Role of activated c‐H‐ras Oncogene in the induction and progression of immortal liver epithelial cell lines derived from normal C3H mice

Abstract

The nature of 15 immortal mouse liver epithelial cell (MLEC) lines established from normal C3H mice was investigated specifically in terms of ras oncogene activation. Neither transforming activity nor point mutation within codon 61 of c-H-ras could be demonstrated in any of the cell lines by DNA transfection in a NIH/3T3 cell system or by the direct sequencing method after polymerase chain reaction, respectively. Acrylamide gel migration analysis of ras p21 products did not show any shift from the normal. Transplantation experiments demonstrated only 2 out of the 15 lines to be tumorigenic in nude mice. When 4 of the non-tumorigenic MLEC lines were transfected with cloned activated c-H-ras containing a point mutation within codon 61, they all became tumorigenic, the resultant neoplasms being hepatocellular carcinomas often associated with albumin mRNA expression. Our results thus indicate that ras activation is not necessary for immortalization or even for transformation of mouse liver cells in culture, and that ras activation may be an event during the progression process in mouse hepatocarcinogenesis in vivo.

Morphology, Proliferation and Apoptosis of Mouse Liver Epithelial Cells Cultured as Spheroids

“…The present study was conducted to assess the histological cell type of MLEC10 cells, known to serve as precursors for hepatocellular carcinomas, [13][14][15] placed in a 3-dimensional environment. In order to determine whether they correspond to altered hepatocytes 11,12) or oval cells 6,7) in vivo, two options were initially considered.…”

“…[13][14][15] The Ras-1 cell line is a clonal tumorigenic variant of MLEC10 obtained by transfection with an activated human H-ras oncogene.…”

“…19,20) Total RNA from fresh spheroids was extracted by the guanidinium thiocyanate method, 21) and 20 µg aliquots were electrophoresed in 1.2% agarose gels containing formaldehyde and stained with ethidium bromide to check equal loading and the intactness of 18S and 28S ribosomal RNAs. The RNA was then transferred onto a nylon filter and hybridized with rat p53, 22) human c-myc, 19) rat albumin, 14) or mouse β-actin (Wako, Osaka) probe cDNA 32 P-labeled by the random prime method. After stringent washing, hybridization signals on the filters were visualized by autoradiography with X-ray film overnight.…”

“…11,12) Previously, we established multiple liver epithelial cell lines from untreated, young C3H/HeN mice, approximately half of which expressed albumin. 13) Although most of the cell lines did not form tumors when backtransplanted into nude mice, they acquired tumorigenicity after transfection with an activated H-ras oncogene 14) or a hepatocyte growth factor gene.…”

“…[13][14][15] Since spontaneous hepatocellular tumors are frequently seen in old C3H/HeN mice, 16) we suggested that the cell lines from C3H/HeN mice may represent spontaneously initiated hepatocytes developing phenotypic immaturity due to long-term culture. 13) Consistent with this idea, liver epithelial cell lines were far more difficult to establish from C57BL/6N mice, which have a low incidence of spontaneous tumors.…”