RESUMEN
Infection with hepadnaviruses and exposure to dietary aflatoxin are considered major risk factors in the development of hepatocellular carcinoma (HCC) both in humans and in animals. Recently, a broad range of mutations in the p53 tumor suppressor gene has been reported in human HCCs, predominantly from hepatitis B virus carriers in areas with either high or low levels of exposure to dietary aflatoxin. To determine whether p53 mutations are common to HCCs of hosts infected with related hepadnaviruses with and without treatment with aflatoxin, we studied the occurrence of mutations in the p53 gene in HCCs of ground squirrels and woodchucks with history of infection with ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus, respectively. Sequencing of wild type p53 genes from ground squirrels and woodchucks revealed remarkable homology between the two species with only a few amino acid differences in exons 4, 8, and 9. Using direct polymerase chain reaction sequencing, we analyzed the state of the p53 gene (exons 4-9) in 20 HCCs from ground squirrels (2 uninfected, 7 with past, and 11 with ongoing infection with GSHV) and in 11 HCCs from woodchucks persistently infected with woodchuck hepatitis virus. Five GSHV carrier and two uninfected ground squirrels received i.p. administration of aflatoxin B1. We detected only one mutation in the p53 gene of the tested animals. This mutation was located in codon 176 of exon 5 in the HCC of a GSHV-positive ground squirrel treated with aflatoxin. Mutation was caused by a G to T transversion in the second position of the codon, resulting in the replacement of cysteine with phenylalanine, and was accompanied by a tumor-specific loss of heterozygosity. p53 allelic amino acid variation with sequences coding for aspartic acid or asparagine was present in codon 61 in the variable region of exon 4 in both HCCs and nonneoplastic tissues of ground squirrels. In view of the considerably lower apparent rate of mutations in comparison to human HCCs, we suggest a less important role for aflatoxin in the induction of p53 mutations in HCCs of ground squirrels. Alternatively, etiological factors other than p53 mutations may be of greater significance in the development of HCC in ground squirrels and woodchucks.
Asunto(s)
Aflatoxina B1 , Carcinoma Hepatocelular/genética , ADN Complementario/genética , Genes p53/genética , Infecciones por Hepadnaviridae/genética , Hepatitis Viral Animal/genética , Mutación/genética , Orthohepadnavirus/genética , Sciuridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/veterinaria , Infecciones por Hepadnaviridae/veterinaria , Hepatitis B/genética , Hepatitis B/microbiología , Hepatitis B/veterinaria , Virus de la Hepatitis B de la Marmota/genética , Hepatitis Viral Animal/microbiología , Marmota/genética , Marmota/microbiología , Datos de Secuencia Molecular , Sciuridae/microbiología , Especificidad de la EspecieRESUMEN
Hepatitis B virus (HBV) is the causative agent of hepatocellular carcinoma (HCC) in man. The HBV genome is a circular partially double-stranded DNA molecule of about 3.2 kb. The HBV genome contains four structural genes coding for the HBV envelope (HBsAg) and core (HBcAg/HBeAg) proteins, endogenous DNA-polymerase with the additional enzymatic activity of a reverse transcriptase and polypeptide X functioning as a trans-activator of cellular and viral genes. HBV DNA integration in the genomes of HCCs and hepatocytes of HBV carriers is an important evidence establishing a relationship between the HBV infection and the development of HCC. The mechanism of HBV DNA integration into the cellular genome and the possible role of integrated HBV DNA sequences in the malignant transformation of hepatocytes are discussed.
Asunto(s)
Carcinoma Hepatocelular/etiología , Hepatitis B/complicaciones , Neoplasias Hepáticas/etiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/microbiología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/microbiologíaRESUMEN
Three fragments of cellular DNA containing integrated hepatitis B virus (HBV) sequences have been cloned from the genomic library of a PLC/PRF/5 cell line. The complete nucleotide sequences of the HBV DNA inserts have been determined, including the S gene and the cellular flanking DNA. Viral sequences were found to be fragmented and rearranged. The nucleotide sequences of the HBV-HBV and HBV-human DNA junctions in two of the clones were precisely the same as described by others for the analogous HBV-DNA inserts, providing direct evidence for the stability of HBV-DNA integration pattern and sequence in the genome of the PLC/PRF/5 line. Two clones contain the HBV surface antigen gene which is well conserved. According to the amino acid sequence it could be related to the adw subtype. HBV DNA and contiguous human sequences in HC217 clone are flanked by the cellular perfect inverted repeat of at least 3.5 kb. Similar sequences have been found in the genome of the original PLC/PRF/5 cell line and human placental DNA.
