Biomedical subjects
Publications and source records attributed to T T PUCK.
An incidence of 0.6 percent of sex-chromosome aberrations in newborns was found during a 5-month period, while no aberrations occurred in similar populations before and after this period. Down's syndrome also exhibited an elevated frequency during this same critical interval. A severe rubella epidemic may have influenced this pattern.
A method for life cycle analysis in mammalian cells which utilizes the collection function has been applied to the Chinese hamster ovary grown in suspension. The following durations were found for the various parts of the life cycle: S, 4.13 hours; G1, 4.71 hours; G2, 2.81 hours; mitosis, 0.81 hours. The cell has a total generation time of 12.4 hours as opposed to 20.1 hours for the S3 HeLa cell. However, the relative lengths of each phase of the life cycle are identical within experimental uncertainty in the two cells.
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Equations are presented describing the accumulation of cells at any part of the life cycle as a result of addition of specific blocking agents. An experimental methodology using these relationships is described which makes possible analysis with relatively high resolution of the distribution of cells throughout the life cycle in normal cultures or those treated with various agents. The action of colcemide on S3 HeLa cells studied by this method revealed that colcemide has no effect on the G1, S, or G2 stages; it blocks cells quantitatively at the metaphase-anaphase region; but it accumulates mitotic figures only from the cells which have not yet entered mitosis at the time of its addition. The technique was also applied to study the efficiency of x-irradiation in delaying the entrance of G2 cells into mitosis. A definite lag was found at the lowest dose studied which was 9 rads. Only the cells confined to a central region of G2 at the time of irradiation are affected by this dose.
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The single cell plating technique has been applied to quantitation of the reproductive killing of mammalian cells by specific antibodies. This method confirms previous demonstrations by other workers of localization of all the killing activity in the gamma-globulin fraction of specific cell antisera but not of normal sera; the need for complement for the killing action in low doses of antibody and the leakage of cell constituents attending cell killing under these conditions. In concentrations of 4 per cent or higher of heated antiserum cell killing occurs without added complement. The cell plating technique permits highly reproducible quantitation of antibody action and demonstrates antibody activity in sera diluted 1:3000. It permits demonstration of very high degrees of species specificity as shown by virtually complete absence of cross-reaction between antisera to Chinese hamster and S3 HeLa cells, respectively. Somatic cells which have been sensitized by absorption of specific antibody lose their sensitization when incubated at 37 degrees unless complement is added within 1 hour.
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Two purified serum protein fractions, fetuin and serum albumin, will replace whole or dialyzed serum in supporting the growth of single S3 HeLa cells in an otherwise chemically defined nutrient solution. In the serum-free medium, single S3 cells will form macroscopic colonies with essentially 100 per cent efficiency. The generation time of S3 cells in the serum-free medium is approximately 50 per cent greater than that observed in an optimal, serum-containing medium. All components of the serum-free medium are available commercially, except fetuin, which can easily be prepared in substantial quantities. The problem of the purity of the protein preparations and of their possible roles in promoting cell growth is discussed.
A methodology designed to eliminate mitotic inhibitor action and involving use of pretested fetal calf serum and careful pH and temperature control has been described by which cells from normal human and animal tissue can be maintained in active growth for long periods in vitro without development of aneuploidy. By means of this procedure, it is possible reliably to establish cell cultures from minute skin biopsies which can be taken from any individual. Clones of mammalian cells with chromosomal markers have been isolated by this means from x-irradiated non-irradiated cell cultures. Application of these techniques to chromosome delineation in large numbers of human subjects; determination of chromosomal sex in patients; spontaneuos and induced genetic changes in somatic mammalian cells in vivo and in vitro; comparison of metabolic differences between normal and cancerous cells and other problems have been indicated.