Reconstitution of wild-type p53 gene in Hep3B cells using adenovirus-mediated transfer

Reconstitution of wild-type p53 gene in Hep3B cells using adenovirus-mediated transfer

HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995 329 AASLD A NOVEL abI-LIKE GENE ASSOCIATED WITH LIVER CIRRHOSIS AND HCC. JY Liubimova~ SE Wilson*~ J Weng*...

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HEPATOLOGY Vol. 22, No. 4, P t . 2, 1995

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AASLD

A NOVEL abI-LIKE GENE ASSOCIATED WITH LIVER CIRRHOSIS AND HCC. JY Liubimova~ SE Wilson*~ J Weng*~ LM Petrovie~ SA Geller~ R Lopez~ A Hoffman~ AA Demetriou. Departments of Surgery, Pathology and Liver Support Unit, Cedars-Sinai Medical Center, Los Angeles, CA, *Department of Ophthalmology, University of Texas S0uthwestem Medical Center, Dallas, TX, USA.

ABSTRACTS

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RECONSTITUTION OF WILD-TYPE P53 GENE IN HEP3B CELLS USING ADENOVIRUS-MEDIATED TRANSFER Reiser M. Zhang WW 1. Lau JYN.

Section of Hepatobiliary Diseases, University of Florida, Gainesvllle, FL; and MD Anderson Cancer Center 1, University of Texas, Houston, TX. ILackm'ouud Mutations/deletion of the tumor suppressor gene, p53, were found to be common in hepatocellular carcinoma (HCC). The HCC-derived cell line, Hep3B, has been shown to have a homozygous p53 deletion. In addition, Hep3B does not express detectable levels of retinoblastoma gene mRNA or protein (RB). Hypothesis Reconstitetion of wild-type p53 to Hep3B may restore the phenotype or cause cell death. Aim To reconstitute wild-type p53 into Hep3B cells using adenovirus-mediated transfer. Methods A recombinant, replication deficient, adenovirus expressing wild-type p53 was constructed by Substituting the adenoviral E1 region with a p53 expression cassette consisting of human CMV promoter, wildtype p53 eDNA and SV40 polyadenylation signal (Ad5CMV-p53). Recombinant adenovirus with CMV promoter and polyadenylation signal was used as a control (Ad5CMV-pA). The titers of the recombinant viruses were determined by plaque assay using 293 cells which complement El. Hep3B cells were transduced with a multiplicity of infection (MOI) of 0, 5, 10, 20, 40 and 100 and assessed on day 3, 5 and 7. Expression of p53 was determined by immunohistochemistry. Cell proliferation was determined by an ELISA based on BrdU incorporation. Cell death was determined by counting the detached cells. Results The control adenovirus showed a dose-dependent increase in toxicity from a MOI of >20 to 100 (D50 at a MOI of 70). Assessment was only possible in the range of MOI 10-40. Expression of p53 p53 is known to have a short half-life. In Hep3B cells infected with Ad5CMVp53 at a MOI of up to 40, only 30% of the cells were p53 positive as determined by immunohistochemistry. The positive signal was detected exelusi~'elyin the cell nucleus. However, all dead ceils were positive for p53, which is likely to represent adenovirus toxicity, since similar proportion of dead cells were obtained with Ad5CMV-pA. Cell Morphology No gross morphological changes were observed in p53 positive Hep3B cells. Cell Proliferation/Death No difference in BrdU incorporation or cell death was observed between AdSCMV-p53 and Ad5CMV-pA (control) infected Hep3B cells from a MOI of 0-100. Conclusions (1) p53 can be reconstituted in Hep3B cells with nuclear localization using adenoviral gene transfer. (2) Even up to a MOI of 40, this approach did not restore the phenotype or induce cell death, which may be related to either (a) the limited therapeutic ratio, or (b) other genes like RB is also playing an important role in carcinogenesis.

IDENTIFICATION OF PROTEINS F R O M A HUMAN L I V E R eDNA L I B R A R Y T H A T B I N D TO W I L D - T Y P E HUMAN p53. Hao Yuwen Edward Tabor. National Cancer Institute, National Institutes of Health, Bethesda, MD. Mutations or deletions in the p53 gene are found in up to 50% of hepatocellular carcinomas from some countries. The role of p53 in controlling the growth of hepatocytes and of transformed hepatocytes might be regulated by other cellular proteins that interact with p53. Methods: A eDNA expression library made from normal human liver was screened in E. coil. Wild-type human p53 produced by means of a recombinant vaceinia virus in CV-I monkey kidney cells was used to detect p53 binding proteins. Binding was detected using monoclonal antibody to p53 (pAB-1801) and the enhanced chemiluminescence (ECL) system. Nueleotide sequences were determined using the dideoxy chain termination method. Results: Bacteriophage containing gene fragments coding for p53 binding proteins were isolated; one of nine such fragments showed extensive homology (98%) with the carboxyl end of a cellular protein that is known to bind to p53 (human double-minute [HDM2] protein). Six others were identified among the nine p53-binding products; one of these was homologous (96%) to the enzyme topoisomerase lib, although this has not yet been confirmed by other in vitro studies. Discussion: These findings are consistent with the concept that wildtype p53 may be regulated by binding by the cellular protein HDM2 (analogous to mdm2 in the mouse model). As in some other human cancers, amplification of the HDM2 gene could interfere with the function of p53 in the absence of a mutation or deletion in p53 and contribute to hepatocarcinogenesis. If the finding of a binding protein related to topoisomerase IIb is confirmed, it would have important implications for concepts of the role of p53 in hepatocarcinogenesis.

To identify genes which may be involved in liver neoplasm development, we studied HGF and its receptor c-met proto-oncogene and their products by RT-PCR and immunofluoreseence in 4 normal livers, 11 cirrhotic livers (HCV, HBV and ALD) and 6 differentiated hepatocellular carcinomas (HCC). RNA extraction and serial sections were made from fresh-frozen human livers. C-met gene was expressed in all normal, 6 of I 1 cirrhotic and all HCC livers. HGF gene expression was found in 4 of 11 cirrhotic livers and all HCC, but not in normal livers. Immunostaining for c-met product was seen in the cytoplasm of bepatocytes and biliary epithelium in normal, cirrhotic livers and in HCC. In normal livers, c-met staining was much weaker than in HCC and cirrhotic livers. Weak and occasional HGF staining was observed in normal blood vessel endothelium. In cirrhoses and HCC, a strong staining wa s seen in the vessel endothelium and bile duct cells. During RT-PCR of the c-met gene, we consistently detected a PCR band of approximately 150 bp in cirrhotic and HCC livers. The band was cut from the gel and sequenced. The 154 bp. product was compared with known sequences in the NCBI Database. No known gene matched this sequence. Partial homology was found with several genes, with the most similarity to human c-abl proto-oncogene. The 112 bp between c-met primers specify an open reading frame. Using the primers for the 101 bp internal fragment of this abl-like gene, we studied its expression in 18 human fresh-frozen livers by RT-PCR: 4 normal, 1 fulminant, 7 cirrhotic and 6 HCC. The abl-like gene was not expressed in normal or fulminant livers. In contrast, all cirrhotic livers and 5 of 6 HCC expressed the gene. This expression pattern did not correspond to that of c-met which was always detected in normal livers. The new gene.might be a mediator of signal transduetion during cell transformation. Work is in progress t o clone and characterize this gene.

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p53 ABNORMALITIES 1N H E P A T O C E L L U L A R

CARCINOMAS F R O M THE UNITED STATES. Y. Kazachkov ~, V.I. Khaoustov2, B. Yoffe2, H. Solomon3, G.B.G. Klintmalm3, E. Tabor ~. ~National Cancer Institute, National Institutes of Health, Bethesda, MD; 2VAMC, Baylor Coll. of Med., Houston, TX; 3Baylor Univ. Med. Center, Dallas, TX. The precise sequences of mutations in the p53 tumor suppressor gene in hepatocellular carcinomas (HCCs) from the United States have only rarely been reported and never for the entire coding region. Methods: The entire coding region (exons 1-11) of p53 was studied in 12 HCCs from the United States. Genomic DNA was subjected to PCR, single strand conformation polymorphism analysis, and sequencing, and was compared to a database of published p53 mutations (Cariello et al, Cancer Res. 1994, 54:445460). Results: p53 mutations were found in 5/12 HCCs, including single-base substitutions at one site in 4 HCCs (at codons 131,267, 278, 282), and three substitutions in a 5th HCC (at codons 56, 118, and the first position of 74, followed by a deletion). Mutations at these eodons are uncommon and have been reported in <1% of HCCs worldwide and in <3.5% of all human tumors. However, mutations at G, preceded by G and followed by C, found in one HCC in the present study, is the most frequently reported pattern of p53 mutation in HCCs in the database (113/288 reported HCCs). (A p53 mutation was found in only 1/10 nontumorous livers available from the same HCC patients.) Conclusions: 1) p53 mutations were found in 40% of HCCs from the United States and at sites uncommon in HCCs from other regions. 2) Analysis of all 11 exons of p53 (not previously reported in HCCs) did not reveal any mutations outside of the highly conserved exons 4-8. 3) No mutations were found at the "hot spot" codon 249.