Karyotypic analysis of cloned human colon carcinoma cells containing enriched numbers of double minute chromosomes or homogeneously staining regions

Abstract

This thesis describes research with the human colon carcinoma cell line, COLO 320-DM, which contains double minute chromosomes (DM) in approximately 47% of the cells. After about 12 months of continuous culture, the number of DMs dropped to approximately 13% and a homogeneously staining region (HSR) appeared in one of the F group chromosomes. Ring-shaped chromosomes have been demonstrated in these cells which may also be related to DMs and HSRs. By using a soft agar overlay technique, which has been used to test for tumorigenic potential, the numbers of DMs per cell have been enriched to the point where 100% of the cells contain DMs. A repeat of this procedure yielded the ring-shaped chromosomes in two clones, as well as DMs and HSRs. Clones obtained from COLO 320-DM contained DMs, whereas clones obtained from the COLO 320, which contained an HSR, also contained an HSR in the same chromosome as the parent line. The presence of DMs, HSRs and ring-shaped chromosomes may offer a selective growth advantage to these cells in soft agar. This technique may be useful for increasing the numbers of cells containing DMs and HSRs. The amplified c-myc oncogenes present in both the COLO 320-DM and the high DM copy number clones obtained could be useful in studying the mechanism of the carcinogenesis. It was necessary to isolate the DMs from the remainder of the COLO 320-DM chromosomes in order to transfect their DNA into a primary cell line, Bloom's syndrome (GM3498B). The first attempt to isolate the DMs using a series of filtrations, ended in grossly contaminated cultures. Then isolation of the DMs by flow cytometry was attempted because of the rapidity, ease and accuracy of this method. Initially, the DNA content of the COLO 320-DM and four clones derived from it was analyzed. The four clones all showed a slightly lower total DNA content than that of the parent line even though they have the same average number of chromosomes and have a greater content of DMs or HSRs. Attempts at obtaining flow karyotypes of COLO 320 were unsuccessful although flow karyotypes were obtained for the CHO line, AA8, and for HeLa cells.