Colcemide

Cultures of cell strains with and without metallothionein were exposed to CdCl2 in doses ranging from 10 mu mol/l to 200 mu mol/l . Cell growth parameters were monitored by flow cytometric DNA-measurements, cell counts and counting of mitoses during the first two days after exposure. CdCl2 inhibited cell growth in a dose dependent way. The cadmium resistant cells were inhibited with concentrations above 100 mu mol/l, the concentration which the metallothionein-containing cells had previously been adjusted to. Microscopy of the cell cultures showed a dose dependent accumulation of cells in the mitotic prophase, whereas the other phases of the cell cycle were unaffected as measured by flow cytometry. When exposed to colcemide, however, the two cell strains showed identical responsiveness.

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.

Sister chromatid exchange (SCE) refers to the interchange of DNA between replication products. The technique for detecting such exchanges takes advantage of the semiconservative nature of DNA synthesis. 5'-bromodeoxyuridine (BrdU) is incorporated into the newly synthesized DNA. Using standard culturing techniques, Colcemid is added to the culture and conventional cytogenetic preparations are made. Differential staining with Hoechst dye and Giemsa allows the newly synthesized DNA within a chromatid to be recognized, since BrdU incorporation results in much weaker staining. SCEs represent a point of DNA template exchange during strand synthesis, visualized as asymmetric chromatid staining or "harlequin" chromosomes.