- Email: [email protected]
Allelic expression of p73 in human ovarian cancers
Annals of Oncology 10: 949-953, 1999. © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Allelic expression ofp73 in human ovarian cancers ;1
A. M. Codegoni, F. Bertoni, C. Patregnani, E. Marinetti, M. D'lncalci & M. Broggini
^Molecular Pharmacology Unit, Department of Oncology, Istituto di Ricerche Farmacologiche 'Mario Negri', Milan; 2Ospedale S. Gerardo, University of Milan, Monza (MI), Italy
Introduction
The recently-described p73 gene has a close structural similarity to the tumor suppressor genep53 [1]. In particular, with respect to the central DNA binding domain of p53 which is responsible for downstream gene activation, its homology with p73 reaches 63% of identity at the amino acid level [1, 2]. The tumor suppressor/755 has been found mutated in almost 50% of human cancers; despite the strong structural similarities between p53 and p73, initial reports on p73 failed to reveal any mutations in different cancer types [3-5]. The p73 gene has been localized on chromosome Ip36, a region frequently found deleted in human cancers [1]. At least three putative tumor suppressor genes could be present in this area and/?73 was considered an attractive candidate suppressor gene. Available data, however, refute a role forp73 as a tumor suppressor [6-8]. In addition,/;73 knock-out mice show specific neuronal disorders and a defective immunological response but do not develop spontaneous tumors, which are invariably found in p53null mice [9,10]. The p73 gene was reported to be monoallelically expressed in normal human tissues [1], and it was found that there was expression of the silent allele in lung cancer, suggesting a possible oncogenic role for p73 [8], p73 binds the same DNA sequence recognized by p53, and we recently reported that/?73 can compete withp53 for binding to Dann, resulting in a generally lower transactivation ability of p53 in the presence of high
concentrations of p73 [11], which would fit with the overexpression of p73 found in cancer cells [6, 8]. The monoallelic expression ofp 73 in normal tissues has not been confirmed by other laboratories [6, 12] although in other tumor types allele switching [13] and monoallelic and biallelic expression [14] of/?73 have been reported. Genetic analyses of ovarian cancers have not yet suggested important genes in their oncogenesis. p53 is in fact found mutated in ovarian cancers, but it is more likely that its role is linked to tumor progression rather than tumor development [4, 15, 16]. In addition, many studies have reported a lack of correlation between p53 status and the survival of ovarian cancer patients [1618]. In 51 patients from whom tumor and normal cells were available, we studied the allelic expression of/?73 in ovarian cancer patients. Tumor allelic expression of this gene was compared in patients with ovarian cancer at different stages with borderline tumors.
Patients and methods Patients, pathology specimens and cell lines Samples from primary ovarian tumours and corresponding peripheral blood samples were obtained from the Department of Gynecological Oncology of the Ospedale San Gerardo, Monza (Italy). Tumour cases were diagnosed according to the World Health Organization Classification [19] and staged according to the International Federation of Gynecology and Obstetrics (FIGO) criteria [20]. Pathology samples were promptly frozen at -80 °C, until the time of analysis.
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
Results: We found an allelic distribution similar to that previously reported. LOH was found in two patients with Background: The p73 gene is structurally related to the tumor ovarian cancer. In one case in which normal ovarian tissue suppressor genep53. The role of p73 in tumor development is was available biallelic expression ofp73 was found. still unclear and no data on ovarian cancer are so far available. Conclusion: In comparisons of ovarian cancers and borderFor this reason we have analyzed, in a panel of ovarian line tumors, no differences in allelic distribution and/or excancers, the allelic distribution and expression ofp73. pression were found, suggesting that p73 does not play an Patients and methods: Fifty-one samples from ovarian important role in the pathogenesis and development of ovarian cancers and five human ovarian cancer cell lines growing in cancer. culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating Key words: allelic expression, borderline tumors, ovarian allelic expression of/?73. cancer, p73, tumor suppressor gene Summary
950
<
<
<
<<
<<
<<
z z z z z z z z z z
N
T
N
T
N
T
N
<
o a c o r r o r r o r r o a :
T 229 b p 157 b p -
U
U
U U •ii6bp( • 84 bp.
72 b p -
Figure 1. Ethidium bromide staining of amplifyed DNA from normal or tumor tissue digested with Sty I to determine the allelic status of p73. In these conditions, the undigested amplified fragment (G/C allele) has a length of 229 bp, while the digested fragments (A/Tallele) have a length of 157 and 72 bp.
Figure 2. Representative gel showing allelic status and expression of p73 determined in DNA and RNA samples, respectively. For DNA, the length of the fragments is as indicated in Figure 1. For fragments amplified from RNA, the undigested (G/C allele) fragment has a length of 116 bp while the Sty digested fragment (A/Tallele) has a length of 84 bp.
The human ovarian cancer cell lines OVCAR-3, SW626, A2780, SKOV-3 and IGROV-1 were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS). The neuroblastoma cell line IMR-32 was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. DNA extraction was performed with the QIAamp Blood kit and with the QIAamp Tissue kit according to the manufacturer's instructions (Qiagen). Total RNA was extracted from untreated or treated cells by the guanidine/caesium chloride gradient method [21].
AT allele. In each experiment, DNA and RNA samples from the human neuroblastoma cell line IMR-32, previously characterized as having the presence and expression of the A/Tallele [1], was used to check the quality of the PCR reaction and enzyme digestion. Analysis of five human ovarian cancer cell lines showed that all presented the G/C; G/C alleles (Table 2). p73 allelic expression was evaluated in the heterozygous samples, in either ovarian carcinomas or in borderline tumors. Both of the two normal ovary samples available showed G/C; G/C homozygosity and therefore could not be used for allelic expression analysis. The same was found for the five human ovarian cancer cell lines analyzed, all showing G/C; G/C homozygosity at DNA level (Table 1). Our analysis focused on the possible differences between ovarian carcinomas and borderline tumors. Of the informative cases of ovarian carcinomas, five showed biallelic and one monoallelic expression (Figure 2). The two remaining cases (reported to have lost one allele at the DNA level) did express the allele retained. As regards the borderline tumors, six out of seven showed biallelic expression of p73, while only one case resulted in monoallelic expression. Paired normal and tumor tissue was obtained from one case only, in which we found biallelic expression of p73 in the normal ovary as well as in the tumor.
Allelic expression of p73 Heterozygous samples were detected by PCR on DNA samples using primers annealing to intronic sequences amplifying a fragment of 229 bp. After 35 cycles of amplification as described [8], the samples were digested with Sty I overnight, separated on agarose or polyacnlamide gels and stained with ethidium bromide. Heterozygous samples were selected for RT-PCR analysis which was performed with primers amplifying the C/T polymorphism. PCR conditions were as above except that a33P-dATP was included in the PCR reactions and, after digestion overnight with Sty I, the samples were resolved on polyacrilamide gels and autoradiographed.
Results Of the 51 specimens examined, 32 were from ovarian cancers, 17 from borderline tumors and two from normal ovaries. The Sty I polymorphism was utilized to study thep73 allele frequency using DNA collected from blood (Figure 1). Overall, the frequency of G/C; G/C homozygosity was 67%, A/T; A/T 2% and G/C; A/T heterozygosity 31%, values similar to those previously reported [1,6]. For the 15 heterozygous informative cases, the allelic frequency was analyzed at tumor DNA level (see Table 1). Thirteen of these (87%) retained both alleles, while apparent allelic loss was observed in two cases. In one case the G/C allele was retained and in the other the
Discussion Thep73 gene is one of the recently discovered members of the p53 family. Unlike p53, however, for which a defined tumor suppressor role has been established [2, 22, 23], the role of/? 75 in cancer progression and development is still unclear. Evidence presented so far tends to exclude a role for this gene as a tumor suppressor, particularly in those tumors with LOH at Iq36, the region
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
72 b p - *
951 Table I. Allelic expression ofp73 in human ovarian cancer samples. Case
Histologic type
Figo stage
Grade
Normal DNA p73 alleles
Tumour DNA p73 alleles
Tumour expressed alleles
126 128 131 132 138 140 145 148 149 150 154 155 156 157 165 167 168 175 176 180 191 191A 193 196 198 200 205 210 216 217 226 225 5 8 11 17A 19A 22A 24 25 42 43A 141A 142 160 164A 181A 209A 220 Nl N2
Serous cystadenocarcinoma Serous cystadenocarcinoma Endometrioid adenocarcinoma Endometrioid adenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Clear cell cystadenocarcinoma Mucinous cystadenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Mucinous cystadenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Unclassified tumor Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Unclassified tumor Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Endometrioid adenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Teratoma Mucinous borderline tumour Serous borderline tumour Serous borderline tumour Serous borderline tumour Mucinous borderline tumour Mucinous borderline tumour Mucinous borderline tumour Serous borderline tumour Mucinous borderline tumour Serous borderline tumour Mucinous borderline tumour Serous borderline tumour Mucinous borderline tumour Serous borderline tumour Serous borderline tumour Serous borderline tumour Serous borderline tumour Normal ovarian tissue Normal ovarian tissue
me me
3 3 2 2 2 3 3 3 1 3 3 3 2 3 3 3 0 3 3 3 3 3 3 3 3 1 1 0 2 2 3 — _ _ _ -
G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; A/T G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; A/T G/C; A/T G/C; A/T G/C; A/T G/C; G/C A/T; A/T G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C
_ G/C;A/T _ G/C; A/T G/C; A/T G/C G/C; A/T G/C; A/T G/C; A/T A/T G/C; A/T G/C; A/T G/C; A/T G/C; A/T G/C; A/T A/T; A/T _ G/C; A/T G/C; A/T _ _ _ -
A/T G/C; _ _ G/C; G/C _ G/C; G/C; G/C, AT G/C; _ G/C; G/C; A/T G/C; — _ _ G/C; G/C; _ _ -
IA IA
me me me lie
IA
me me me me me lie
me me me me me me me me
IA IC IB IC IA IC IA IB
me IC IA IA IA IA IA
me
IC IC IA IIIB IA IC IIIB _ -
A/T
A/T
A/T
A/T
A/T A/T A/T A/T
A/T A/T
in which the/?73 gene has been mapped [1]. In all of the conditions in which monoallelic expression in the nortumor samples examined so far, no mutations in thep73 mal adjacent tissue was found [8,13]. The possibility that gene have been found, as opposed to the high percentage p73 could be an imprinted gene remains, however, at of mutations found in the p53 gene [5, 12, 14]. Contrast- least debatable and more data on the possible loss of ing results have been obtained on the expression of the imprinting in cancer cells are needed to clarify this p73 gene. It was initially reported to be monoallelically point. In this study we analyzed the allelic distribution expressed [1], a result not confirmed by other authors and expression of p73 in ovarian cancer, a tumor for who reported a biallelic expression in normal subjects which alterations in the chromosomal region mapped by [6, 12]. In tumor samples, evidence of biallelic expres- p73 have been reported [24]. sion of p73 was reported in lung and renal cancers, in Taking advantage of a polymorphic site present in
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
me me
A/T
952 Table 2. Allelic status of/?75 in human ovarian cancer cell lines.
Acknowledgements
Cell line
Tissue type
p73 alleles status
Expressed allele
OVCAR-3 SW626 A2780 IGROV SKOV-3 1MR32
Ovarian cancer Ovarian cancer Ovarian cancer Ovarian cancer Ovarian cancer Neuroblastoma
G/C; G/C; G/C; G/C; G/C; A/T
The generous contributions of the Italian Association for Cancer Research (AIRC) and of the Nerina and Mario Mattioli Foundation are gratefully acknowledged.
A/T
G/C G/C G/C G/C G/C
1. Kaghad M, Bonnet H, Yang A et al. Monoallelically expressed gene related to p53 at Ip36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997; 90: 809-19. 2. Oren M. Lonely no more:p53 finds its kin in a tumor suppressor haven. Cell 1997; 90: 829-32. 3. Hollstein M, Soussi T, Thomas G et al. p53 gene alterations in human tumors: Perspectives for cancer control. Recent Results Cancer Res 1997; 143: 369-89. 4. Harris CC. The 1995 Walter Hubert Lecture - molecular epidemiology of human cancer: Insights from the mutational analysis of thep53 tumour-suppressor gene. Br J Cancer 1996; 73: 261-9. 5. Nimura Y, Mihara M, Ichimiya S et al. p73, a gene related top53, is not mutated in esophageal carcinomas. Int J Cancer 1998; 78: 437-40. 6. Kovalev S, Marchenko N, Swendeman S et al. Expression level, allelic origin, and mutation analysis of the p73 gene in neuroblastoma tumors and cell lines. Cell Growth Differ 1998; 9: 897903. 7. Kroiss MM, Bosserhoff AK, Vogt T e t al. Loss of expression or mutations in thep73 tumour suppressor gene are not involved in the pathogenesis of malignant melanomas. Melanoma Res 1998; 8: 504-9. 8. Mai M.Yokomizo A, Qian C et al. Activation of p73 silent allele in lung cancer. Cancer Res 1998; 58: 2347-9. 9. Kaghad M, Yang A, Bonnet H et al. p53 family: What isp73 for? 9thp53 workshop 1998; 65 (Abstr). 10. Donehower LA, Harvey M, Slagle BL et al. Mice deficient torp53 are developmentally normal but susceptible to spontaneous tumours. Nature 1992; 356: 215-21. 11. Vikhanskaya F, Dincalci M, Broggini M. p73 competes withp53 and attenuate its response in a human ovarian cancer cell line. Nucleic Acids Res 1999 (submitted). 12. Nomoto S, Haruki N, Kondo M et al. Search for mutations and examination of allelic expression imbalance of the p73 gene at Ip36.33 in human lung cancers. Cancer Res 1998; 58: 1380-3. 13. Mai M, Qian C, Yokomizo A et al. Loss of imprinting and allele switching of p73 in renal cell carcinoma. Oncogene 1998; 17: 1739-41. 14. Takahashi H, Ichimiya S, Nimura Yet al. Mutation, allelotyping, and transcription analyses of thep73 gene in prostatic carcinoma. Cancer Res 1998; 58: 2076-7. 15. Harris CC, Hollstein M. Clinical implications of the p53 tumorsuppressor gene. N Engl J Med 1993; 329: 1318-27. 16. Mazars R, Pujol P, MaudelondeTet a\.p53 mutations in ovarian cancer: A late event? Oncogene 1991; 6: 1685-90. 17. Wertheim I, Muto MG, Welch WR et al. p53 gene mutation in human borderline epithelian ovarian tumors. J Natl Cancer Inst 1994; 86: 1549-51. 18. Hollstein M, Sidransky D, Vogelstein B et al. p53 mutations in human cancers. Science 1991; 253: 49-53. 19. World Health Organization. International Histological Classification of Tumours, Second Edition. Geneva: World Health Organization. Berlin: Springer-Verlag 1998. 20. UICC TNM Classification of Malignant Tumours, Fifth Edition. New York: John Wiley & Sons 1997. 21. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989. 22. Vogelstein B, Kinzler K.W. p53 function and d>sfunction. Cell 1992; 70: 523-6.
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
exon 2 of the gene, we determined, from DNA obtained from blood of cancer patients, a percentage of allelic distribution similar to that previously reported [1, 6]. LOH was found in two cases, one retaining the A/T allele and the other the G/C allele. We did not determine the extent of allelic loss in these two subjects but the results clearly showed loss of part of the/?73 gene in one allele. Comparison of the allelic expression of p73 in normal versus tumor tissues was not possible in the present study because the two normal ovarian tissues we obtained showed homozygosity at DNA level and could not be used for expression analysis. However, the only case in which it was possible to analyse DNA and RNA from both tumor and normal tissue showed biallelic expression in both the normal and tumor tissue. We decided therefore to try to distinguish between allelic expression on ovarian carcinomas versus borderline tumors. The available data from the literature in which ovarian carcinomas and borderline tumors were analysed, showed that the two classes always behave differently, and distinct gene expression levels could often be found [17, 25-27]. It was therefore reasonable to compare these two classes, and the results obtained did not reveal substantial differences in the expression of/? 73. In the majority of ovarian carcinomas as well as in borderline tumors, we found a biallelic expression of p73. Considering the accumulating data reporting biallelic expression of p73 in normal tissues [6, 12], our results tend to exclude a role for p73 in the pathogenesis or progression of ovarian carcinoma. We cannot, however, rule out the possibility that a specific monoallelic expression of p73 could be present in normal ovarian tissue, although the only informative normal tissue studied showed a biallelic expression. Another point to be considered is the relative expression of p73 in tumor tissue compared to normal tissue. The recent data reporting that p73 could compete with p53 for binding to DNA and therefore reduce the potential activity of p53 [11], suggest the possibility that tumor cells could overexpress p73 as an alternative way to inactivate p53, although there is still no direct evidence of these possible effects. Taken together, these data, like those reported for other tumor types, tend to refute the possibility of a role for/? 73 in ovarian cancer development.
References
953 23. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991; 351: 453-6. 24. Thompson FH,TaetIe R,Trent JMet al. Band Ip36 abnormalities and t(l;17) in ovarian carcinoma. Cancer Genet Cytogenet 1997; 96: 106-10. 25. Shelling AN, Cooke IE, Ganesan TS. The genetic analysis of ovarian cancer. Br J Cancer 1995; 72: 521-7. 26. Gallion HH, Pieretti M, DePriest PD et al. The molecular basis of ovarian cancer. Cancer 1995; 76: 1992-7. 27. Kristensen GB, Trope C. Epithelial ovarian cancer. Lancet 1997; 349: 113-7.
Received 26 March 1999; accepted 26 May 1999. Correspondence to: Dr M. Broggini Molecular Pharmacology Unit Istituto di Ricerche farmacologiche 'Mario Negri' via Eritrea 62 20157 Milan Italy E-mail: [email protected]
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
Original article Allelic expression ofp73 in human ovarian cancers ;1
A. M. Codegoni, F. Bertoni, C. Patregnani, E. Marinetti, M. D'lncalci & M. Broggini
^Molecular Pharmacology Unit, Department of Oncology, Istituto di Ricerche Farmacologiche 'Mario Negri', Milan; 2Ospedale S. Gerardo, University of Milan, Monza (MI), Italy
Introduction
The recently-described p73 gene has a close structural similarity to the tumor suppressor genep53 [1]. In particular, with respect to the central DNA binding domain of p53 which is responsible for downstream gene activation, its homology with p73 reaches 63% of identity at the amino acid level [1, 2]. The tumor suppressor/755 has been found mutated in almost 50% of human cancers; despite the strong structural similarities between p53 and p73, initial reports on p73 failed to reveal any mutations in different cancer types [3-5]. The p73 gene has been localized on chromosome Ip36, a region frequently found deleted in human cancers [1]. At least three putative tumor suppressor genes could be present in this area and/?73 was considered an attractive candidate suppressor gene. Available data, however, refute a role forp73 as a tumor suppressor [6-8]. In addition,/;73 knock-out mice show specific neuronal disorders and a defective immunological response but do not develop spontaneous tumors, which are invariably found in p53null mice [9,10]. The p73 gene was reported to be monoallelically expressed in normal human tissues [1], and it was found that there was expression of the silent allele in lung cancer, suggesting a possible oncogenic role for p73 [8], p73 binds the same DNA sequence recognized by p53, and we recently reported that/?73 can compete withp53 for binding to Dann, resulting in a generally lower transactivation ability of p53 in the presence of high
concentrations of p73 [11], which would fit with the overexpression of p73 found in cancer cells [6, 8]. The monoallelic expression ofp 73 in normal tissues has not been confirmed by other laboratories [6, 12] although in other tumor types allele switching [13] and monoallelic and biallelic expression [14] of/?73 have been reported. Genetic analyses of ovarian cancers have not yet suggested important genes in their oncogenesis. p53 is in fact found mutated in ovarian cancers, but it is more likely that its role is linked to tumor progression rather than tumor development [4, 15, 16]. In addition, many studies have reported a lack of correlation between p53 status and the survival of ovarian cancer patients [1618]. In 51 patients from whom tumor and normal cells were available, we studied the allelic expression of/?73 in ovarian cancer patients. Tumor allelic expression of this gene was compared in patients with ovarian cancer at different stages with borderline tumors.
Patients and methods Patients, pathology specimens and cell lines Samples from primary ovarian tumours and corresponding peripheral blood samples were obtained from the Department of Gynecological Oncology of the Ospedale San Gerardo, Monza (Italy). Tumour cases were diagnosed according to the World Health Organization Classification [19] and staged according to the International Federation of Gynecology and Obstetrics (FIGO) criteria [20]. Pathology samples were promptly frozen at -80 °C, until the time of analysis.
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
Results: We found an allelic distribution similar to that previously reported. LOH was found in two patients with Background: The p73 gene is structurally related to the tumor ovarian cancer. In one case in which normal ovarian tissue suppressor genep53. The role of p73 in tumor development is was available biallelic expression ofp73 was found. still unclear and no data on ovarian cancer are so far available. Conclusion: In comparisons of ovarian cancers and borderFor this reason we have analyzed, in a panel of ovarian line tumors, no differences in allelic distribution and/or excancers, the allelic distribution and expression ofp73. pression were found, suggesting that p73 does not play an Patients and methods: Fifty-one samples from ovarian important role in the pathogenesis and development of ovarian cancers and five human ovarian cancer cell lines growing in cancer. culture were analyzed. Allelic origin was analyzed by PCR after digestion with the restriction enzyme Sty I. Heterozygous, informative cases were selected for studies aimed at evaluating Key words: allelic expression, borderline tumors, ovarian allelic expression of/?73. cancer, p73, tumor suppressor gene Summary
950
<
<
<
<<
<<
<<
z z z z z z z z z z
N
T
N
T
N
T
N
<
o a c o r r o r r o r r o a :
T 229 b p 157 b p -
U
U
U U •ii6bp( • 84 bp.
72 b p -
Figure 1. Ethidium bromide staining of amplifyed DNA from normal or tumor tissue digested with Sty I to determine the allelic status of p73. In these conditions, the undigested amplified fragment (G/C allele) has a length of 229 bp, while the digested fragments (A/Tallele) have a length of 157 and 72 bp.
Figure 2. Representative gel showing allelic status and expression of p73 determined in DNA and RNA samples, respectively. For DNA, the length of the fragments is as indicated in Figure 1. For fragments amplified from RNA, the undigested (G/C allele) fragment has a length of 116 bp while the Sty digested fragment (A/Tallele) has a length of 84 bp.
The human ovarian cancer cell lines OVCAR-3, SW626, A2780, SKOV-3 and IGROV-1 were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS). The neuroblastoma cell line IMR-32 was grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FCS. DNA extraction was performed with the QIAamp Blood kit and with the QIAamp Tissue kit according to the manufacturer's instructions (Qiagen). Total RNA was extracted from untreated or treated cells by the guanidine/caesium chloride gradient method [21].
AT allele. In each experiment, DNA and RNA samples from the human neuroblastoma cell line IMR-32, previously characterized as having the presence and expression of the A/Tallele [1], was used to check the quality of the PCR reaction and enzyme digestion. Analysis of five human ovarian cancer cell lines showed that all presented the G/C; G/C alleles (Table 2). p73 allelic expression was evaluated in the heterozygous samples, in either ovarian carcinomas or in borderline tumors. Both of the two normal ovary samples available showed G/C; G/C homozygosity and therefore could not be used for allelic expression analysis. The same was found for the five human ovarian cancer cell lines analyzed, all showing G/C; G/C homozygosity at DNA level (Table 1). Our analysis focused on the possible differences between ovarian carcinomas and borderline tumors. Of the informative cases of ovarian carcinomas, five showed biallelic and one monoallelic expression (Figure 2). The two remaining cases (reported to have lost one allele at the DNA level) did express the allele retained. As regards the borderline tumors, six out of seven showed biallelic expression of p73, while only one case resulted in monoallelic expression. Paired normal and tumor tissue was obtained from one case only, in which we found biallelic expression of p73 in the normal ovary as well as in the tumor.
Allelic expression of p73 Heterozygous samples were detected by PCR on DNA samples using primers annealing to intronic sequences amplifying a fragment of 229 bp. After 35 cycles of amplification as described [8], the samples were digested with Sty I overnight, separated on agarose or polyacnlamide gels and stained with ethidium bromide. Heterozygous samples were selected for RT-PCR analysis which was performed with primers amplifying the C/T polymorphism. PCR conditions were as above except that a33P-dATP was included in the PCR reactions and, after digestion overnight with Sty I, the samples were resolved on polyacrilamide gels and autoradiographed.
Results Of the 51 specimens examined, 32 were from ovarian cancers, 17 from borderline tumors and two from normal ovaries. The Sty I polymorphism was utilized to study thep73 allele frequency using DNA collected from blood (Figure 1). Overall, the frequency of G/C; G/C homozygosity was 67%, A/T; A/T 2% and G/C; A/T heterozygosity 31%, values similar to those previously reported [1,6]. For the 15 heterozygous informative cases, the allelic frequency was analyzed at tumor DNA level (see Table 1). Thirteen of these (87%) retained both alleles, while apparent allelic loss was observed in two cases. In one case the G/C allele was retained and in the other the
Discussion Thep73 gene is one of the recently discovered members of the p53 family. Unlike p53, however, for which a defined tumor suppressor role has been established [2, 22, 23], the role of/? 75 in cancer progression and development is still unclear. Evidence presented so far tends to exclude a role for this gene as a tumor suppressor, particularly in those tumors with LOH at Iq36, the region
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
72 b p - *
951 Table I. Allelic expression ofp73 in human ovarian cancer samples. Case
Histologic type
Figo stage
Grade
Normal DNA p73 alleles
Tumour DNA p73 alleles
Tumour expressed alleles
126 128 131 132 138 140 145 148 149 150 154 155 156 157 165 167 168 175 176 180 191 191A 193 196 198 200 205 210 216 217 226 225 5 8 11 17A 19A 22A 24 25 42 43A 141A 142 160 164A 181A 209A 220 Nl N2
Serous cystadenocarcinoma Serous cystadenocarcinoma Endometrioid adenocarcinoma Endometrioid adenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Clear cell cystadenocarcinoma Mucinous cystadenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Mucinous cystadenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Unclassified tumor Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Unclassified tumor Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Endometrioid adenocarcinoma Endometrioid adenocarcinoma Serous cystadenocarcinoma Serous cystadenocarcinoma Teratoma Mucinous borderline tumour Serous borderline tumour Serous borderline tumour Serous borderline tumour Mucinous borderline tumour Mucinous borderline tumour Mucinous borderline tumour Serous borderline tumour Mucinous borderline tumour Serous borderline tumour Mucinous borderline tumour Serous borderline tumour Mucinous borderline tumour Serous borderline tumour Serous borderline tumour Serous borderline tumour Serous borderline tumour Normal ovarian tissue Normal ovarian tissue
me me
3 3 2 2 2 3 3 3 1 3 3 3 2 3 3 3 0 3 3 3 3 3 3 3 3 1 1 0 2 2 3 — _ _ _ -
G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; A/T G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; G/C G/C; A/T G/C; G/C G/C; A/T G/C; A/T G/C; A/T G/C; A/T G/C; G/C A/T; A/T G/C; G/C G/C; G/C G/C; G/C G/C; A/T G/C; A/T G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C G/C; G/C
_ G/C;A/T _ G/C; A/T G/C; A/T G/C G/C; A/T G/C; A/T G/C; A/T A/T G/C; A/T G/C; A/T G/C; A/T G/C; A/T G/C; A/T A/T; A/T _ G/C; A/T G/C; A/T _ _ _ -
A/T G/C; _ _ G/C; G/C _ G/C; G/C; G/C, AT G/C; _ G/C; G/C; A/T G/C; — _ _ G/C; G/C; _ _ -
IA IA
me me me lie
IA
me me me me me lie
me me me me me me me me
IA IC IB IC IA IC IA IB
me IC IA IA IA IA IA
me
IC IC IA IIIB IA IC IIIB _ -
A/T
A/T
A/T
A/T
A/T A/T A/T A/T
A/T A/T
in which the/?73 gene has been mapped [1]. In all of the conditions in which monoallelic expression in the nortumor samples examined so far, no mutations in thep73 mal adjacent tissue was found [8,13]. The possibility that gene have been found, as opposed to the high percentage p73 could be an imprinted gene remains, however, at of mutations found in the p53 gene [5, 12, 14]. Contrast- least debatable and more data on the possible loss of ing results have been obtained on the expression of the imprinting in cancer cells are needed to clarify this p73 gene. It was initially reported to be monoallelically point. In this study we analyzed the allelic distribution expressed [1], a result not confirmed by other authors and expression of p73 in ovarian cancer, a tumor for who reported a biallelic expression in normal subjects which alterations in the chromosomal region mapped by [6, 12]. In tumor samples, evidence of biallelic expres- p73 have been reported [24]. sion of p73 was reported in lung and renal cancers, in Taking advantage of a polymorphic site present in
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
me me
A/T
952 Table 2. Allelic status of/?75 in human ovarian cancer cell lines.
Acknowledgements
Cell line
Tissue type
p73 alleles status
Expressed allele
OVCAR-3 SW626 A2780 IGROV SKOV-3 1MR32
Ovarian cancer Ovarian cancer Ovarian cancer Ovarian cancer Ovarian cancer Neuroblastoma
G/C; G/C; G/C; G/C; G/C; A/T
The generous contributions of the Italian Association for Cancer Research (AIRC) and of the Nerina and Mario Mattioli Foundation are gratefully acknowledged.
A/T
G/C G/C G/C G/C G/C
1. Kaghad M, Bonnet H, Yang A et al. Monoallelically expressed gene related to p53 at Ip36, a region frequently deleted in neuroblastoma and other human cancers. Cell 1997; 90: 809-19. 2. Oren M. Lonely no more:p53 finds its kin in a tumor suppressor haven. Cell 1997; 90: 829-32. 3. Hollstein M, Soussi T, Thomas G et al. p53 gene alterations in human tumors: Perspectives for cancer control. Recent Results Cancer Res 1997; 143: 369-89. 4. Harris CC. The 1995 Walter Hubert Lecture - molecular epidemiology of human cancer: Insights from the mutational analysis of thep53 tumour-suppressor gene. Br J Cancer 1996; 73: 261-9. 5. Nimura Y, Mihara M, Ichimiya S et al. p73, a gene related top53, is not mutated in esophageal carcinomas. Int J Cancer 1998; 78: 437-40. 6. Kovalev S, Marchenko N, Swendeman S et al. Expression level, allelic origin, and mutation analysis of the p73 gene in neuroblastoma tumors and cell lines. Cell Growth Differ 1998; 9: 897903. 7. Kroiss MM, Bosserhoff AK, Vogt T e t al. Loss of expression or mutations in thep73 tumour suppressor gene are not involved in the pathogenesis of malignant melanomas. Melanoma Res 1998; 8: 504-9. 8. Mai M.Yokomizo A, Qian C et al. Activation of p73 silent allele in lung cancer. Cancer Res 1998; 58: 2347-9. 9. Kaghad M, Yang A, Bonnet H et al. p53 family: What isp73 for? 9thp53 workshop 1998; 65 (Abstr). 10. Donehower LA, Harvey M, Slagle BL et al. Mice deficient torp53 are developmentally normal but susceptible to spontaneous tumours. Nature 1992; 356: 215-21. 11. Vikhanskaya F, Dincalci M, Broggini M. p73 competes withp53 and attenuate its response in a human ovarian cancer cell line. Nucleic Acids Res 1999 (submitted). 12. Nomoto S, Haruki N, Kondo M et al. Search for mutations and examination of allelic expression imbalance of the p73 gene at Ip36.33 in human lung cancers. Cancer Res 1998; 58: 1380-3. 13. Mai M, Qian C, Yokomizo A et al. Loss of imprinting and allele switching of p73 in renal cell carcinoma. Oncogene 1998; 17: 1739-41. 14. Takahashi H, Ichimiya S, Nimura Yet al. Mutation, allelotyping, and transcription analyses of thep73 gene in prostatic carcinoma. Cancer Res 1998; 58: 2076-7. 15. Harris CC, Hollstein M. Clinical implications of the p53 tumorsuppressor gene. N Engl J Med 1993; 329: 1318-27. 16. Mazars R, Pujol P, MaudelondeTet a\.p53 mutations in ovarian cancer: A late event? Oncogene 1991; 6: 1685-90. 17. Wertheim I, Muto MG, Welch WR et al. p53 gene mutation in human borderline epithelian ovarian tumors. J Natl Cancer Inst 1994; 86: 1549-51. 18. Hollstein M, Sidransky D, Vogelstein B et al. p53 mutations in human cancers. Science 1991; 253: 49-53. 19. World Health Organization. International Histological Classification of Tumours, Second Edition. Geneva: World Health Organization. Berlin: Springer-Verlag 1998. 20. UICC TNM Classification of Malignant Tumours, Fifth Edition. New York: John Wiley & Sons 1997. 21. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989. 22. Vogelstein B, Kinzler K.W. p53 function and d>sfunction. Cell 1992; 70: 523-6.
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019
exon 2 of the gene, we determined, from DNA obtained from blood of cancer patients, a percentage of allelic distribution similar to that previously reported [1, 6]. LOH was found in two cases, one retaining the A/T allele and the other the G/C allele. We did not determine the extent of allelic loss in these two subjects but the results clearly showed loss of part of the/?73 gene in one allele. Comparison of the allelic expression of p73 in normal versus tumor tissues was not possible in the present study because the two normal ovarian tissues we obtained showed homozygosity at DNA level and could not be used for expression analysis. However, the only case in which it was possible to analyse DNA and RNA from both tumor and normal tissue showed biallelic expression in both the normal and tumor tissue. We decided therefore to try to distinguish between allelic expression on ovarian carcinomas versus borderline tumors. The available data from the literature in which ovarian carcinomas and borderline tumors were analysed, showed that the two classes always behave differently, and distinct gene expression levels could often be found [17, 25-27]. It was therefore reasonable to compare these two classes, and the results obtained did not reveal substantial differences in the expression of/? 73. In the majority of ovarian carcinomas as well as in borderline tumors, we found a biallelic expression of p73. Considering the accumulating data reporting biallelic expression of p73 in normal tissues [6, 12], our results tend to exclude a role for p73 in the pathogenesis or progression of ovarian carcinoma. We cannot, however, rule out the possibility that a specific monoallelic expression of p73 could be present in normal ovarian tissue, although the only informative normal tissue studied showed a biallelic expression. Another point to be considered is the relative expression of p73 in tumor tissue compared to normal tissue. The recent data reporting that p73 could compete with p53 for binding to DNA and therefore reduce the potential activity of p53 [11], suggest the possibility that tumor cells could overexpress p73 as an alternative way to inactivate p53, although there is still no direct evidence of these possible effects. Taken together, these data, like those reported for other tumor types, tend to refute the possibility of a role for/? 73 in ovarian cancer development.
References
953 23. Levine AJ, Momand J, Finlay CA. The p53 tumour suppressor gene. Nature 1991; 351: 453-6. 24. Thompson FH,TaetIe R,Trent JMet al. Band Ip36 abnormalities and t(l;17) in ovarian carcinoma. Cancer Genet Cytogenet 1997; 96: 106-10. 25. Shelling AN, Cooke IE, Ganesan TS. The genetic analysis of ovarian cancer. Br J Cancer 1995; 72: 521-7. 26. Gallion HH, Pieretti M, DePriest PD et al. The molecular basis of ovarian cancer. Cancer 1995; 76: 1992-7. 27. Kristensen GB, Trope C. Epithelial ovarian cancer. Lancet 1997; 349: 113-7.
Received 26 March 1999; accepted 26 May 1999. Correspondence to: Dr M. Broggini Molecular Pharmacology Unit Istituto di Ricerche farmacologiche 'Mario Negri' via Eritrea 62 20157 Milan Italy E-mail: [email protected]
Downloaded from https://academic.oup.com/annonc/article-abstract/10/8/949/200325 by guest on 29 January 2019