells were seeded at a density of 2 103 (H1299 and HT29) cells per well in six-well culture plates and were infected with the different Ad (AdCO1, wtAdBRCA1, truncated AdBRCA1 (Ad1025), Adp21WAF1 wt Adp53) at an MOI of 1000 PFU/cell. After 10–14 days, the cells were fixed and stained with 0.2% crystal violet for 20 min. The colonies were scored and plotted as a percentage of the number of colonies compared to mock-infected cells. A lower number of colonies indicated inhibition of cell growth. Cell cycle assays Cells infected with different Ad (AdCO1, Adp21WAF1 wtAdBRCA1, truncated Ad BRCA1 (Ad1025)) at an MOI of 600 PFU/cell were collected 2 days postinfection, washed twice in PBS and fixed with 70% ethanol at 201C. After centrifugation at 4000 r.p.m. and two washes in PBS, cells were suspended in PBS containing 100 mg/ml of RNase A and 50 mg/ml of propidium iodide. A total of 10 000 cells was analyzed by flow cytometry. Tumorigenicity in nude mice All of the operative procedures and animal care were in conformity with the Guidelines of the French Government (decree 87–848 of October, the 19th 1987, license 7593 and A38071). The capacity of wtBRCA1 to interfere Gene Therapy

with tumor growth in vivo was assayed in the two settings. Tumors were generated by subcutaneous injection of 2.5 106 H1299 or HT29 cells into the dorsa of 6-week-old nude mice. Tumors of approximately 50–100 mm3 in size were injected three times at 4-day intervals with 2 109 PFU of recombinant Ad. In each setting, tumor sizes were monitored weekly. Comparisons of the mean tumor size between the animal groups were analyzed by the ANOVA test of covariance.

Histopathological and immunohistochemical analysis Tumor tissues were fixed in ethanol and embedded in paraffin. Sections (5 mm) treated with toluene and rehydrated were microwaved three times for 5 min in 10 mM citrate buffer (pH 6.0) and quenched by 3% H2O2 for 5 min. Slides were stained with hematoxylin–eosin. Expression of Ki-67 was analyzed by immunohistochemistry. Quantification of stained nuclei (stained blood vessels) on histological slides was achieved using PixCyt, a software package for computer-assisted image analysis, designed by the Groupe Re´gional d’Etudes sur le Cancer, Centre François Baclesse, Caen (Dr Paulette Herlin). Signal evaluation was performed after digitization of the whole histological slide using a dedicated scanner.41 Apoptotic cells within sections were detected by a kit using a TUNEL (Boehringer Mannheim). The number of apoptotic cells was scored for each tumor (three 200 fields of view) as described.42

Abbreviations Ad, adenovirus; ERa or b, estrogen receptor a or b; AR, androgen receptor; MOI, multiplicity of infection.

Acknowledgements We thank Jeannine Cabannes for her technical assistance, Patrice Ardouin for animal care and Jean François Savouret (Paris) for the gift of the PR antibody. We also thank Lorna Saint Ange for editing. This work is supported by CRC contracts from the Institut GustaveRoussy.

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