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p73 Gene Expression in Ovarian Cancer Tissues and Cell Lines

Department of Obstetrics and Gynecology, The University of Hong Kong, Hong Kong, S. A. R., China

	ABSTRACT

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The p73 gene, a homology of p53, is a new candidate of imprinting and tumor suppressor gene. To investigate the role of p73 in ovarian cancer, we studied the allelic expression in 56 cases of ovarian cancer using StyI polymorphism analysis. We also examined p73 expression by semi-quantitative reverse transcription-PCR as well as by Western blot analysis and DNA methylation study of the CpG island in exon 1 in ovarian cancer tissues and cell lines. Loss of heterozygosity was found in 8.3% (2 of 24) of the cases. Biallelic expression was demonstrated in 91.7% (22 of 24) of the tumor samples, in 70.8% (17 of 24) of the normal samples, and in 1 ovarian cancer cell line. Imbalanced expression and monoallelic expression were found in three and two pairs of matched samples, respectively. Overexpression of p73 was found in advanced ovarian cancer rather than in early-stage disease or in borderline ovarian tumor. No significant difference was found in the p53 expression. Three cell lines with absent p73 protein expression and one tumor sample with monoallelic expression were methylated in the CpG island. Demethylation in SKOV3 cell line using 5-azacytidine can reactivate the expression of this gene in both the mRNA and the protein level. Our results indicated that p73 was not imprinted in most of the ovarian cancer and normal tissues, but it could be involved in the advanced ovarian cancer through overexpression. DNA methylation may contribute to the lack of p73 expression.

	INTRODUCTION

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Genetic alteration of p53 plays important role in the development of human cancer (1) . p73, a novel family member of p53, has recently been identified and found to activate p21Waf1/Cip1 and to induce apoptosis. (2) . The p73 gene was also considered as a candidate of the imprinted tumor suppressor gene because: (a) p73 was mapped on 1p36.33, a region frequently found to be deleted in the maternal chromosome of neuroblastoma and other tumors (3 , 4) ; (b) monoallelic expression of this gene was found in numerous cancer cell lines and in normal tissues (2) ; and (c) activation of the silent allele was found in renal cell carcinoma and lung cancer (5 , 6) that is similar to LOI. However, biallelic expression of this gene was also observed in some normal tissues and lymphocytes as well as in both bladder cancer and normal bladder (2 , 7) . These data suggest that p73 is not monoallelically expressed in all human tissues, and it could be tissue dependent (7) .

In addition, overexpression of p73 was found in several cancers such as lung, bladder, prostate, and colorectal (6, 7, 8, 9) . But no mutations were revealed in most of the cancer cells (7, 8, 9, 10, 11, 12) . The expression of p73 was increased in lung cancer independent of p53 gene mutation (13) . Transcriptional silencing of p73 in leukemia was associated with 5' CpG island methylation (14 , 15) . However, there was no evidence of methylation in any of the solid tumors analyzed, including breast, renal, and colon cancers (14) .

In ovarian cancer, several genetic alterations have been identified, including mutations of p53 tumor suppressor, Ras, BRAC1, BRAC2, C-erb2, and Bcl-2 oncogenes. LOI of IGF2 and H19 genes was also involved in the genesis of ovarian cancer (16 , 17) . To date, the molecular mechanisms of ovarian cancer development are not well established. The new candidate tumor suppressor, p73, which is implicated in the pathogenesis of many types of cancer, may also be involved in the development of ovarian cancer. To elucidate the role of p73 in the development of ovarian cancer and the allelic-specific expression as a potential imprinted tumor suppressor, we investigated the specific allelic expression of the p73 gene in 56 cases of ovarian cancer, borderline ovarian tumor, and matched normal tissues. We also compared the expression level of p73 by semi-quantitative RT-PCR² with p53. To further understand the expression of p73, we studied the p73 expression at the mRNA level and at the protein level as well as elucidating the methylation status of p73 gene in ovarian cancer tissues and cell lines.

	MATERIALS AND METHODS

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Characteristics of Patients.
Ovarian tumors and adjacent normal tissues including ovary, cervix, endometrium, and peripheral blood were collected from 56 patients with ovarian cancer at Queen Mary Hospital, Hong Kong. The tissues were frozen and stored in liquid nitrogen until analysis. Histological diagnosis and clinical staging were performed according to the International Federation of Gynecologists and Obstetricians criteria. Among the 56 patients, 16 were in stage I (28.7%), 15 were in stage II (26.8%), 18 were in stage III (32.1%), and 7 were in stage IV (12.5%). The histology included 20 serous cystadenocarcinomas, 12 ovarian mucinous cystadenocarcinomas, 8 endometrioid adenocarcinomas, 9 clear-cell carcinomas, 5 borderline mucinous tumors, and 2 borderline serous cystadenomas. Five ovarian cancer cell lines, OVCA3, SKOV3, A2780s, A2780cp, and OV2008, were also studied.

DNA, RNA, and Protein Extraction and Purification.
Genomic DNA and RNA were isolated from frozen tissues and lymphocytes. The tissues were pulverized in liquid nitrogen and the powder was transferred to tubes with 75 mM NaCl and 25 mM EDTA. DNA was extracted by proteinase K-phenol/chloroform methods. RNA and protein were isolated using Tripure Isolation Reagent (Boehringer Mannheim) according to the manufacturer’s protocol.

Alleleic Expression of p73.
To identify heterozygous samples, primers for exon 2 of the p73 gene were used. PCR was performed using 100 ng of tumor and normal matched DNA under the following conditions: (a) 95°C for 5 min; (b) 35 cycles of 95°C for 20 s; (c) 62°C for 15 s; (d) 72°C for 30 s; and (d) a final extension for 4 min at 72°C. A specific 229-bp fragment was obtained. Five µl of PCR product was digested with 10 units of StyI restriction enzyme at 37°C overnight. Ten µl of product was electrophoresed on 3% NuSieve (3:1) agarose gel and stained with ethidium bromide. The size of the undigested DNA band 229 bp and the StyI digested band 157 bp were named as a and b allele, respectively. A known sample of b allele was used as a control to ensure complete digestion for each experiment. The primers used were as follows: primer 1, 5'-CAGGAGGACAGAGCACGAG-3'; and primer 2, 5'-CGAAGGTGGCTGAGGCTAG-3'. Heterozygous samples were chosen for RT-PCR using cDNA primers flanking the GC/AT polymorphism and StyI RFLP. Two µg of total RNA was reverse-transcribed by Superscript II (Life Technologies, Inc.). One-twentieth of the cDNA volume was used for PCR amplification. The PCR reaction was performed using primer 3 and primer 5. A specific band of 285 bp was obtained. One-fiftieth of the first PCR product was used for the nested PCR with primer 3 and primer 4. The primers used were as follows: primer 3, 5'-GGGCTGCGACGGCTGCAGAGC-3; and primer 4, 5'-GAGAGCTCCAGAG GTGCTC-3'; and primer 5, 5'-ACCAGATGAGCAGCCGCG-3'. The final size of the band is 116 bp. Each of the amplifications was 25 cycles using the previous PCR conditions. Contamination by genomic DNA in total RNA was determined by the presence of DNA bands of different sizes and by the experiments without reverse transcriptase. No band was seen when reverse transcriptase was omitted. After RT-PCR, the StyI digestion was performed as described previously, and a size of 84 bp was seen for complete digestion that represented A/T polymorphism.

Semi-quantitative RT-PCR.
Because advanced ovarian cancer tends to spread bilaterally and both ovaries are affected, it’s difficult to collect both tumor and normal ovarian tissue samples from the same patient. As a result, only three matched normal ovarian and tumor samples were obtained. The other normal tissues were either normal endometrium or normal cervix from the same patient. We first compared the expression level of p73 in two patients with normal endometrium, cervix, ovary, or lymphocytes. The expression level of p73 was nearly the same in the adjacent normal tissues in the two patients and a little bit lower in the lymphocytes. So, we investigated the expression level of p73 in 14 advanced ovarian cancer specimens (stages III and IV), in 9 samples of early stages (stages I and II), and in 6 borderline ovarian tumors. All of them were compared with the expression level in normal tissues. PCR was performed according to conditions stated previously, except that 28 cycles were used for p73 with primers 6 and 7 and 24 cycles for ß-actin using primers 8 and 9. The conditions for p53 were the same, except that the annealing temperature was 54°C and 28 cycles with primers 10 and 11. The primers used were as follows: primer 6, 5'-AACGCTGCCCCAACCACGAG-3' and primer 7, 5'-GCCGGTTCATGCCCCCTACA-3' (for p73); primer 8, 5'-ATCTGGCACCACACCTTC TACAATGAGCTGCG-3' and primer 9, 5'-CGTCATACTCCTGCTTGCTGATCCACA TCTGC-3' (for ß-actin); and primer 10, 5'-CTGAGGTTGGCTCTGACTGTACCACCATCC-3' and primer 11, 5'-CTCATTCAGCTCTCGGAACATCTCGAAGCG-3' (for p53). The cycles were determined by the standard curve amplified from 16, 20, 24, 28, 32, 36, and 40. Conditions were chosen to give a linear relationship between the amount of amplified product and the input RNA (data not shown). A 231-bp band was seen for p73, 373 bp for p53, and 400 bp for ß-actin. Ten µl of PCR product was electrophoresed on a 2.5% agarose gel and stained with ethidium bromide. The software UVP Gel Works 1.0 for Windows was used to analyze the expression levels of p73 and p53 in both tumor and normal specimens with ß-actin as a control. Each PCR and electrophoresis procedure was repeated twice. We first calculated the average of p73 and ß-actin expression for each sample, then we calculated the ratio of p73 and ß-actin in both tumor and normal tissues, and finally we compared the p73 expression ratio between tumor and normal tissues (T/Tß/N/Nß). Student’s t test was used for statistical analysis. A P <0.05 was considered a significant difference. The same procedure was performed for p53 expression.

Western Blot and Methylation Analysis of the p73 Gene in Ovarian Cancer.
Ovarian cancer cell lines were cultured in DMEM medium supplemented with 10% FCS. Western blot was performed in ovarian cancer cell lines using 50 µg of protein and a goat antihuman polyclonal anti-p73 antibody (Santa Cruz Biotechnology, Inc.) with a dilution of 1:500 and visualized with the ECL chemiluminescent detection kit (Amersham). The filters were also reprobed with ß-actin (Sigma). Seven advanced ovarian cancer samples, five early-stage, and three borderline ovarian tumor specimens and matched normal tissues were also studied by Western blot using 20 µg of protein. The methylation state of p73 in ovarian cancer cell lines and tissues was detected as described previously (14) . Two-tenths µg of genomic DNA was digested with 20 units of either methylcytosine-sensitive enzyme HpaII (Promega) or its methylation-resistant isoschizomer, MspI (Promega), for 3 h at 37°C, then phenol/chloroform-extracted, ethanol-precipitated, dried, and resuspended to 10 µl of Tris-EDTA buffer. One µl (20 ng) was amplified by PCR using primers 5'-GGGGACGCAGCGAAACCG-3' and 5'-CTGCAGCCGTCGCAGCC-3', which amplified the CpG island in exon 1 with a 77-bp band. Normal placenta DNA was used as a negative control. The band existed after HpaII digestion, but not if there were methylation alleles.

	RESULTS

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Frequent Biallelic Expression of the p73 Gene in Ovarian Cancer Tissues, Normal Ovaries, and Adjacent Tissues or Lymphocytes.
Of the 56 samples, 24 were heterozygous. LOH was found in 2 of 24 informative samples (8.3%). Among the 24 heterozygous samples, biallelic expression of p73 was found in 22 tumor samples (91.7%) including 2 LOH specimens. Nineteen matched normal samples were also proved to be biallelically expressed (79.2%). Eight of them were lymphocytes. Only three samples showed imbalanced expression of p73, with biallelic expression in tumors and monoallelic expression in normal specimens. Among these three samples, two were lymphocytes and one was endometrium. The remaining two samples showed monoallelic expression in both tumor and normal specimens. Interestingly, two samples with LOH also showed biallelic expression for both tumor and normal tissues. One informative cell line, OVCA3, was also expressed biallelically. The results are summarized in Tables 1 and 2 and in Fig. 1 .

View larger version (24K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Analysis of allelic expression of p73 in tumor (T) and normal (N) samples. p73 allelic expression was analyzed in eight matched samples and one ovarian cancer cell line (OVCA3) using a C/T polymorphism in exon 2. The size of the band (116 bp) was seen after nested RT-PCR. Biallelic expression was identified for both 116 bp and 84 bp after StyI digestion. Biallelic expression of the p73 gene in both tumor (T) and normal (N) samples was found in samples 1, 14, 17, 20, 30, and 33. Imbalanced expression was shown in sample 13, with biallelic expression in tumor and monoallelic expression in normal lymphocytes. Both T21 and N21 remained monoallelic in expression. OVCA3 also showed biallelic expression. Ctr, control of complete digestion.

View larger version (54K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. p73 expression in tumor and normal ovaries. A, three matched tumor (T) and normal (N) ovary samples were examined for p73 expression by semi-quantitative RT-PCR and Western blot. ß-actin was used as a control. For RT-PCR, the cycles were 28 for p73 (231 bp) and 24 for ß-actin (400 bp), respectively, according to the standard curve. Western blot was performed using 20 µg of protein and a goat antihuman polyclonal anti-p73 antibody (Santa Cruz Biotechnology, Inc.) with a dilution of 1:500 and visualized with the ECL chemiluminescent detection kit (Amersham). The filters were also reprobed with ß-actin (Sigma). The expression ratio between tumor (T) and normal (N) ovary was 1.8:1, 4.8:1, and 3.7:1 for mRNA and 1.6:1, 4.7:1, and 4.5:1 for protein in T48, T55, and T59, respectively. T55 and T59 were advanced stage, T48 was stage Ia1. B, p73 mRNA and protein expression in different stages of ovarian cancer and in normal tissues. T24, T41, and T44 were advanced-stage cancer, T54 and T45 were early stage, and T4 was a borderline ovarian tumor.

Loss of p73 Protein Expression Was Related to CpG Island Methylation in Ovarian Cancer Cell Lines.
Among the five ovarian cancer cell lines studied, p73 protein was detected in two, OV2008 and A2780cp. We hypothesized that the absence of p73 protein might be caused by methylation of the CpG islands. The results showed that the protein-negative cell lines OVCA3, A2780s, and SKOV3 were methylated in exon 1. In OV2008 and A2780cp with p73 expression, exon 1 was not methylated (Fig. 3) . SKOV3, exhibiting no p73 expression in mRNA and protein level, was treated with 10 µM of 5-aza for 8 days. RT-PCR and Western blot showed reexpression of this gene at both the mRNA and protein levels, as shown in Fig. 4 . No evidence of methylation was found in ovarian cancer tissues and normal tissues, except that one cancer sample (T21) with monoallelic expression was methylated.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. p73 methylation in the CpG island of exon 1 in ovarian cancer cell lines. DNA from the cell lines was digested with 20 units of either methylcytosine-sensitive enzyme HpaII (Promega) or its methylation-resistant isoschizomer, MspI (Promega), for 3 h at 37°C, then phenol/chloroform-extracted, ethanol-precipitated, dried, and resuspended to 10 µl of Tris-EDTA buffer. One µl (20 ng) was amplified by PCR using primers 5'-GGGGACGCAGCGAAACCG-3' and 5'-CTGCAGCCGTCGCAGCC-3', which amplified the CpG island in exon 1 (77bp). Normal ovary DNA was used as a negative control. U, uncut; H, HpaII; M, MspI. The PCR band was shown in the uncut. The HpaII digestion band can be seen in SKOV3, OVCA3, and A2780s, which is shown as methylation. No band was found in OV2008.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. p73 expression in ovarian cancer cell lines by Western blot and RT-PCR. Western blot using 50 µg of protein and goat antihuman polyclonal anti-p73 antibody (Santa Cruz) was shown in ovarian cancer cell lines OVCA3, A2780s, A2780cp, OV2008, SKOV3, and SKOV3 after 5-aza treatment for 8 days. p73 protein was identified in OV2008 and A2780cp. After 5-aza treatment, p73 protein was detected in the SKOV3 cell line. A similar result was shown using RT-PCR in the same ovarian cancer cell lines. RT-PCR was performed using 28 cycles for p73 and ß-actin; the annealing temperature was 62°C.

	DISCUSSION

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Monoallelic expression of p73 has been reported previously, suggesting that p73 may be a candidate of the imprinting gene (2 , 5) . In contrast to previous findings, we observed 91.7% (22 of 24) of the ovarian cancer and 70.8% (17 of 24) of the normal endometrium and normal cervix expressed biallelically. Even lymphocytes were biallelic expressed in eight of the twelve samples. Our results are in line with several other studies that biallelic expression of this gene was found in 25 of 26 normal lung specimens (18) , in both bladder cancer and normal bladder (7) , in 3 peripheral lymphocyte cell lines, 1 colon cancer cell line, and melanoma cell lines (12) . These results supported the conclusion that biallelic expression was involved in most of the ovarian cancer and normal tissues. On the other hand, in our study 2 of 24 informative samples maintained monoallelic expression in both tumor and normal specimens. Imbalanced allelic expression was identified in 3 of 24 (12.5%) samples with biallelic expression in tumor and monoallelic expression in normal tissues. This phenomenon looks like LOI. The same phenomenon was found in (5 of 5) lung cancer (6) , (1 of 26) lung cancer (19) and (7 of 11) renal carcinoma (5) . Genomic imprinting has been suggested to play a role in many human cancers (20 , 21) . Activation of the silent allele with biallelic expression in cancer cells and monoallelic in normal tissues was considered as LOI. LOI has been reported in many types of tumors (21 , 22) . As shown by others (7 , 12 , 18) , our results showed that p73 was evidently biallelically expressed in both normal tissues and ovarian cancer. Therefore, the p73 gene was not imprinted in ovarian cancer and LOI of this gene was not an important step in the tumorigenesis when compared with renal cell carcinoma and lung cancer (5 , 6) . It seems that allelic expression of p73 may be tissue specific or individual specific.

In this study, we showed that expression level of p73 is higher in advanced ovarian cancer than in normal tissues. However, our present study failed to show any correlation in the mRNA level between p73 and p53. Overexpression of p73 gene has also been reported in lung cancer, bladder cancer, and colorectal cancer without mutation (6 , 7 , 9) . It is possible that wild type p73, not mutant p73, is overexpresssed in tumors. The wild type p73 functions as a security guard when overexpressed and works to arrest the cell cycle as a tumor suppressor gene. However, another possibility is that p73 functions as an oncogene in the up-regulation of cell growth (19) and silences p53 function by binding to its functional binding site (23) .

To elucidate the imprinting and expression of p73 gene, we studied the methylation of promoter CpG island in ovarian cancer tissues and normal tissues. Finding no evidence of methylation in these samples further suggests that it is not an imprinting gene. However, in ovarian cancer cell lines the lack of p73 protein expression with evidence of methylation supports that methylation of p73 may play a role. Reactivating the expression by 5-aza also confirms the role of methylation in the control of expression of this gene. Our results in the ovarian cell lines support that epigenetic silencing of tumor suppressor genes via methylation of the promoter CpG island is involved in the ovarian malignancy. Although it is difficult to explain the high level of both mRNA and protein in advanced ovarian cancer and the absence of protein expression in ovarian cancer cell lines, this is the first report that loss of p73 expression is attributable to methylation in ovarian cancer.

Our study revealed that LOH of the p73 gene occurred in a low frequency in ovarian cancer. Biallelic expression of p73 was found in the majority of ovarian cancer and normal tissues. Overexpression of p73 was associated with advanced ovarian cancer when compared with the early-stage and borderline ovarian tumors, and we showed that p73 overexpression was independent of p53. Lack of expression of p73 was associated with CpG island methylation. Demethylation using 5-aza in SKOV3 can reactivate the expression of this gene in both the mRNA and protein levels. We conclude that the p73 gene is not an imprinting gene in ovarian cancer. Overexpression of p73 in both the transcriptional and translational levels is associated with ovarian cancer in advanced stages. DNA methylation is involved in the p73 inactivation in ovarian cancer cell lines.

	FOOTNOTES

 
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.

¹ To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, The University of Hong Kong, Hong Kong. Phone: 852-28554684; Fax: 852-28550947; E-mail: hysngan{at}hkucc.hku.hk

² The abbreviations used are: RT-PCR, reverse transcription-PCR; LOI, loss of imprinting; LOH, loss of heterozygosity; 5-aza, 5-azacytidine.

Received 4/ 3/00; revised 7/17/00; accepted 7/17/00.

	REFERENCES

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