The analysis of malignancy by cell fusion. VII. cytogenetic analysis of hybrids between malignant and diploid cells and of tumours derived from them

ABSTRACT A cytogenetic analysis was made of a series of hybrids between malignant and diploid mouse cells and of the tumours produced by the injection of these hybrids into immune-suppressed syngeneic animals. The malignant cells were crossed with diploid fibroblasts and lymphocytes. As we had found previously with other such crosses, the tumorigenicity of the malignant cells was profoundly depressed when they were fused with the diploid cells; fibro-blasts were more effective in this respect than lymphocytes. The ability to generate progressive tumours reappeared in almost all the hybrid cell populations on continued cultivation in vitro. The most informative crosses were those made between diploid cells and two clonal derivatives of a malignant melanoma. The adaptive chromosomal changes produced by growth in vitro were found to be very similar in a number of independently derived hybrid clones; and the tumours arising from the injection of these clones showed remarkably consistent patterns of chromosome elimination. In biparental crosses between the melanoma cells and diploid cells, only those hybrids in which one copy of chromosome 15 had undergone duplication succeeded in generating proliferating colonies. It did not seem to matter whether the duplicated chromosome 15 was derived from the malignant or the diploid parent cell. This requirement for an increased dosage of some gene or genes located on chromosome 15 was not a peculiarity of the melanoma crosses; it was also seen in other hybrids between malignant and diploid cells. Of the systematic chromosome losses seen in the melanoma × diploid cell hybrids during the period of cultivation in vitro, the most striking was the regular elimination of one copy of chromosome 4. By the use of appropriate cytogenetic and enzyme markers, it could be shown that the selection pressure against this chromosome was directed specifically against the homologues derived from the diploid parent. All tumours arising from the injection of these hybrids were found to have eliminated both copies of the diploid chromosome 4; both copies of the malignant chromosome 4 were, however, retained. Selection pressure directed specifically against the diploid homologues of chromosome 4 could also be demonstrated in tumours arising from hybrids between diploid cells and the cells of a fibroblastic sarcoma. Analysis of crosses between the melanoma derivatives and diploid cells in which either one or both copies of chromosome 4 were involved in cytologically recognizable rearrangements permitted more precise mapping of the genetic locus against which the selection pressure was directed: this locus was found to be situated in the lower part of the upper half of chromosome 4. Since, in the hybrid cells, the selection pressure against chromosome 4 discriminates categorically between the diploid and the malignant homologues, it can be concluded that in both the melanoma and the fibrosarcoma the activity of the critical locus on this chromosome is in some way altered. A similar discrimination between diploid and malignant homologues could not be demonstrated for any other chromosome. The elimination of both copies of the diploid chromosome 4 from hybrids in which malignancy had been suppressed was not, however, enough in itself to induce the reappearance of the malignant phenotype; some hybrids from which both diploid chromosomes 4 had already been eliminated in vitro remained unable to generate progressive tumours. The reappearance of malignancy evidently required the intervention of some additional event. This additional event could not be correlated with the systematic elimination of any one diploid chromosome.