A Quest for the Mechanism of “Spontaneous” Malignant Transformation in Culture With Associated Advances in Culture Technology2

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Abstract
The discovery of “spontaneous” malignant transformation of mouse cells in culture raised questions answerable only by obtaining a better control of culture conditions through technologic advances. One of these advances was the introduction of cellophane and later glass as a substrate to replace the plasma clot matrix, widely used in tissue culture at that time. Removal of cells from plasma clot and culture under cellophane or on glass not only increased cell yield but also allowed the manipulation of individual cells in suspension. Other advances included the design and technology of three-dimensional substrate cultures, the establishment for the first time of clonal cell populations from single isolated cells, the development of instrumentation and quantitative techniques for measuring cell proliferation, the introduction of the suspension culture of cells of “fixed tissue” origin, and the formulation of serum-free, antibiotic-free, chemically characterized culture media that could support the continuous proliferation of numerous cell lines from different tissues and species. These technologic advances promoted the application of microbial techniques to vertebrate tissue cells, marked the beginning of modern quantitative cell culture, and provided the tools for recent developments in virology, vaccine production, genetics, molecular biology, and other areas of biomedical research. These technologic advances were associated with a quest for an explanation of spontaneous malignant transformation. In this quest it was found that normal tissue cells of various rodent species after prolonged culture in the absence of added carcinogens or viruses and even in serum-free, chemically defined medium undergo malignant transformation. Certain differences between the normal cell and its malignant derivative were identified, and their reliability as in vitro criteria of malignancy was shown. Certain observations support the hypothesis that aberrant methylation of nucleic acids may be a chemical event in carcinogenesis, and methyltransferases were discovered in all RNA retroviruses examined. Three environmental agents were found to enhance or induce malignant transformation: the type of serum used, molecular oxygen, and repeated exposure to low-intensity fluorescent light. All three agents produce chromosomal DNA damage. Experimental results indicate that the DNA damage produced by light results directly or indirectly from the intracellular generation of hydrogen peroxide and/or the derivative free hydroxyl radical. Thus the malignant transformation of rodent cells is not spontaneous but is induced by specific environmental agents that produce chromosomal DNA damage. The progression to malignancy in mouse cells and the susceptibility of human cells to malignant transformation appear to be associated with an impaired capacity to repair the DNA damage produced by these environmental agents.

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