A human hepatocellular carcinoma 3.0-kilobase DNA sequence transforms both rat liver cells and NIH3T3 fibroblasts and encodes a 52-kilodalton protein.

Neoplastic transformation of rat liver cells in vitro by DNA-mediated gene transfer with an oncogene, hhcM, derived from human (Mahlavu) hepatocellular carcinoma, is described and compared with that of NIH3T3 cells. hhcM was cloned in a neomycin-resistant simian virus 40 promoter vector (pNeor/S) and was designated pNrpM-1. BRL-1 or NIH3T3 cells, transfected with pNrpM-1 DNA, showed significant morphological changes, loss of contact inhibition, and anchorage-independent growth. They became highly tumorigenic in nude rats and nu/nu mice. Control BRL-1 and NIH3T3 cells, whether transfected with pNeor/S DNA or not, remained contact inhibited and nontumorigenic. Both the transformants and the tumor cells contained integrated hhcM DNA as shown by Southern blot hybridization. The complete nucleotide sequence of the hhcM 3.0-kilobase DNA was also determined, and it consisted of a possible open reading frame for a protein of 52 kilodaltons (467 amino acids). The high-level production of a slightly modified form of this 52-kilodalton protein in a bacterial expression system has been successfully achieved. The bacteria-produced protein was similar in electrophoretic behavior to the 52- to 53-kilodalton protein synthesized in a cell-free translation system using rabbit reticulocyte lysate programmed with hybrid-selected hhcM-specific mRNA from Mahlavu hepatocellular carcinoma cells.

Transforming DNA sequences of human hepatocellular carcinomas, their distribution and relationship with hepatitis B virus sequence in human hepatomas.

Several related human transforming DNA sequences, hhc, and a putative normal liver homologue, c-hhc, have been molecularly cloned from the genomic DNAs of individual African and Asian hepatomas and from normal liver respectively. hhcM (Mahlavu) and hhcK3 (Korean), but not c-hhc, transformed NIH3T3 cells in DNA-mediated gene transfer assays. Transformed cells were found tumorigenic in athymic NIH Swiss nu/nu mice. In view of recent epidemiological studies implicating hepatitis B virus (HBV) infection early in life as causative for the eventual development of primary hepatocellular carcinoma in humans in Southeast Asia, the Far-East, and certain areas of Africa, we hereby analyzed the relationship between these hhcs and HBV in a survey of 20 hepatomas for DNA sequences homologous to hhcM and HBV by sequential hybridizations against [32p]hhcM and [32p]HBV probes. hhcM related DNA sequence were found highly amplified in 80% of the 20 hepatomas but HBV DNA sequence was rare or low. hhcM lends itself as a marker for human hepatomas. However, overall results indicated that patients with integrated HBV DNA sequences showed high copy number of hhcM sequence. Furthermore, EcoR1-restricted hepatoma DNAs showed that HBV and hhcM DNA sequences resided at different fragments in hepatomas. Our results suggest that HBV contributes to hepatocarcinogenesis probably via an activation mechanism involving possibly an integration or transient interaction of HBV DNA with hepatocyte DNA sequences, leading to recombination and eventual amplifications of the hhcM sequence in Mahlavu.

Dose dependency of aflatoxin B1 binding on human high molecular weight DNA in the activation of proto-oncogene.

The binding of aflatoxin B1, AFB1, a potent hepatocarcinogen, to various high molecular weight (HMW) DNAs from human normal liver and two liver cancer cell lines, Alexander primary liver carcinoma (PLC) and Mahlavu hepatocellular carcinoma (hHC) and from NIH/3T3 cell have been investigated. The kinetics of AFB1 binding to these DNAs showed similar initial rates but the extents of binding to the PLC and hHC DNAs seemed to be slightly higher. Preferential AFB1 bindings were identified in both PLC and hHC DNAs compared to normal liver DNA when analyzed by restriction endonuclease digestions and agarose gel electrophoresis. A critical AFB1 binding dosage, ranging 100 to 460 fmole/microgram DNA, was found to activate the carcinogenic effect of the Mahlavu hHC HMW DNA, but not normal liver HMW DNA, rendering it capable of inducing focal transformation in NIH/3T3 cell. Excessive AFB1 binding on the hHC and PLC HMW DNAs resulted in an "over-kill" of both cell transformation capability and templating activity of the DNA.