Observations of de novo clones of cytogentically aberrant cells in primary fibroblast cell strains from phenotypically normal women

Abstract

In a recent study of chromosome breakage frequencies in 36 primary fibroblast cell strains derived from skin from 10 phenotypically normal women, we observed seven different clones of cells having consistent chromosomal abnormalities. Five of the stem lines were noted in cultures from "control" women and two in fibroblasts from women taking oral contraceptives. We observed aneuploid clones as well as stem lines bearing structural abnormalities (e.g., translocation, inversions). The various aberrant clones were found in cultures ranging in age from 41 to 144 days and comprised varying percentages of the cell populations ranging from 0.8% to virtually 100%. The possible evolution in culture of clones of cells having aberrant karyotypes should be considered in interpreting findings from fibroblast cultures initiated for clinical evaluation.

PIP: At about 3-6 month intervals, a minimum of 4 sets of blood and skin biopsy samples were obtained from each of 5 women who were taking oral c ontraceptives and from 5 others who were not. Subjects were aged 20-40 with no known phenotypic abnormalities of family history of congenital m alformations. Primary fibroblast cultures were made from 2 mm punch bio psies. Cell strains were maintained in RPMI 1640 culture medium supplem ented with 20% calf serum and antibiotics. When sufficient cells had accumulated for cytogenic studies, replicate flasks from each primary cell strain were exposed to a medium containing either fetal calf serum, autologous serum, homologous serum, or serum from a woman taking oral contraceptives. 24 hours later the cultures were studied for cytogenetic evaluation. From each subculture, 100 metaphases were recor ded for induced chromosomal aberrations, including chromated and slides from each fibroblast preparation were evaluated for clones of cells bearing morphologically identical lesions. All metaphases with question able structural rearrangements were photographed and karyotyped. If a specific chromosomality was seen in 3 or more metaphases from a replicate series of cultures from a single biopsy, these cells were considered to be members of a stem line. Fibroblast cultures from 19 biopsies from the 5 control women and 17 biopsies from the contraceptive users were successful. From the replicate subcultures 10,202 metaphases were examined. Significant differences in breakage frequencies within replicate cultures were not observed, nor among fibroblasts from control women as compared with oral contraceptive users. In the 36 sets of cultures, 7 different clones of aberrant cells were seen. Of these, 5 were from cultures of control women and 2 in preparations from oral contraceptive users. The lesions were classified only on the basis of morphology. Karyotypically aberrant stem lines were observed in cultures from 41 to 144 days old. These stem lines comprised from 1% to almost 100% of the cell population. A description of the aberrant clones is given. Lymphocyte cultures from all the women were normal. Results show that under cultural conditions denovo clones may evolve in fibroblast cell strains from clinically normal persons and in some instances may comprise almost 100% of the recoverable cell population. Clones of cells resulting from nondisjunction as well as structural rearrangements may arise in culture. These findings should be considered in interpretations of data from clinical fibroblast cultures.