P73 Overexpression Is Associated with Resistance to Treatment With

[CANCER RESEARCH 61, 935–938, February 1, 2001] Advances in Brief p73␣ Overexpression Is Associated with Resistance to Treatment with DNA-damaging Agents in a Human Ovarian Cancer Cell Line1

Faina Vikhanskaya,2 Sergio Marchini,2 Mirko Marabese, Emanuela Galliera, and Massimo Broggini3 Laboratory of Molecular Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche “Mario Negri,” 20157 Milan, Italy

Abstract changes in gene expression and the response of these clones to different chemical and physical DNA-damaging agents. We examined the consequences of p73␣ overexpression on gene expression and cellular response to anticancer agents in clones from the human Materials and Methods ovarian cancer cell line A2780. Using microarray filters, the expression of ␣ 588 genes in two clones overexpressing p73 (A2780/p73.4 and A2780/ Cell Culture and Treatment. Clones derived from the human ovarian p73.5) in comparison with empty vector-transfected cells was evaluated. carcinoma cell lines A2780, A2780/pCDNA3, A2780/p73.4, and A2780/p73.5 There were clear differences in gene expression profiles. Both of the clones were obtained and grown as reported previously (12). DDP4 was obtained from showed a marked increase in the expression of genes involved in DNA Bristol Myers Squibb (Syracuse, NY). Topotecan was obtained from Smith, repair, including genes participating in nucleotide excision repair and Kline & Beecham (Brentford, United Kingdom) and MNNG was purchased mismatch repair. This was confirmed by reverse transcription-PCR and from Sigma (Milan, Italy). For clonogenic assay, cells were plated at 250 Northern blot analysis and was associated with an increase in the ability cells/well. After treatment with different drug concentrations or UV doses, of p73␣-expressing clones to repair two different DDP (cis-dichlorodiam- plates were incubated for 8–10 days in drug-free medium, and the number of mine platinum)-damaged plasmids in a host reactivation assay. p73␣ colonies formed were stained with 10% cristal violet in 20% ethanol and overexpressing clones were less sensitive than parental cells to alkylating automatically counted on an image analyzer (Immagini & Computer, Milan, agents treatment or UV radiation but equally sensitive to the topoisomer- Italy). The data of the survival curves were plotted as percentages of untreated ase I inhibitor topotecan, which indicated that the increase in expression controls. IC50 values were calculated from three-four independent experiments, of DNA repair genes has implications for the response to DNA damaging each consisting of at least three replicates. Plating efficiency was ϳ80% in all agents. three of the cell lines. Microarray Analysis of Gene Expression. Total RNA was isolated from Introduction exponentially growing cells using the Qiamp RNA kit (Qiagen, Milan, Italy) followed by in column treatment with RNase-free DNase (Qiagen). Five ␮gof The p53-related gene p73 shares high sequence homology with p53 total RNA were retrotranscribed to cDNAs in the presence of [32P]dATP, in critical regions including the DNA binding domain (1). As a result, (Amersham, Milan, Italy) using a mixture of specific oligonucleotides (Clon- p73 can transcriptionally activate p53 target genes such as p21, BAX, tech, Firenze, Italy). Equal amounts of labeled cDNAs were hybridized to 14-3-3␴, and GADD45, although with different specificity (1, 2). filters containing 588 genes (Clontech; ATLAS human cancer). After washing as recommended, filters were autoradiographed and gene expression patterns Although in some cell lines in vitro, the introduction of p73 arrests the evaluated using the ATLAS Image Software 1.0 (Clontech). growth (1, 3), the role of p73 as a tumor suppressor is still uncertain. RT-PCR and Northern Blot Analysis. Semiquantitative RT-PCR analysis Mutational analysis of p73 gene in different human cancers did not was done using the RT-PCR core kit (Perkin-Elmer, Milan, Italy) with a reveal any significant tumor-associated mutations (4–6); p73 knock- reduced number of cycles and coamplification of actin as internal control. out mice show specific neuronal disorders and a defective immuno- Northern blot analysis was done according to standard procedures (16). Filters logical response (7) but do not develop spontaneous tumors, which are were hybridized with 32P-labeled cDNAs using a Rediprime kit (Amersham). invariably found in p53 null mice (8). Hybridizations were done in 50% formamide, 10% dextran sulfate, 1% SDS, In certain tumor types, the expression of p73 is greater than in 1 M NaCl at 42°C for 16 h, followed by two 10-min washes at room temperature with 2ϫ SSC and one 30-min wash at 65°C in 2ϫ SSC-1% SDS. normal tissue (4, 9–11). This could drastically influence the cell Cell Reactivation of a DDP-damaged Plasmid. Purified pGL2 and responses to stress, because p73␣ overexpression can reduce the pG13Luc plasmids were treated with DDP (20 or 200 ␮M) for 10 min at room transcriptional activity of p53 (12, 13). temperature, as described previously (17), and were precipitated and washed in p53 is one of the important factors in determining cellular sensi- 70% ethanol. Cells were transfected with 5 ␮g of pGL2 or pG13Luc and 0.5 tivity to anticancer agents (14, 15). The role of p73, if any, in cellular ␮g of untreated pRL-SV40 used for internal normalization with the calcium responses to stress still needs to be elucidated. phosphate technique. Reporter gene activities were evaluated after 24 h using We investigated the consequences of p73␣ overexpression using the Dual Luciferase system (Promega, Milan, Italy). Results are expressed as clones established after transfection of p73␣ in the wild-type p53- the percentage of the control luciferase reported activity normalized by the renilla activity value. The mean Ϯ SD of three independent experiments is expressing human ovarian cancer cell line A2780; we analyzed shown.

Received 3/31/00; accepted 12/13/00. Results The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with ␣ 18 U.S.C. Section 1734 solely to indicate this fact. Overexpression of p73 did not affect the growth rate of A2780 1 F. V. is a visiting scientist from Institute of Cytology (Russian Academy of Scien- cells; in fact A2780/p73.4 and A2780/p73.5 showed doubling times of ces), St. Petersburg, Russia. S. M. is recipient of a fellowship from the Italian Foundation for Cancer Research (F.I.R.C.). 2 4 F. V. and S. M. contributed equally to this work. The abbreviations used are: DDP, cis-dichlorodiammine platinum; IC50, concentra- 3 To whom requests for reprints should be addressed, at Laboratory of Molecular tions inhibiting colony formation by 50%; SSC, 150 mM NaCl-15 mM sodium citrate; Pharmacology, Department of Oncology, Istituto di Ricerche Farmacologiche “Mario DNA-PK, DNA-dependent protein kinase; NER, nucleotide excision repair; ATM, ataxia Negri,” via Eritrea 62, 20157 Milan, Italy. Fax: 39-02-3546277; E-mail: broggini@ telengectasia mutated; MMR, mismatch repair; MNNG, methyl-nitro-nitroso-guanidine; marionegri.it. MGMT, O6-alkylguanine-DNA-alkyltransferase; RT-PCR, reverse transcription-PCR. 935

Fig. 1. Scatter plot of log-transformed expression data for A2780/p73.4 and A2780/pCDNA3 cells (left panel) and for A2780/p73.5 and A2780/pCDNA3 cells (right panel). Solid line, identity between the two cell populations. Points below or above the two dashed lines, those for which a Ͼ2-fold change in gene expression has been found.

40 and 50 h respectively, not significantly different from the 42 h for clear change in the overall pattern of expression. Although we de- empty vector-transfected cells (data not shown). tected changes in the expression of some genes involved either in cell The gene expression profile, analyzed using microarray technology cycle regulation or in apoptosis or belonging to the family of growth in the clones overexpressing p73␣, showed a clear difference com- factors, there was no similar change in gene expression between the pared with empty vector-transfected cells (Fig. 1). As it can be seen two clones, as was found for DNA repair and DNA damage response from this figure, many genes showed a more than 2-fold difference in genes (Table 1). Some differences were also found when we analyzed the expression in the two clones when compared with empty vector- the expression of genes involved in cell-cell interactions. For some of transfected cells. these genes, in fact, A2780/p73.4 showed a relatively modest decrease A2780/p73.4 and A2780/p73.5 clones showed a similar level of in the levels compared with control cells, whereas A2780/p73.5 cells p73 mRNA, respectively, 10 and 11 times higher than in the empty showed an increase (Table 1); the gene expression data obtained for vector transfected cells (Table 1). Similarly, the basal level of genes the two p73 overexpressing clones and not reported here are available known to be transcriptionally responsive to p53 (and to p73) and on the Internet.5 Overall we could detect changes in gene expression present in the filter, such as GADD45, BAX , and some of the PIG (with values greater than 2) in ϳ22 and 16% of genes for A2780/ genes were higher in the two clones than in control cells (Table 1). p73.4 and A2780/p73.5, respectively. Thirty-five (6%) of 588 genes These findings confirmed the previously reported increase in basal were consistently changed in both of the clones, and the majority of levels of p53-responsive genes in these clones, determined by North- these were up-regulated. ern blot analysis (12) and provide a good internal control for the For some of the genes whose expression was changed by microar- analysis of gene expression patterns. The most striking changes in ray analysis (including XPA, XPG, XPD, DNA-PK, b-RAF), we con- gene expression profile were detected in the DNA repair gene section, firmed the changes by using RT/PCR and/or Northern blot analysis in which both clones showed an increase in the expression of many (data not shown). genes including members of the NER and MMR pathways, GADD45, We used the host cell reactivation assay to evaluate whether the DNA-PK, and ATM (Table 1). marked changes in the expression of genes involved in DNA repair For the other groups of genes on the filter, we could not pick up any pathways found in both of the p73-expressing clones could result in differences in their DNA repair ability. Two DDP-damaged luciferase-encoding plasmids, one under the control of SV40 promoter and Table 1 Quantitative changes in gene expression in A2780/p73.4 and A2780/p73.5 cells compared with controls the other under the control of 13 copies of a strong p53-responsive element, were transfected in A2780/pCDNA3, A2780/p73.4, or Up-regulated genes Down-regulated genes A2780/p73.5 cells. Fig. 2 reports the relative luciferase activity with Gene A2780/p73.4 A2780/p73.5 Gene A2780/p73.4 A2780/p73.5 the two plasmids in these cell lines. The two p73-transfected clones p73 10 11 Rho C 0.41 1.35 seemed to repair both of the DDP-damaged plasmids better, in agree- BAX 2.1 2.3 Collagen III 0.62 0.42 GADD45 3.45 2.8 Desmoplak III 0.4 1.54 ment with the levels of expression for the DNA repair genes. We PIG3 1.43 2.6 MMP2 0.34 0.42 found more extensive repair with the plasmid containing the p53- PIG11 1 1.8 PDGFR ␤ 0.31 1.04 responsive element, as shown previously (18). PIG12 2.26 1.8 Paxillin 0.49 1.79 b-RAF 2.4 0.8 Stromelysin1 0.24 0.2 Considering the reported effects of p73 overexpression on p53 DNA-PK 56TIMP-2 0.71 0.29 function in A2780 cells (12) and the gene expression and DNA repair ATM 3.2 2.3 MEKK3 0.67 0.57 data obtained here, we checked whether this could translate in differ- XRCC6 2.2 3.5 Cadherin 14 0.32 5.04 XPD 3.9 4.4 ences in the cellular responses to DNA-damaging agents. Fig. 3A XPG 2.3 2.05 reports four independent experiments performed treating the three cell XPB 4.5 3.3 XRCC1 2.5 1 lines with DDP. Both of the p73-overexpressing clones were less MGMT 2.7 2.6 hMLH1 1.7 2 5 Internet address: ftp://ftp.marionegri.it/download/microarray. 936

Fig. 2. Host cell reactivation assay in A2780/pCDNA3 (f), A2780/p73.4 (F), and A2780/p73.5 (E) cells. Values represent the mean Ϯ SD of at least three determinations.

sensitive than empty vector-transfected cells with IC50s approxi- UV (left panel). In particular, for GADD45, we found an increased mately 3 and 4 times higher for A2780/p73.4 and A2780/p73.5 clones, expression after DNA damage both in parental and p73-overespress- respectively, than for A2780/pCDNA3 cells. Similarly, both of the ing cells. The differences found in the basal levels of GADD45 were, p73-overexpressing clones showed lower sensitivity (2- to 3-fold) to however, maintained after induction. another type of DNA damage such as that induced by UV radiation (Fig. 3B) or by treatment with a methylating agent such as MNNG Discussion (Fig. 3C). A drug not directly acting on DNA, such as the topoisomer- ␣ ase I inhibitor topotecan, showed, instead, similar activity in the two In the present study, overexpression of p73 in a human ovarian clones and in the empty vector-transfected cells (Fig. 3D). cancer cell line resulted in marked up-regulation of genes encoding Finally we checked whether the expression of some of the genes for DNA repair proteins, thus modifying the ability of cells to repair involved in DNA repair was still higher in p73-overexpressing clones DNA damage and to become less susceptible to DNA-damaging after treatment with DNA damaging agents. Fig. 4 reports the results agents. obtained with GADD45, XPD, XPB, and XPG. In general the differ- The approach used to investigate the changes in gene expression ␣ ences in expression found in the basal levels were maintained evalu- related to p73 overexpression was to assess a large number of genes ating the levels 24 h after treatment with both DDP (right panel) and by microarray technology. Although several other genes appeared to be affected by p73␣ overexpression, we focused subsequent studies on DNA repair genes, which showed in fact the most striking differences. Because the genes encoding for the enzymes involved in NER were consistently up-regulated we examined two DNA-damaging agents, DDP and UV radiation, for which there is convincing evidence that NER plays a crucial role in repairing their cytotoxic DNA lesions (19–21). To our knowledge, this is the first study analyzing the consequences of p73 overexpression on gene expression and response to DNA- damaging agents. The product of the tumor suppressor gene p53 has been widely reported to affect the sensitivity of different cancer cell lines to anticancer drugs treatment (14, 17, 22). Unlike p53, p73 does not seem to be activated after DNA damage (1, 3, 12). In different tumors, however, levels of p73 were higher than in normal tissues (4, 9–11), and this could be an important determinant of response to treatment with anticancer agents. The decreased response of p73-overexpressing clones to DNA damage could also be attributable to overexpression of GADD45 gene observed in both p73 overexpressing clones. In fact GADD45 has been reported to participate in DNA repair (23). GADD45 is a p53- responsive gene whose transcription can also be partially activated by DNA damage through a p53-independent mechanisms (24). The observed increase in GADD45 levels after treatment with DDP and UV in all of the three cell lines used is likely to reflect both of the mechanisms and is in good agreement with previous results obtained in these clones with waf1/p21 gene (12). The fact that even after induction by DDP and UV, the levels of GADD45 (as well as those of Fig. 3. Cell growth inhibition induced by DDP (A), UV (B), MNNG (C), and topotecan the other genes tested, i.e., XPG, XPD, and XPB) are higher in the two (D) in empty vector-transfected and p73-overexpressing clones. Values reported are the p73-overexpressing clones further support the idea that these genes mean Ϯ SD from at least three independent experiments, each consisting of four replicates. f, A2780/pCDNA3; Ⅺ, A2780/p73.4; E, A2780/p73.5. Cells were treated for 2 h play a role in the relative resistance observed in these two clones. with DDP and for 1 h with MNNG and topotecan. With regard to the MMR status, defects in this system have been 937

p73 have been found to be higher in many human tumors than in normal tissues (4, 9–11).

Acknowledgments

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Faina Vikhanskaya, Sergio Marchini, Mirko Marabese, et al.

Cancer Res 2001;61:935-938.

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