Loss of heterozygosity (LOH) at 1p36 occurs in a number of solid tumors including hepatocellular carcinoma (HCC). Recently, a novel gene, p73, has been identified at 1p36.33. p73 is structurally and functionally related to p53 located at 17p13.1, which is a target for inactivation in HCCs. p73 produces at least two splicing variants, p73โฃ and โค, and a polymorphism in exon 2 results in two alleles, GC or AT. Initially, only the AT allele and p73โฃ transcripts were identified in malignant cell lines, suggesting a role for these in the malignant phenotype. The aims of this study were to determine the extent of LOH at 1p36 and 17p13.1 in HCCs from Australia and South Africa, and to identify patterns of p73 mRNA and p73 and p53 protein expression. LOH at 1p36 was found in 8 of 25 Australian and 6 of 10 South African cases. p73 mRNA expression occurred in 8 HCCs, but not in nonmalignant liver tissue. Two of these 8 HCCs had LOH of 1p36. Both โฃ and โค transcripts were observed in GC/GC homozygotes and GC/AT heterozygotes. No p73 protein expression was observed by immunohistochemistry in nonmalignant liver tissue or in HCC. p53 inactivation appeared to be associated with up-regulation of p73 expression, suggesting a compensatory role for p73 in this situation. The LOH at 1p36 implies a liver-specific tumor suppressor gene is in this region. However, the upregulation of p73 mRNA suggests p73 is not the target of this loss. (HEPATOLOGY 2000;31:601-605.)
Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, with an estimated annual incidence of 1 million cases. 1,2 The molecular mechanisms involved in hepatic carcinogenesis are poorly understood. Inactivation of the p53 tumor suppressor gene (TSG) is one of the most common abnormalities in HCC, particularly in those from Sub-Saharan Africa and Asian countries where hepatitis B infection is endemic and dietary exposure to aflatoxin is significant. 3,4 p53 is located at chromosome 17p13.1 5 and plays a role in the recognition and response to DNA damage that includes initiation of cell-cycle arrest by activation of p21 waf and/or apoptosis. 6 Recently, a putative TSG, designated p73, was identified at 1p36.33, 7 a common region of loss in neuroblastoma and other cancers including HCC. p73 shows significant sequence homology with p53, implying that it may have similar biological activity. 7 There are at least 2 transcripts of p73, p73โฃ and p73โค, with p73โค lacking exon 13. 7 When overexpressed in cell culture, both p73โฃ and p73โค have a functional role that is similar to p53, activating the cell-cycle inhibitor, p21 waf , and inducing apoptosis. 7,8 However, p73 exhibits different regulatory responses to DNA damage. 9 Demonstration of inactivation of p73 by mutation or decreased expression would provide additional support for p73 being a TSG. Several studies have attempted to identify mutations of p73 in a variety of malignancies, [10][11][12][13][14][15][16] and to date, a number of polymorphisms have been described, 14 but only one mutation, and that in only one breast cancer. 11 Two studies have found evidence that the paternal allele of p73 is silenced by imprinting. 7,17 In this situation, loss of the maternal allele would inactivate p73 function and support its role as a TSG. However, in a variety of malignancies, p73 expression is biallelic. 10,18 In exon 2 of p73, there is an allelic polymorphism consisting of a double-nucleotide substitution at positions 4 and 14. In DNA, this double-nucleotide substitution results in either a GC or AT allele. This sequence is immediately proximal to the initiation codon for p73 and potentially gives rise to a stem loop structure that could influence protein expression. 7 In the neuroblastoma cell lines, but not in peripheral blood lymphocytes from blood donors, only the AT allele was expressed, and in this situation, only p73โฃ transcripts were identified, suggesting that there was an interaction between the AT allele, p73โฃ expression, and the malignant phenotype. 7 The importance of p53 as a TSG in HCC, the structural and functional similarity of p73 and p53, and the location of p73 in a region of significant chromosomal loss in HCC suggest p73 may function as a TSG in the liver. The aim of this study was to assess the role of p73 in HCC, specifically the evidence in support of its role as a TSG, and the relationship between inactivation of p73 and p53. Loss of heterozygosity (LOH) of p53 and p73 was examined in a series of 25 Australian and 10 South African HCCs, to identify the rates of LOH in these regions, and the extent of loss on 1p36.3 in the vicinity of p73. The GC/AT exon 2 polymorphism and reversetranscription polymerase chain reaction (RT-PCR) were used to determine the relationship between these alleles and p73โฃ
Abbreviations: HCC, hepatocellular carcinoma; TSG, tumor suppressor gene; LOH, loss of heterozygosity; RT-PCR, reverse-transcription polymerase chain reaction; TBS, Tris-buffered saline.