DNA content and chromosome number of a heteroploid human tumour cell line.

In this paper investigations concerning the relation between variability of chromosome number and variability of DNA content within the cells of a tumour stemline are reported. A highly heteroploid human tumour cell line was used, which was derived from a chondrosarcoma. Flow cytometrical and scanning cytophotometrical measurements confirmed the heteroploid nature of the original cell line and of several subclones. Measurement of the DNA content per metaphase showed a linear relation between chromosome number and DNA content of heteroploid cells. This finding is discussed with regard to its implications for the mechanism of heteroploidy in tumour cells.     

Evidence for altered cell-cycle traverse of the non-modal cells of the heteroploid MCa-11 line

Classic stem cell theory states that the growth of heteroploid cell populations is due to the proliferation of 'main stemline'cells with modal DNA content and chromosome number. Cells with non-modal DNA content and chromosome number are thought to be blocked and/or destroyed at mitosis. To test this, we studied two chromo-somally stable cell populations (mouse bone marrow and WCHE-5 cells) and one heteroploid, chromosomally diverse cell line (MCa-11). The heteroploid MCa-11 cells showed significant [³H]dT labelling for cells with DNA contents below the modal G_o/G₁ peak and above the modal G₂ peaks ( P <0.001). This was consistent with the presence of cells with the non-modal DNA content that were engaged in replicative DNA synthesis. A percentage labelled mitosis analysis showed that MCa-11 cells with non-modal DNA content and chromosome number were able to complete mitosis, although with prolonged pre-karyokinetic time. These results suggest that many non-modal cells present in heteroploid cell populations are capable of continued proliferation.     

Non-random distribution of abnormal mitoses in heteroploid cell lines.

We have performed absorption-cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa-11 and HL-60 lines, as well as the WCHE-5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase-cell pairs of the heteroploid MCa-11 and HL-60 lines than in those of the chromosomally stable WCHE-5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non-random, unblanced divisions of heteroploid cells with non-modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.     

Improved sensitivity in comparative genomic hybridization analysis of DNA heteroploid cell mixtures after pre-enrichment of subpopulations by fluorescence activated cell sorting.

Cytogenetic analysis of solid tumors with comparative genomic hybridization (CGH) is hampered by the dilution of DNA from individual tumor subpopulations with DNA from other cells. We investigated to what extent this dilution effect can be alleviated using fluorescence activated cell sorting (flow sorting) of experimental DNA heteroploid cell mixtures prior to CGH. From mixtures of normal lymphocytes with triploid K-562 cells the individual components were sorted according to stemline DNA content and processed by CGH in comparison with pure K-562 samples and the original mixtures. Compared with 30 autosome copy number imbalances found in pure K-562 samples, a mixture with 32% K-562 cells showed 16 imbalances, and none were detected in mixtures with 13% or 5% K-562 cells. In contrast, 29, 22 and 23 imbalances were detected in K-562 nuclei sorted from the 32%, 13% and 5% mixtures, respectively. This indicate that CGH analysis of flow sorted DNA aneuploid subpopulations enables a specific cytogenetic analysis of the individual subclones in a DNA heteroploid cell population.     

Difference between diploid and aneuploid Chinese hamster cells in replication at mid-S-phase

Following partial synchronization of the heteroploid Chinese hamster cell line V-79 and of normal diploid lung fibroblasts of the Chinese hamster in culture, their DNA replication during S-phase was compared by means of a BrdU-incorporation/thymidine pulse technique and Hoechst-Giemsa differential staining of metaphase chromosomes. This comparison indirectly shows the S-phase of the heteroploid cells of V-79 to be 2 h shorter than the diploid cell S-phase. When the thymidine pulse is applied to diploid lung fibroblasts at mid-S-phase, differential staining colours metaphase chromosomes a pale blue. Performing the corresponding experiment with V-79 cells, neither a pale blue nor dark red staining is obtained, but rather an intermediate shade, showing prominently dark staining regions in parts. The pause in DNA synthesis observed at mid-S-phase of the diploid Chinese hamster lung fibroblasts seems to be omitted at mid-S-phase of the V-79 cells.     

A new human breast carcinoma cell line resistant to DNA-damaging drugs

To investigate the phenomenon of active dissociation of the vital dye, Hoechst 33342 (Ho342), from DNA (DNA clearing), a new MCF7HoeR-7 human breast carcinoma cell line was isolated from parent MCF7 cells by step-wise selection with increasing concentrations of Ho342. This cell line possesses an enhanced ability for DNA clearing. The MCF7HoeR-7 line is characterised in detail and compared with the parental MCF7 line and a typical P-glycoprotein-mediated multidrug resistant (MDR) cell line, MCF7/Adr. MCF7HoeR-7 cells have an increased population growth rate, a lower DNA content and a reduced number of chromosomes. Enhanced DNA clearing in MCF7HoeR-7 cells is associated with the high resistance of the cells to the toxic effects of Ho342 and cross-resistance to etoposide, a topoisomerase II inhibitor in clinical use. The MCF7HoeR-7 and parent MCF7 cell lines have similar expression levels of transport proteins. The results obtained confirm that DNA clearing is an atypical MDR mechanism in tumour cells.     

The fate of the primary diploid population during spontaneous transformation of growth factor-supplemented murine cell cultures

Primary cultures derived from lung and renal tissue of the newborn harvest mouse (Micromys minutus) were serially passaged in media supplemented with epidermal growth factor, hydrocortisone, transferrin, insulin, and triiodothyronine. Although these growth factor supplements eliminated the growth crisis commonly encountered during the initial stages of murine primary cultures, the original diploid cell fraction clearly underwent such a "crisis"; the truly diploid cells invariably disappeared as these cultures reached 20 to 40 population doublings. They were replaced, either gradually or precipitously, by various heteroploid cell fractions. In three of four independent cultures, these "established" cells were hypotetraploid and appeared to be derived from a small number of progenitors already present during the very early (precrisis) culture stages. In contrast to rather frequent DNA changes displayed by clones and subclones derived from the various heteroploid cell lineages, the predominant components of the established mass cultures displayed a highly constant DNA fluorescence pattern. Our results suggest that primary murine cell cultures develop heteroploid cell lineages even if the initial growth crisis is mitigated by growth factor supplements. These heteroploid cells appear to respond more efficiently to stimulation by various growth factors than the primary diploid cell population.     

Localization of ribosomal RNA genes on human acrocentric chromosomes

Summary Clonal derivatives of a human heteroploid cell line, with different numbers of acrocentric chromosomes, show different rDNA contents. A linear relationship has been found between the rDNA content and the relative mass of the acrocentric chromosomes (D+G) expressed as the ratio between the mass of their DNA and the mass of the DNA of the whole chromosomal complement. The results suggest that human rRNA genes are located exclusively on the chromosomes of the groups D and G and that all these chromosomes contain rRNA genes.     

Characteristics of a new rapidly proliferating rabbit cell line

Summary A new tissue culture cell line (Calg-ARLC) has been established from an explant culture of adult rabbit lung. The Calg-ARLC line has been characterized with respect to morphology, chromosome constitution, tissue culture requirements, proliferative capacity, cell cycle, attainable synchrony, radioisotopically labeled precursor incorporation into nucleic acids and protein, and radioisotope sensitivity. The cells are fibroblast-like in appearance with a stabilized heteroploid chromosomal modal number of 35. They grow exponentially from high split ratios in several commercially available defined media with a generation time of 12 hr and are easily synchronized. Although sensitive to some isotopically labeled precursors, high specific activity nucleic acids have been isolated. The ARLC line is especially useful for the isolation of high specific activity nucleic acids and proteins of rabbit origin. The Calg-ARLC line should be invaluable in the fractionation of reiterated DNA sequences since no very rapidly reassociating DNA sequences such as those found in mouse are evident. This work was supported by operating grants from the National Cancer Institute of Canada and the National Research Council of Canada.     

A quantitative study of growth variability of tumour cell clones in vitro

Abstract.   Objectives : In this study, we quantify growth variability of tumour cell clones from a human leukaemia cell line. Materials and methods : We have used microplate spectrophotometry to measure growth kinetics of hundreds of individual cell clones from the Molt3 cell line. Growth rate of each clonal population has been estimated by fitting experimental data with the logistic equation. Results : Growth rates were observed to vary between different clones. Up to six clones with growth rates above or below mean growth rate of the parent population were further cloned and growth rates of their offspring were measured. Distribution of growth rates of the subclones did not significantly differ from that of the parent population, thus suggesting that growth variability has an epigenetic origin. To explain observed distributions of clonal growth rates, we have developed a probabilistic model, assuming that fluctuation in the number of mitochondria through successive cell cycles is the leading cause of growth variability. For fitting purposes, we have estimated experimentally by flow cytometry the average maximum number of mitochondria in Molt3 cells. The model fits nicely observed distributions in growth rates; however, cells in which mitochondria were rendered non-functional (ρ⁰ cells) showed only 30% reduction in clonal growth variability with respect to normal cells. Conclusions : A tumour cell population is a dynamic ensemble of clones with highly variable growth rates. At least part of this variability is due to fluctuations in the initial number of mitochondria in daughter cells.     

Effect of laminin on numerical karyotype variability of kangaroo rat kidney cell lines

The numerical karyotypic variability has been investigated in "markerless" epithelial-like Rat kangaroo kidney cell lines NBL-3-11 and NBL-3-17 on cultivation on a laminin-2/4 coated surface. In cell line NBL-3-17, cultivated on the laminin-coated surface for 2, 4 and 12 days, the character of numerical karyotypic variability has changed. In 2 days the general character of cell distribution for the chromosome number did not change, but the frequency of cells with modal number of chromosomes decreases significantly, while that of cells with lower chromosome number show a tendency to increase. At a prolongation of cultivation time to 4 and 12 days, the numerical karyotypic heterogeneity in cell population increases due to a significant change in the general character of cell distribution for the chromosome number, which is caused by a significant decrease in the frequency of cells with the modal number of chromosomes, and by an increase in the frequency of cells with lower chromosome number. The analysis of distribution of individual chromosomes showed that the number of types of additional structural variants of the karyotype (SVK) increases significantly on cultivation on laminin for 2-12 days. In cell line NBL-3-11, cultivated on the laminin-coated surface for 2 and 4 days, the character of numerical karyotypic variability did not change compared to control variants. Possible reasons of the observed changes of numerical karyotypic variability in cell line NBL-3-17 is discussed. The reason of differences in the character of numerical karyotypic variability between cell lines NBL-3-11 and NBL-3-17 possibly consists in the change of gene expression, namely in a dose of certain functioning genes. The polymerase chain reaction with arbitrary primers revealed no differences between DNA patterns of cell lines NBL-3-17 and NBL-3-11. This can reflect a similarity in the primary DNA structure of both cell lines. Hence, these lines differ only in the number of homologous chromosomes (hypotriploid and hypodiploid).     

Kinetics of herpes simplex virus photoinhibition by haematoporphyrin in both diploid and heteroploid cells

The kinetics of inhibition of herpes simplex virus type 1 (HSV 1) on both diploid (CEF) and heteroploid cells (HEp2) by light-irradiated haematoporphyrin (HP) was studied. The inactivation of HSV1 by HP was drug-dose dependent and light-irradiation dependent; the viruses grown in heteroploid cells being in all cases more sensitive to inhibition than viruses grown in diploid cells. Cell toxicity by HP was markedly more evident on HEp2 cells than on CEF. The highest viral sensitivity to photodynamic inactivation by HP was found to be between the 4th and the 5th hour after cell infection, when the viral DNA synthesis is at its peak and before it is incorporated into complete virions. Microfluorometric and spectrofluorometric assays revealed that virus infected cells always take up more HP than uninfected cells, and heteroploid cells incorporated more HP than diploid cells. The possibility that an increased uptake of HP and modifications of the cell micro-environment in virus infected cells could account for the viral-inhibiting properties of HP, is discussed.     

Quantitative determination of nuclear pore complexes in cycling cells with differing DNA content

The number of pore complexes per nucleus was determined for a wide variety of cultured cells selected for their variable DNA content over a range of 1-5,6000. The pore number was compared to DNA content, nuclear surface area, and nuclear volume. Values for pore frequency (pores/square micrometer) were relatively constant in the species studied. When the pore to DNA ratio was plotted against the DNA content, there was a remarkable correlation which decreased exponentially for the cells of vertebrae origin. Exceptions were the heteroploid mammalian cells which had the same ratio as the diploid mammalian cells despite higher DNA content. The results are interpreted to mean that neither the nuclear surface, the nuclear volume, nor the DNA content alone determines the pore number of the nucleus, but rather an as yet undetermined combination of different factors. The surface and volume of vertebrate nuclei do not decrease with decreasing DNA content below a given value. The following speculation is suggested to account for the anomalous size changes of the nucleus relative to DNA content in vertebrates. Species with small DNA complements have a relatively large proportion of active chromatin which determines the limits of the physical parameters of the nucleus. The amount of active chromatin maybe the same for at least the vertebrates with low DNA content, At high DNA content, the nuclear parameters may be determined by the relatively high proportion of inactive condensed chromatin which increases the nuclear surface and volume.     

The effect of gamma-linolenic acid and zinc supplementation on the growth of normal and tumour cells in vitro

The effects of zinc, gamma-linolenic acid (GLA) and zinc combined and GLA supplementations on the growth of a benign monkey kidney, cell line (LLCMK) and a malignant tumour murine melanoma, cell line (BL-6) cells in vitro were studied. Cell growth was indicated by both cell counts and 3H-thymidine incorporation into DNA. The addition of zinc to the cells resulted in a general trend of overall reduction in the growth of tumour cells but not in the normal cells. The addition of GLA at high concentrations resulted in a general decrease in cell growth of both the benign and malignant tumour cells while the addition of lower concentrations of GLA had less effect. The combined effect of supplementary zinc and GLA resulted in an inhibitory effect on the growth of the malignant cells while a less and variable effect on the non-malignant cells was found. Some interaction between zinc and GLA in reducing tumour cell growth is suggested by the results.     

New cell line of mouse ependymoblastoma

A new cell line derived from transplanted mouse ependimoblastoma consists of typical fibroblast-like cells with well-defined pleomorphism. The line is characterized by a rapid increase and decrease of proliferative activity, by a short stationary phase and early degeneration. The average time of themitotic cycle was determined as being 22-24, tG1-11-12, ts-8-9, tG2-3 and tM-near 0.9 hours. The cytophotometrical evaluation of DNA content revealed two cell subpopulations with different ploidy, both in culture and in tumor in vivo. As compared to the original tumor cells, the cultured cells differ in wider variability of DNA content and a higher average ploidy per nucleus.     

Population of murine rhabdomyosarcoma MKh-53 clones under different proliferation conditions studied by flow cytometry

Clones of rhabdomyosarcoma cells were obtained due to implantation of the tumor cells in the eye anterior chamber, in subcutaneous connective tissue and in lungs of mice. The DNA contents in the clone cells were measured using flow cytometry. Diploid indices of clones were calculated from the ratio of the modal content of DNA in G1-cells to the DNA content in lymphocytes contaminating these clones. The diploid indices of various clones varied from 1.4 to 2.3. The mean diploid index calculated for clones that grew under given conditions of proliferation varied within 1.85-1.88. Only one of the 66 clones examined displayed a two-peak distribution of cell according to their DNA content in phase G1, which may suggest a karyotypic instability of the progeny of the tumor clonogenic cell. No correlation was revealed between the diploid indices of clones and the following parameters, such as: the portion of tumor cells being at different phases of cell cycle; the number of normal (stromal) cells contaminating the clones, coefficients of variation of DNA contents in tumor cell clones. A positive correlation was observed between the coefficients of variation of DNA contents in the normal (stromal) cells contaminating the clones and those of tumor clone cells being in phase G1. It is concluded that the variability of results of the flow cytometric measurement of DNA in G1-cells may reflect the variability of cells in respect not only to their DNA contents but also to their capacity of dye sorbtion.     

Isolation of a line of immortal chicken embryo fibroblasts after transfection with the nuclei of Rous sarcoma virus-transformed Chinese hamster cells

Secondary cultures of chicken embryo fibroblasts were transfected with purified nuclei from lysed cells of a clonal line of temperature-sensitive Rous sarcoma virus (tsRSV)-transformed Chinese hamster fibroblasts. After propagation for 3 months an established cell line designated ChR32 was obtained in one chicken cell culture. The cells of this line have been propagated so far for 18 months, whereas normal chicken embryo fibroblasts died after 2 months. The established cells were heteroploid with a diploid modal number of macrochromosomes and two Z chromosomes. No Chinese hamster chromosomes could be identified. Southern blot analysis of DNA from the uncloned ChR32 cells and the clones provided evidence that these established cells were, in fact, clonal in origin and contained full-length RSV proviruses and no defective proviruses. Furthermore, they contained, at the 3' end proviral-cellular junction, Bg/II, HpaI, KpnI, SacI, and XbaI fragments of the same size as the Chinese hamster donor cells, suggesting that the cellular sequence adjacent to the provirus is of Chinese hamster origin. The cells after establishment were able to grow continuously at 37 degrees or 41 degrees C and produce a large amount of ts sarcoma virus particles. A corollary finding was that these virus particles were non-leaky for the transforming function at the non-permissive temperature.     

Differential sensitivities to gamma radiation of human bladder and testicular tumour cell lines

Gamma radiation sensitivities of continuous cell lines from nine human tumours were measured, comparing four derived from transitional cell carcinomas of the bladder with five from non-seminomatous germ cell tumours of the testis. The testicular cells were significantly more radiosensitive than the bladder cells, corresponding to the response to therapy of these tumour types in patients. These observations indicate that radiosensitivity is retained in vitro and is an inherent property of the testicular tumour cells. These gamma radiation sensitivities were compared with those of SV40-transformed fibroblasts derived from a normal individual and one with the heritable disease, ataxia-telangiectasia (A-T). The bladder cells had gamma radiation sensitivities similar to that of the SV40-transformed normal line. The testicular cells were hypersensitive to gamma radiation, although not as sensitive as the SV40-transformed A-T line. A-T cells, unlike those derived from normal individuals, continue to synthesize DNA at a normal rate following radiation exposure, prompting a comparison of the kinetics of DNA synthesis in three bladder and three testicular tumour cell lines. One of the bladder and two testicular lines showed a reduced inhibition when compared to the other tumour cell lines and the SV40-transformed normal line. Thus there was no clear association between DNA synthesis inhibition and radiosensitivity.     

Differential temperature sensitivity of normal and cancer cells in culture

Serially-propagated growing heteroploid and growing diploid cell cultures do not survive incubation at 42° for 24 hours, whereas contact-inhibited diploid monolayers are still viable after at least nine days at this elevated temperature. Heat-treated heteroploid HeLa cells and growing diploid cells exhibit a variety of morphologic abnormalities, but contact-inhibited cells are only minimally affected. A similar differential temperature sensitivity exists in the synthesis of cellular macromolecular components such as DNA, RNA, and protein: incorporation of radioactive precursors is drastically reduced in growing diploid and heteroploid cells after 24 hours at 42°, but not in contact-inhibited cells. Incorporation of labelled glucose, choline, or linolenic acid is actually enhanced in heat-treated contact-inhibited cells.