ras oncogene activation does not induce sensitivity to natural killer cell-mediated lysis in human melanoma.

An important phenomenon in tumor immunology that has come under recent attention is the impact of oncogene activation in tumor cells on the sensitivity to lysis by immune effector cells. Several studies suggested that transfer of an activated ras oncogene into cultured rodent fibroblasts induces susceptibility to natural killer cell (NK)-mediated lysis. Experiments using human tumor cells, however, have produced conflicting data on the effect of ras activation in this respect. In studying the activation of the oncogene c-myc, which is often overexpressed in human melanoma, we have found that in cell lines expressing high levels of Myc protein, the sensitivity to lysis by NK cells was dramatically increased due to reduced expression of Human Leukocyte Antigen B locus products. Since the N-ras oncogene was found to be activated in 15% of human melanomas, we examined the possibility that in melanoma, in analogy to the murine systems, the mutated ras oncogene may influence NK susceptibility of human melanoma cells. Two N-ras genes harboring frequently found mutations were cloned into an expression vector. Transfection of the IGR39D melanoma cell line with wildtype and mutant N-ras constructs yielded several ras-expressing clones that were tested for NK sensitivity. Neither high expression of the wildtype N-ras protein, nor expression of two mutant proteins (N61-arg, N61-lys) was shown to result in enhanced NK-mediated lysis. We conclude that activation of ras oncogenes does not lead to the induction of an NK-sensitive phenotype in human melanoma cells.

Activation of the mas oncogene involves coupling to human alphoid sequences.

We have shown previously that mouse NIH3T3 cells transfected with DNA from a human ovarian carcinoma were rendered tumourigenic by an activated mas oncogene in four independent transfection experiments. In all cases the 5'-noncoding region was rearranged in comparison to the original ovarian tumour DNA. We now report that in all four transfectants the newly acquired sequences consist of human centromeric alpha satellite repeat DNA. In at least three transfectants the alphoid DNA originates from the centromere of chromosome three. Analysis of the sequences of the recombination site in one transfectant revealed that a homologous sequence of five base pairs (CAGCA) is present in both parental strands, and might thus have contributed to the recombinational event. To establish a conclusive role for alphoid DNA in the activation of mas, we performed a co-transfection experiment in NIH3T3 cells with cloned alphoid DNA and the mas coding sequence. We show that the transfectants expressing a transformed phenotype contain amplified mas linked to alphoid DNA. NIH3T3 cells transfected with plasmids that contained alphoid sequences cloned directly upstream of the mas coding sequence, and injected into nude mice, gave rise to tumours with amplified mas sequences (7/7). In six of these tumours the alphoid sequences were amplified as well. Our data suggest a novel mechanism of oncogene activation: recombination with normal alphoid repeat DNA resulting in amplification of the oncogene.

Establishment and characterization of 7 ovarian carcinoma cell lines and one granulosa tumor cell line: growth features and cytogenetics.

The characteristics of 7 newly established ovarian carcinoma cell lines and one granulosa tumor cell line obtained from tumor samples of 7 patients with varying histology of the primary tumor are reported. The cell lines were isolated from 5 serous carcinomas, a mucinous carcinoma, an endometrioid carcinoma and a granulosa cell tumor. All cell lines were passaged at least 25 times and showed stable growth rates. Colony-forming efficiency varied on plastic from 2 to 57% and in agar from 0.01 to 9.30%. The DNA index of the granulosa tumor cell line was diploid, while the ovarian carcinoma cell lines were all aneuploid. In 2 cell lines polyploidisation occurred during culturing. A thorough cytogenetic analysis of 7 cell lines revealed that the granulosa tumor cell line has only minor cytogenetic abnormalities (+5, 22q+). In contrast, the epithelial ovarian-cancer cell lines gave very complex karyotypes with numerous markers and structurally rearranged chromosomes. The chromosomes most often in excess were 15 and 20. Structural rearrangements of chromosomes 1, 3, 7 and 11 were prominent in all ovarian cell lines. In addition, we found changes in chromosomes X, 5, 8 and 13 that have rarely been described before.