Spontaneous and mutagen-induced loss of DNA mismatch repair in Msh2-heterozygous mammalian cells

We have developed a simple procedure that enables the efficient selection of cells that are deficient for DNA mismatch repair (MMR). This selection procedure was used to investigate the frequency of fortuitous MMR-deficient cells in a mouse embryonic stem cell line, heterozygous for the MMR gene Msh2. We found a surprisingly high frequency (3 x 10(-4)) of Msh2-deficient cells. The wild type Msh2 allele was almost invariably lost by loss of heterozygosity. Single treatments with the genotoxic agents ethylnitrosourea, UVC light and mitomycin C resulted in a further increase of the number of Msh2-/- cells in the heterozygous cell line. This increase was not only due to induced loss of the wild type allele but also to a selective growth advantage of preexisting Msh2-/- cells to ethylnitrosourea and UVC. Mitomycin C, in contrast to ethylnitrosourea and UVC, uniquely induced loss of heterozygosity at Msh2. These mechanistically different ways of loss of the wild type Msh2 allele reflect the different repair pathways processing these damages. Heterozygous germ line defects in one of the MMR genes underlie the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. Based on the results described here we hypothesize that mutagen-induced loss of MMR in the intestine of these patients contributes to the tissue specificity of carcinogenesis in HNPCC patients.     

Spontaneous and mutagen-induced sister chromatid exchange in motor neurone disease

Spontaneous and mutagen-induced sister-chromatid exchange frequencies have been studied in the peripheral blood lymphocytes of 6 patients with motor neurone disease. Their values were compared with those obtained in age- and sex-matched healthy controls. No significant differences were observed between the 2 groups. These results do not support the hypothesis of a defect in the repair of DNA damage as the primary abnormality in the development of the disease.     

Segregation studies in CHO hybrid cells: I. Spontaneous and mutagen-induced segregation events of two recessive drug-resistant loci

The process of segreation or phenotypic expression of two recessive drug-resistant loci from heterozygous Chinese hamster ovary hybrid lines is examined. The spontaneous segregation rates of phytohaemagglutinin resistance (Phar) and a temperature-dependent 8-azaguanine-resistant locus (Azarts) from heterozygous quasitetraploid lines using Luria-Delbruck fluctuation analysis were 5 X 10(-5) and 10(-5) events/cell/generation, respectively. In quasihexaploid lines, the latter rates increased 40- and 200-fold, respectively, and were dependent on the number of presumptive drug-sensitive allelel. The mutagens EMS, MNNG, ICR-170, ICR-191, and gamma rays significantly increased the frequency of segregation events. The mutagen-induced frequency of dominant mutations to ouabain (Ouar) and alpha-amanitin (Amar) rsistance in the same hybrid line was much lower in comparison to segregation events and was mutagen specific. The chromosome number per metaphase cell was more variable than DNA content in quasitetraploid lines. These properties of marker segregation are consistent with mechanisms of either restricted chromosome loss, rearrangement, or mutation.     

Genetic complementation in hybrid cells derived from mutagen-induced mouse clones deficient in HGPRT activity.

Cultured mouse clonal cells, H-5, were treated with two different mutagens, ethyl methanesulfonate (EMS) and N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Then two selective procedures using 8-azaguanine (8-AZ) or 6-thioguanine (6-TG) were compared in an effort to isolate hypoxanthine-guanine phosphoribosyl-transferase (HGPRT)-deficient cells containing different gene alterations. While many 8-AZ resistant cells were induced by EMS treatment, considerably more 6-TG resistant cells were induced by the same treatment. MNNG treatment induced many 8-AZ resistant mutants but induced hardly any 6-TG resistant mutants. After a fusion experiment of 91 sets involving 13 HGPRT-deficient mouse clones, 7 of which were resistant to 8 AZ and 6 of which were resistant to 6TG with subsequent selection on HAT medium, complementation occurred only in those hybrid mixtures formed between 8-AZ- and 6-TG-resistant clones, while it did not occur at all in hybrid mixtures formed between different 8-AZ-resistant clones and mixtures formed between different 6-TG-resistant clones. The clonally isolated HGPRT-positive cells, characterized by tetraploid karyology, had an apparent activity of HGPRT ranging from 25 to 30% of that of the wild-type parental cells. Heat-inactivation of HGPRT at 65 °C revealed that HGPRT from wild-type cells was heat stable and HGPRT from some 8-AZ-resistant clones were heat labile, while HGPRT from hybrid cells had intermediate stability. These results indicate that there would be alterations in the structural gene of HGPRT in the 8-AZ- or 6-TG-resistant mutants, and also that two selective procedures with 8-AZ or 6-TG alone can isolate different alterations in the structural gene of HGPRT. Moreover, this indicates that some of these gene alterations were mutually complementary. It is most likely that there would be at least 3 cistrons in the locus responsible for HGPRT activity in the mouse cells.     

Spontaneous formation of tumorigenic hybrids between human omental adipose-derived stromal cells and endometrial cancer cells increased motility and heterogeneity of cancer cells

Recent reports indicate that mesenchymal stem cells (MSCs) can fuse with cancer cells to promote cancer progression. Omental adipose-derived stromal cells (O-ASCs) are similar to MSCs, which could be recruited to the stroma in endometrial cancer. The aim of our study was to investigate whether O-ASCs can fuse with endometrial cancer cells to influence cancer cells biological characteristics. We isolated O-ASCs from patients with endometrial cancer. O-ASCs and endometrial cancer cells were labeled with different fluorescent tags and directly co-cultured in an Opera high-throughput spinning-disk confocal microscopy system to observe the processes involved in the fusion, division and migration of hybrid cells. Immunofluorescence and high-throughput imaging analyzes were performed to evaluate proteins related to epithelial-mesenchymal transition (EMT).We found O-ASCs could spontaneously fuse with endometrial cancer cells, including cytomembrane and nuclear fusion. After fusion, endometrial cancer cells assume an elongated and fibroblast-like appearance that exhibit mesenchymal phenotypes. The hybrid cells proliferated through bipolar and multipolar divisions and exhibited more rapid migratory speeds than were observed in the parental cells (P < 0.01), potentially because of their EMT-associated changes, including the down-regulation of E-cadherin and up-regulation of Vimentin. Our results collectively suggest that tumorigenic hybrids spontaneously formed between human O-ASCs and endometrial cancer cells, and that the resulting cells enhanced cancer mobility and heterogeneity by accelerated migration and undergoing multipolar divisions. These data provide a new avenue for investigating the roles of O-ASCs in endometrial cancer.     

Some immunological properties of high and low tumorigenic cellular variants of c-H-ras transformed 3T3 cells

NIH 3T3 cells transformed in vitro with the c-H-ras oncogene were subcloned. The resulting subclones were assayed for in vivo tumorigenicity in nude and in immunocompetent mice. The response of two high tumorigenic and two low tumorigenic clones to mediators of natural immunity was analyzed. The clones did not differ in sensitivity to NK cell-mediated lysis. However, compared to low tumorigenic clones, the high tumorigenic ones had a down-regulated expression of a membrane determinant recognized by a certain monoclonal naturally occurring antibody. The determinants recognized by other monoclonal naturally occurring antibodies available in the laboratory were equally expressed on the high and low tumorigenic clones. The high tumorigenic cells showed an increased resistance to cytotoxicity mediated by lymphotoxin. These results suggest that naturally occurring antibodies and lymphotoxin may participate in controlling the tumorigenicity of transformed cells. The high tumorigenic clones but not the low tumorigenic ones contained a novel 3.5-kb ras mRNA.     

Cloning of mouse myeloma cells and detection of rare variants

Cultured mouse myeloma cells have been cloned in soft agar using a modification of the method established by Pluznik and Sachs ('65, '66) and by Bradley and Metcalf ('66). A linear relationship existed between the number of cells plated and the number of colonies produced. Conditions for obtaining optimum cloning efficiency and colony size were determined for the MPC-11 cell line. Feeder cells of mouse, human and rabbit origin and conditioned growth medium obtained from mouse cultures and had an enhancing effect on colony formation. Immunoglobulin production by cloned cells was detected by overlaying the clones with anti-immunoglobulin antiserum. The antiserum had no adverse effect on cloning efficiency or colony size. A reconstruction experiment was performed to show that the plate assay could reliably detect rare variants of immunoglobulin producing cells. The plate assay was validated by studying immunoglobulin production following recovery of clones from dishes and their growth to mass suspension culture. Immunoglobulin formation in these cultures was assessed by a Ouchterlony immunodiffusion of the supernatant medium, and by incubating the cells with radioactive amino acids and analyzing the intracellular and secreted immunoglobulin on polyacrylamide gels.     

Lack of correlation between mutagen-induced chromosomal univalency and aneuploidy in mouse spermatocytes

Aneuploidy represents the predominant type of chromosomal abnormality found in human newborns with birth defects. The concern that environmental agents may cause aneuploidy in germ cells has prompted development of assay systems for detection of potentially aneuploidy-producing agents. One of the most frequently used methods involves cytogenetic analysis of murine spermatogenic cells at the stages of meiotic metaphases. However, criteria for aneuploidy induction have not been standardized in this test system. Many investigators consider the ability of an agent to induce univalents an appropriate measure of its potential to induce aneuploidy. In the present study, the relationship between univalency and aneuploidy was examined in mouse spermatocytes after various mutagen treatments. 45 Swiss mice were treated with 4 different agents; viz., adriamycin vinblastine sulfate, cytosine arabinoside, and radiation (cobalt 60) and 10 untreated animals served as controls. From each animal, 50–200 MIs were examined for both sex-chromosomal and autosomal univalency (X-Y U and AU), and equal numbers of MIIs were examined for aneuploidy (hyperhaploidy). No significant correlations between univalency (either X-Y U or AU) and aneuploidy were found in the mutagen-treated mice; nor were they found in the untreated animals. These results indicate that induction of univalents by a mutagen is not necessarily predictive of the aneuploidy-inducing ability of his agent.     

Stimulation of anchorage independent proliferation of human adrenocortical carcinoma cells by inhibition of cholesterol biosynthesis

Proliferation of SW13 human adrenocortical carcinoma cells under anchorage independent conditions was stimulated in a dose-dependent manner by treatment with the cholesterol biosynthesis inhibitor mevinolin. Induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was observed in mevinolin treated cultures. The growth stimulatory effect of mevinolin, but not that of epithelial transforming growth factor, a polypeptide growth factor for SW13 cells, was reversed by exogenous mevalonic acid. However, neither dolichol nor low density lipoprotein supplementation affected the response of SW13 cells to mevinolin. The results suggest that mevalonic acid metabolites may participate in the regulation of anchorage independent growth of SW13 cells.     

Microarray proteomic analysis discriminates tumorigenic mouse ovarian surface epithelial cells of divergent aggressive potential

Cancer is an intrinsically heterogeneous disease. Tumors classified under the same etiology and histological type may display divergent growth and invasion properties, resulting in different progression rates and clinical outcomes. Here, we approached this subject in a syngeneic mouse model of ovarian cancer. Antibody microarrays were applied to obtain the proteomic profiles of IF5 and IG10, two spontaneously transformed mouse ovarian surface epithelial (MOSE) cell lines of cognate clonal origin but different tumorigenic behavior in vivo. Repeated dye-swap allowed filter out about 40% of inconsistent signals from a total of 224 arrayed antibodies. Two-class comparison tests resulted in 31 differentially expressed proteins (adjusted p < 0.05). Proteins of the ErbB and focal adhesion signaling pathways showed higher levels in IG10, the most aggressive cell. In contrast, the less aggressive IF5 cell was enriched in proteins related to nuclear chromatin organization and cell-cycle. Additionally, comparison between protein levels and mRNA levels of MOSE cells resulted in a positive rank correlation for 50-60% of protein-mRNA pairs (p < 1.7 × 10(-5)). Importantly, the protein profile of IG10 is linked to invasion and chemotherapy response in human ovarian tumors while the IF5 profile is associated to growth control. The minimal IG10 network contained the proteins Jun, Smad4, Myc, Atf2, and Pak1 as major nodes while Chek2, Mdm2 and Ccna2 were the predominant nodes of the IF5 network. The molecular basis accounting for a high aggressive potential not necessarily related to an increased tumor growth capacity is discussed on a pathway-network basis.     

DNA repair in mouse embryo fibroblasts. II. Responses of nontransformed, preneoplastic and tumorigenic cells to ultraviolet irradiation

Ultraviolet light-induced excision repair, as measured by single-strand DNA-break accumulation in the presence of hydroxyurea and 1-beta-D-arabinofuranosylcytosine, undergoes an apparent decline concomitant with spontaneous transformation of mouse cells in vitro. This decline is seen in preneoplastic transformed cells as well as tumorigenic cells, suggesting that it is an early event in transformation. The difference between nontransformed and transformed mouse cells in apparent incision rates is greatest at short times after irradiation when nontransformed cells show a transient phase of rapid incision. No gross differences in the effects of UV on replicative DNA synthesis, bulk RNA synthesis, cell proliferation or clonal survival in nontransformed and transformed cells were seen, in spite of the reduced incision capacity of the latter. Taken together the results suggest that transformed cells are capable of growth in the presence of significantly increased amounts of DNA damage. A decreased ability of nontumorigenic cells to remove DNA lesions, coupled with unrestricted growth, may be responsible for genetic alterations which increase the probability of a cell becoming tumorigenic.     

Isolation of metabolic cooperation-defective variants from mouse embryonal carcinoma cells

We describe the isolation from the HGPRT^? embryonal carcinoma cell line PC13TG8 of a variant, R5/3, defective in metabolic cooperation. This was achieved in two stages via an intermediate, R2/1, using a selective system in which the HGPRT^? embryonal carcinoma cells were co-cultured with HGPRT⁺ cells in 6-thioguanine. R5/3 cells show increased survival compared with PC13TG8 when retested under selection conditions, and a reduction in grain count index when tested by autoradiography as recipients of [³H]hypoxanthine-labelled nucleotides from wild-type donors and as donors of [³H]thymidine- and [³H]adenine-labelled nucleotides to suitably marked recipients. However, a low residual fraction of heavily labelled recipients is found in all autoradiographic experiments with R5/3 cells. This is not due to heterogeneity of either donor or recipient populations. We also describe the development of a colony-formation assay for metabolic cooperation based on the “kiss of life” phenomenon, in which R5/3 shows very poor survival compared with PC13TG8. R2/1 shows behaviour intermediate between PC13TG8 and R5/3 in all the tests described above. We conclude that two steps can be identified in the change of phenotype by which R5/3 is derived from PC13TG8, and that both steps modify the ability of the cells to form permeable junctions.     

Co-cultivation of tumorigenic mouse melanoma cells with cells of a non-tumorigenic subclone inhibits plasminogen activator expression by the melanoma cells.

Clone B559 mouse melanoma cells are highly tumorigenic and produce plasminogen activator. Cells of clone C3471, a line obtained by continued growth of B559 cells in medium containing 5-bromodeoxyuridine (1 microgram/ml), have no plasminogen activator and are non-tumorigenic. When B559 cells are co-cultivated with C3471 cells, the ability of B559 cells to activate plasminogen is suppressed. Under these conditions cell fusion occurs. Lack of expression of plasminogen activators is not a consequence of cell fusion, inhibition of cell division or release of soluble inhibitors of either plasminogen activators or plasmin. No inhibitors of plasminogen activators could be demonstrated in association with sub cellular fractions of C3471 cells or with the C-type viral particles released from C3471 cells. Close contact between cells of the two lines is shown to be essential for suppression of plasminogen activation.     

Association of mouse adenovirus-induced cell surface antigen(s) with histocompatibility antigens

The topological relationship on the mouse adenovirus (M-Ad)-infected cell surface between virus-induced specific cell surface(s) antigens and serologically defined major histocompatibility antigens (H-2) was analyzed by the cap formation technique. Rhodamine-isothiocyanate (RITC)-labeled anti-S serum failed to stain the surface of virus-infected lymphoid cells which were pretreated with anti-H-2 serum and fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin serum (anti-M-Ig) to cap the appropriate H-2 antigens. Conversely, the capping of the S antigens by pretreatment with anti-S followed by FITC anti-M-Ig serum induced cocapping of H-2 antigens. The β₂ microglobulins (β₂m) were also shown to be cocapped with S antigens by anti-β₂m or by anti-S serum. The S antigens, however, did not cocap with mouse-immunoglobulins or Thyl. 2 antigens on virus-infected B or T lymphocytes, respectively. To further elucidate the molecular relationship between S and H-2 antigens, radio-iodinated virus-infected cells were solubilized with Nonidet P40 (NP40) and S antigens were precipitated with anti-S serum. When the precipitates were analysed with sodium dodecyl sulfate Polyacrylamide gel electrophoresis, two major peaks were seen at positions of molecules of about 45,000 and 12,000 daltons both of which corresponded with molecules which were observed when NP40 extracts of virus-infected or uninfected cells were precipitated with anti-H-2 serum. Sequential immunoprecipitation analysis of infected cell extracts showed that S antigens were coprecipitated with either H-2K or H-2D antigens. These results suggest that the S antigens are somehow associated with H-2K or H-2D antigens separately.     

Immunomagnetic separation and analysis of non-malignant variants and parental malignant mouse lymphoma cells

We have devised conditions whereby non-tumorigenic, immunogenic cell variants of S49 mouse lymphoma were analyzed and separated from parental tumorigenic lymphoma cells. This was carried out using polyclonal antibodies (raised against the immunogenic variants) and immunomagnetic beads. The efficacy of the procedure depended on the amount of polyclonal antiserum, the immunobead to cell ratio, incubation time and the number of repetitions of the procedure. Experiments with mixed tumorigenic and non-tumorigenic cells have resulted in an enrichment of up to 200-fold of the non-tumorigenic, immunogenic cells in the population. These findings indicate the potential use of this procedure (in conjunction with other approaches) to isolate from a population of tumorigenic cells those variant cells that might be used to immunize against the parental tumor.