Spontaneous neoplastic evolution of Chinese hamster cells in culture: multistep progression of phenotype.

Chinese hamster cell strains, each initiated from a separate fetus, were carried in culture and tested for tumorigenicity and in vitro indicators of neoplasia at various passage levels. In the absence of any treatment, all 20 such lineages yielded permanent cell lines and showed other indications of neoplastic progression. A preimplanted gelatin sponge assay method was used to monitor tumorigenicity in nude mice. The process of spontaneous neoplastic progression in vitro could be divided into four stages. The rate of progression through these stages, as measured by passage level, was extremely variable between independent lineages. Detailed studies of one lineage showed that transformation indicators, in general, correlated with tumorigenicity but did not indicate whether or not a preneoplastic, permanent lineage would rapidly progress.

Spontaneous neoplastic evolution of Chinese hamster cells in culture: multistep progression of karyotype.

Chromosomes from successive passages of a Chinese hamster cell strain (WCHE/5) that spontaneously progressed from a euploid primary cell culture to a heteroploid tumorigenic cell line were isolated and analyzed by Giemsa banding and high-resolution flow karyotype analysis. The frequency and identification of aneuploid and marker chromosomes were determined at both pre- and postcrisis culture stages and pre- and posttumorigenic stages. The combination of Giemsa banding and flow karyotypes provided detailed analysis of karyotype instability at each stage of cell culture progression. Aneuploidy (trisomy of chromosome 5) preceded the appearance of tumorigenicity in nude mice as well as in vitro indicators of neoplasia. The four stages of neoplastic progression defined in the previous paper correlated with a steady progression in karyotypic instability, including, in sequence: trisomy of chromosome 5; an 8q marker chromosome; a 3q+ insertion; and trisomy of chromosome 8. Additional changes continued to appear as the cells acquired classical properties of in vitro transformation.

Flow cytometric analysis of the effect 5-bromodeoxyuridine on mouse teratocarcinoma cells.

Several flow cytometric techniques were used to study the effects of 5-bromodeoxyuridine (BrdUrd) on the growth and differentiation of mouse teratocarcinoma cells. The teratocarcinoma system mimics embryogenesis, and the stem cells of this tumor, termed embryonal carcinoma (EC) cells, spontaneously differentiate in vitro to progeny that are morphologically, biochemically and biologically distinct from the EC cells. When cultures of EC cells were treated with 30 microM BrdUrd, a pleiotropic response was observed; i.e., certain parameters mimicked spontaneous differentiation but occurred 2-3 times more rapidly while others were inconsistent with, or diametric to, spontaneous differentiation. In one line of diploid EC cells in which tetraploidy accompanies spontaneous differentiation, the BrdUrd accelerated the appearance of the tetraploid population. Analysis of cell cycle distributions during the course of the BrdUrd treatment showed that virtually all of the cells had cycled 2 or more times and incorporated BrdUrd into both strands of DNA. Multiangle light-scattering and electrical impedance analyses indicated that with respect to these two parameters, cultures treated with BrdUrd for 3 days resembled spontaneously differentiating cultures of 6-8 days. However, BrdUrd treatment did not induce increases in the kinetics of esterase (EC3.1.1.) activities in teratocarcinoma cells that normally accompany spontaneous differentiation. Analysis of alkaline phosphatase (APase) EC (3.1.3.1) activities showed that unlike the decrease in APase activities which accompany spontaneous differentiation, BrdUrd treatment causes an increase in APase activities.

Clonal variation of cadmium response in human tumor cell lines.

Subpopulations of human tumor-derived cell lines A101D, A204, and A549 were screened for Cd2+ cytotoxic response. Three of six A549, two of seven A101D, and four of seven A204 subpopulations were found to differ significantly from the parental line. A variant subpopulation of A101D (T3) was shown by flow cytometry to be comprised of cells having two distinct DNA histograms. One histogram, type 1, resembles that of normal human fibroblasts. The other, type 2, represents cells with one-third more DNA. Early passage T3 clonal populations were comprised primarily of type 1 cells. With passage, type 1 cells decreased relative to type 2 so that by passage 47 the culture was predominantly type 2. Correspondingly, the A101D T3 subpopulation became more Cd2+ sensitive with time in culture. Subclones having only type 1 DNA histograms were found to be Cd2+ resistant relative to subclones with type 2 histograms, and treatment of A101D T3 cultures having approximately equal amounts of type 1 and 2 cells with 2 microM Cd2+ resulted in the selection of type 1 cells. The enhanced Cd2+ resistance phenotype shown by A101D T3 type 1 cells correlated with reduced Cd2+ uptake and is not attributable to enhanced metallothionein synthesis.