Spontaneous immortalization rate of cultured Chinese hamster cells.

Chinese hamster cell cultures derived from either fetal cell suspensions or adult ear clippings invariably became permanent cell lines during conventional subcultivation. The immortal cell cultures arose from rare spontaneous cellular events during the in vitro cultivation of cells with limited proliferative capacity. Immortality was not related to rare, precommitted cells from the animals. The expansion of clones of cells with limited life-span to form permanent cell lines was routinely successful only when the initial, unsubdivided culture achieved a total number in excess of 10(6) cells. On the basis of this observation, a serial clonogenicity assay was developed for determining the life-span of the cells with limited proliferative capacity and for determining whether a cell population is immortal. In addition, the technique of clonal expansion was used for a fluctuation analysis to determine the rate of immortalization. This analysis yielded a rate of 1.9 X 10(6) per cell per generation.

Microfluoremetric analysis of DNA content changes in murine teratocarcinoma.

The multipotential stem cell of the murine tetratocarcinoma, embryonal carcinoma (EC), is capable of differentiation in vivo and in vitro to nonneoplastic progeny. Undifferentiated EC cells, spontaneously differentiating tetratocarcinoma cells, and differentiated cells derived from EC cells were analyzed for DNA content and chromosome number distributions. Flow microfluorometric and fluorescence cytophotometric analysis of DNA content showed that EC cells had a characteristic diploid (2c) distribution, whereas several differentiated cell lines derived from EC cells had 4c DNA distributions. The tetraploid cell populations studied were capable of cell division but had restricted differentiative potential and were either of low tumorigenicity or non-tumorigenic. In vivo teratocarcinomas, comprised of both EC cells and differentiated cell types, contained diploid and tetraploid populations. Chromosomally, EC cells were neardiploid (39 chromosomes) and differentiated cells were near-tetraploid (62 to 76 chromosomes). The teratocarcinoma provides a model for studying the basic mechanisms that control the growth dynamics of the rapidly and slowly proliferating cell populations present in many tumors.

Preneoplastic phenotype and chromosome changes of cultured human Bloom syndrome fibroblasts (strain GM 1492).

The Bloom syndrome fibroblast strain, GM 1492, was examined for phenotypic properties generally associated with neoplastic cells. A serial clonogenicity assay indicated that these cells can proliferate in culture, achieving approximately twice the number of population doublings as compared to normal human skin fibroblasts. Strain GM 1492 appeared to be partially transformed in that these cells showed a slight degree of anchorage independence when grown in methylcellulose, and also appeared to have relaxed growth requirements compared to normal fibroblasts. GM 1492 cells are heteroploid, with 20 to 80 chromosomes/cell and a modal chromosome number of 44. Cytogenetic analysis of G-banded metaphase chromosomes indicated that most cells contained at least one copy of each normal human chromosome, and many cells exhibited only aneuploidies with no detectable chromosomal rearrangements. Minute chromosomes were seen in a few of the metaphase cells examined. GM 1492 cells did not form tumors in athymic nude mice. Since many of the characteristics of GM 1492 cells are similar to those seen only in tumor cells, but the strain is nontumorigenic, we suggest that GM 1492 cells are preneoplastic and thus represent an ideal system for the in vitro study of human neoplastic progression.

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.

Differential fluorochromasia of human lymphocytes as measured by flow cytometry.

Peripheral human lymphocytes reacted with fluorescein diacetate and analyzed by flow cytometry produced a bimodal fluorescence distribution that was shown to be attributable to the differential staining of T and B lymphocytes. Lymphocytes were fractionated into rosetting (T cell) and nonrosetting (B cell) populations. Both subfractions were reacted with fluorescein diacetate and analyzed by flow cytometry. The rosetting fraction was more fluorescent than the nonrosetting fraction, and the analysis of an appropriate mixture of the subfractionated populations produced a fluorescence distribution very similar to that obtained with unfractionated lymphocytes.

Fluorescence polarization and pulse width analysis of chromosomes by a flow system.

Isolated Chinese hamster chromosomes have been analyzed using a multiparameter computer-controlled cell sorter to obtain information about unique properties of individual chromosomes. Parameters other than DNA content were sought that would further aid in distinguishing among chromosomes. The polarized emission of the DNA-specific bis-benzimidazole dye Hoechst 33342 was measured for each class of chromosomes identified by a distinct peak, i.e., differeing in DNA content. The emission anisotropy values for all chromosome classes was constant (emission anisotropy = 0.30), and the same value was obtained for purified DNA in solution. Pulse width was found to be a good parameter for resolving chromosomes as a function of total emission in the case of the smaller chromosomes and orientation (i.e., arm length) for large chromosomes. A simple theoretical model for predicting the pulse shapes generated by arbitrarily oriented, thin, rigid rods was developed and applied to the evaluation of the experimental data.

Flow microfluorometric quantitation of the blastogenic response of lymphocytes.

A method for quantitating the specific stimulation of peripheral lymphocytes has been developed using the techniques of flow microfluorometry. Peripheral bovine lymphocytes were collected and specifically stained for deoxyribonucleic acid (DNA) content using a low-salt propidium iodide procedure. Flow microfluorometry was used to determine, on the basis of DNA content, the percentage of cells in a population that was stimulated. Extremely uniform staining of the lymphocytes (coefficient of variation of less than 2%) provides a high resolution between proliferating and nonproliferating cells. The method provides a rapid, highly repoducible technique for determing the fraction of lymphocytes stimulated in response to tuberculin antigens based on an increase in cellular DNA content. Specific and nonspecific stimulation by a defined antigen can be measured and resolved.

Spontaneous in vitro neoplastic evolution of cultured Chinese hamster cells. Nucleolus organizing region activity.

Silver staining to demonstrate active nucleolus organizing regions (NORs) was performed at four different stages of the spontaneous tumorigenic progression in vitro of Chinese hamster WCHE/5 cells. The number of active NORs increased for fully transformed, highly tumorigenic, late passage cells. The increase of NOR material was due to additional NOR-bearing chromosomes or chromosome arms, i.e., trisomy 5, trisomy 8, and the marker chromosome i(3q). Intermediate stages of the neoplastic evolution showed changing patterns of NOR activity, but not an overall increase. We postulate that the increase of active rDNA enhances cell growth and provides undefined selective advantage, and that this supports our previous conclusion that selectable karyotype changes provide competitive advantages rather than being essential for neoplastic evolution in vitro.

Spontaneous in vitro neoplastic evolution: recurrent chromosome changes of newly immortalized Chinese hamster cells.

Spontaneous neoplastic progression in cultured Chinese hamster cells was studied at the earliest stage possible. Eighteen independent newly immortalized cell populations (from six individual Chinese hamsters) were characterized for karyotype instability. Colonies were selected from initial sparse platings of adult or fetal cells and were expanded for study. The chromosomes from these newly established cell lines were studied using a combination of G-banding and flow karyotype analysis. At a slightly later passage, the 18 cell lines were tested for tumorigenicity in nude mice. Frequent recurring chromosome changes were observed in the karyotypes. The most frequent changes were either total or partial trisomy of chromosome #3 (83%) and trisomy of chromosome #5 (61%). Only 4 of 18 clones (22%) were tumorigenic at the time of testing, and these had long latent periods. The presence of recurrent chromosome changes did not obligate these cell lines to become tumorigenic, but the karyotype instability appeared to be an indicator of the ongoing process of neoplasia.

Spontaneous in vitro neoplastic evolution: selection of specific karyotypes in Chinese hamster cells.

Recurrent cytogenetic changes occurred reproducibly in vitro during the spontaneous neoplastic evolution of cultured Chinese hamster cells. In particular, excess 3q material appeared shortly after immortalization in numerous independent trials. By contrast, when clones were isolated at the earliest possible time after immortalization, a wide spectrum of types of cytogenetic evolution followed, which also resulted in transformed and tumorigenic cells. Clones with stable distinct colonial morphologies were used to demonstrate growth rate interactions when subpopulations compete. We conclude that specific recurring karyotypes are associated with specific stem lines with transient growth advantage during the early stages of in vitro carcinogenesis. Stem lines with other karyotypic change or no detectable karyotypic change are almost equally capable of undergoing the entire spontaneous neoplastic process in vitro.

Cultured Bloom's syndrome substrains: a relationship between growth in low serum and the expression of double minute chromosomes.

Four Bloom's syndrome fibroblast strains were examined for chromosome changes, immortalization, tumorigenicity, anchorage independent growth, and the ability to grow in low serum. Only one strain (GM 1492) exhibited some of these characteristics, which are generally associated with neoplastic cells. Strain GM 1492 also exhibited a low frequency of cells that contained double minute chromosomes. Substrains from GM 1492 were cloned, and showed that some of the above characteristics were expressed as a heritable trait. Karyotype instability (heteroploidy) was seen in all clones studied, with modal chromosome numbers ranging from near-diploid to near-tetraploid, as confirmed by DNA content distributions. Although no clones studied were tumorigenic in nude mice or immortalized, some showed an increased cellular proliferative capacity. Several clones were isolated after growth in 1% fetal bovine serum, and these all showed an increased expression of double minute chromosomes. Two of these 1% serum-isolated clones, (1492/clone 3-1% and clone 8-1%) were further studied by G-banding analysis, and found to show specific chromosomal anomalies. Clone 3 showed monosomy for chromosomes #4, #13, and #19, along with the absence of both copies of chromosome #7 but with both i(7p) and i(7q) chromosomes consistently seen. Clone 8 showed monosomy for chromosome #13. Preliminary experiments using DNA slot blots indicated that erb a and b, v-fes, v-mos, c-myc, n-myc, v-src, and v-sis showed no sequence amplification in GM 1492 cells or subclones.

Karyotype evolution in a simian virus 40-transformed tumorigenic human cell line.

Normal human foreskin fibroblasts (HSF4) were transfected using the pSV3-neo plasmid. A pool of 10 G418-resistant colonies, HSF4-T12, showed a progressive increase in the expression of a number of in vitro transformation markers with passage in culture and became immortalized. Although no tumors were formed when cells were injected subcutaneously into nude mice, this cell line produced progressive tumors when cells were injected into preimplanted Gelfoam sponges in the mice. When these tumors were cultured in vitro and subsequently injected subcutaneously, progressive tumors were produced with median latency periods as short as 4 weeks. Three phases of cytogenetic change could be distinguished. At early passages after transfection. HSF4-T12 exhibited many random chromosomal changes. At a time just after immortalization, both flow karyotype and G-banded analyses showed the appearance of balanced clonal rearrangements. These included t(2;4), t(2;14), t(3;?), 6p-, i(6p), 8p-, t(14;15), i(15), and t(18;?). These clonal rearrangements were stable with passage in culture, and less variability from cell to cell was noted. The only consistent chromosomal loss observed was -Y. Analysis of three independent tumors showed characteristic loss of chromosomal material rather than balanced chromosomal rearrangements. Frequent loss of 6q and chromosomes #13, 15, 20, and Y was noted.

Flow cytogenetics and chromosome sorting.

This review of flow cytogenetics and chromosome sorting provides an overview of general information in the field and describes recent developments in more detail. From the early developments of chromosome analysis involving single parameter or one color analysis to the latest developments in slit scanning of single chromosomes in a flow stream, the field has progressed rapidly and most importantly has served as an important enabling technology for the human genome project. Technological innovations that advanced flow cytogenetics are described and referenced. Applications in basic cell biology, molecular biology, and clinical investigations are presented. The necessary characteristics for large number chromosome sorting are highlighted. References to recent review articles are provided as a starting point for locating individual references that provide more detail. Specific references are provided for recent developments.

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

The clinical usefulness of chromosome analysis by flow cytometry.

Historical and clinical aspects of chromosome analysis by flow cytometric methods are reviewed. A new method of preparing small samples (1.0 mL of blood) of peripheral lymphocytes for flow karyotype analysis using phytohemagglutinin and interleukin-2 is presented in detail. Figures of flow karyotypes, partial banded karyotypes, and idiograms of patients with inv(8), rec dup(8), rob t(14;21), and t(1;22) are presented, as well as examples of univariate and bivariate flow histograms from other researchers' published works. The clinical utility of these techniques is explored, with specific reference to recent work in chromosome polymorphisms and cultured amniocyte lines. We conclude that, although flow karyotyping is not a replacement for classical banded chromosome analysis, used as an adjunct, this new technique has some clinical usefulness relating to its capability to resolve small differences in chromosomal DNA content.