Detection of transforming oncogenes in rat colon tumors induced by direct perfusion with N-methyl-N-nitrosourea.

We assayed rat colon tumors induced by N-methyl-N-nitrosourea (MNU) for transforming oncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA from 3 of 3 adenomas and 3 of 5 carcinomas induced transformed foci on NIH 3T3 cells. DNA from 2 of 3 primary foci also possessed focus-forming activity, and rat-specific sequences were observed in secondary focus DNAs. Furthermore, NIH 3T3 cells transfected with DNA from a carcinoma and from a primary focus derived from it, both positive in the focus-forming assay, induced tumors in nude mice. We found no evidence for rat H-ras, K-ras, or N-ras sequences in the DNA of any of 16 primary foci derived from 6 rat tumors; thus, in contrast to other animal tumor models induced by MNU, activation of the ras genes does not appear to predominantly occur in MNU-induced rat colon tumors. We also did not observe, in any of these foci, sequences corresponding to the rat neu, raf, fms, met, or hst genes, thus indicating that none of these is the transforming oncogene in our model. These results suggest that an as yet unidentified transforming oncogene may be activated in rat colon tumors induced by MNU.     

Establishment of human retinal pigment epithelial cell lines by oncogenes

The primary human retinal pigment epithelial cells were transfected with oncogenic sequences derived from viruses and cellular homologues of retroviral oncogenes 'protooncogenes' linked to simian virus 40 (SV-40) and retroviral promoters. Foci of cells were noted between 2 to 4 weeks after transfection. Individual colonies of cells were expanded from cultures transfected with SV-40 virion DNA, SV-40 large T antigen gene, Ha-ras oncogene, human and mouse c-myc and adenovirus E1A gene. Established cell lines tested were positive for the specific oncogene sequences by Southern hybridization and also expressed the protein as assayed by immunofluorescence and immunoblot analysis. Cell lines established with SV-40 large T antigen, and SV-40 virion DNA, exhibited epithelioid morphology up to the 25th passage and later became more rounded. However, all cell lines established with other oncogenes continued to retain epithelial morphology. Functional analysis of the cell lines demonstrated the presence of polarity and the ability to phagocytize rod outer segments, characteristics of retinal pigment epithelial cells. The use of oncogenes with immortalization/transformation potential may allow the establishment of cell lines from ocular tissues for analysing the biochemical basis of a disease like retinitis pigmentosa.     

Correlation of growth capacity of cells in hard agarose with successful transfection by the activated c-Ha-ras oncogene and in vivo proliferative capacity at metastatic sites

The purpose of this study was to determine whether the degree of anchorage-independent growth of rodent or human cells in increasing concentrations of agarose correlated with successful transfection of the cells with an activated c-Ha-ras oncogene and tumorigenicity in nude mice. NIH 3T3 cells, C3H 10T1/2 fibroblasts, four clones of the murine K-1735 melanoma with different metastatic capacities and the TE85 human osteogenic sarcoma line were transfected with plasmids containing the 6.6-kilobase BamHI fragment of the mutant human c-Ha-ras gene and the neo gene, which confers resistance to neomycin (pSV2-neoEJ). Cells transfected with pSV2-neo, a plasmid containing the neo gene, served as controls. Cells from parental or transfected lines (selected by Geneticin) were plated into medium containing 0.3%, 0.6% 0.9%, or 1.2% agarose. These cells were also injected subcutaneously and intravenously into nude mice. The production of tumor cell colonies in dense agarose (greater than or equal to 0.6%) correlated with successful transfection with pSV2-neoEJ and production of experimental metastases in the lung of nude mice. We conclude that the degree of anchorage-independent growth of cells predicts successful transfection with activated c-Ha-ras oncogene and tumorigenic behavior in vivo. Thus this technique may be useful for the detection of cells transfected with transforming oncogenes.     

Thyroid epithelial cell transformation by a retroviral vector expressing SV40 large T

A recombinant murine retroviral vector encoding the SV40 virus large T antigen was used to infect stably an immortal line of differentiated rat thyroid epithelial cells, FRTL-5. Expression of SV40 T transformed these cells to anchorage independence and tumorigenicity but did not alter morphology or abolish tissue-specific functions and growth factor requirements. The resulting phenotype provides a model of well-differentiated human thyroid cancer.     

A simple, general method for detecting retroviral RNAs expressed in cells

A simple, efficient method has been developed for detecting retroviral RNAs expressed in cells. In this method, total RNAs or poly A+ RNAs extracted from various human cells are separated by electrophoresis and hybridized with synthetic oligonucleotides corresponding to the 3'-terminal 18 nucleotides of various tRNAs. Genomic and subgenomic RNAs of HTLV-I and HTLV-II in virus-infected cells and of xenotropic murine leukemia virus expressed in human lung cancer cells were easily detected with the tRNA(Pro)-derived oligonucleotide probe. This technique can be used to search for unidentified retroviruses expressed in human cancer cells and tissues.     

Retroviral heterogeneity in mouse lymphomas

Provirus modifications which can occur in tumors may lead to tumor antigenic variants. To investigate this possibility we compared the restriction fragment length patterns of mouse mammary tumor virus (MMTV), murine leukemia virus and xenotropic and mink cell-focus inducing (MCF)-related sequences, using Southern blot analysis, in a panel of Balb/c and C57BL/6 lymphomas, some of which were known to express novel histocompatibility (H) antigens. The results indicate differences in amplification and novel integration sites of retroviral sequences in all tumors analyzed, the number of new sequences depending on the specific retroviral sequence. A relationship has been found between integration of new MMTV sequences and a known susceptibility to lysis by syngeneic alloactivated lymphocytes in the same group of tumors. The possibility that retroviruses can induce the expression of novel histocompatibility antigens on tumors is discussed.     

Neoplastic Transformation of Rat Colon Epithelial Cells by Expression of Activated Human K-ras

Somatic mutations of the K- ras oncogene play an important role in colorectal carcinogenesis. We determined whether rat colon epithelial cells could be transformed by introducing retroviruses carrying the activated human K- ras oncogene alone. Primary epithelial cells from the rat distal colon were infected with retroviruses carrying wild-type and two types of activated K- ras (asp and val at codon 12) cDNAs. Cells infected with the wild-type K- ras virus showed no change in morphology and died within 3 weeks, whereas the activated K- ras virus-infected cells underwent morphological changes within 3 days and continued to proliferate. From these cells, several cell lines were subsequently established. Epithelial cells transformed by activated K- ras formed colonies in soft agar culture and tumors in athymic nude mice. Multiple copies of human K- ras genes and large amounts of K- ras mRNAs and proteins were found in the transformed cells. These data suggest that overexpression of activated K- ras transforms rat colon epithelial cells.