Detection of in vitro tetraploidy in heritable colon cancer syndromes. Confirmation by three different assays.

Abstract

The incidence of in vitro tetraploidy during logarithmic growth in dermal fibroblast monolayer cultures from normals without a family cancer history and affected with heritable colon cancer syndromes, familial polyposis coli (FPC) and the Gardner syndrome (GS) was assayed by (1) examination of metaphase preparations for percentage of tetraploids (4N nuclei); (2) determination of [3H]thymidine incorporation (average cpm/cell); and (3) measurement by flow cytophotometry of the percentage of cells with greater than 4C DNA content. For each monolayer culture assayed, if cell density was standardized, concurrence was observed between the first two assays and with the third assay when done. In cultures of individuals with a low level of in vitro tetraploidy (37 normals without a family cancer history, 9 FPC, and 1 GS patient) incidence of this parameter, as measured by all three assays, was not significantly influenced by variation in cell density. However, in cultures with increased in vitro tetraploidy (17 FPC and 6 GS patients) all three assays revealed an inverse relationship between the incidence of tetraploidy and cell density, i.e., tetraploidy increased with decreasing cell density. Only at cell densities below 4.0 X 10(3) cells/cm2 growth area was increased tetraploidy consistently observed by any of the three assays. Above this density its incidence was indistinguishable in all cultures. The use of these three independent assays (two of which are automated) for the determination of tetraploidy in duplicate subcultures from the same dermal monolayer culture should allow reliable detection of this in vitro expression of some cancer-related genes in extensive human kindreds.