Spindle disturbances in mammalian cells II. Induction of viable aneuploid/polyploid cells and multiple chromatid exchanges after treatment of V79 Chinese hamster cells with carbaryl modifying effects of glutathione and S9

Treatment of V79 cells with carbaryl increased the frequency of cells with elevated chromosome numbers (> 22). This effect of carbaryl was inhibited by simultaneous addition of glutathione or S9. Although selective forces seemed to act against cells with increased chromosome numbers, such cells were still significantly more frequent in treated compared with control cultures 74 h after treatment. In addition, a high adaptive value for these remaining aneuploid/polyploid cells was indicated because they were able to go through 2 successive rounds of replication immediately before fixation to the same extent as cells with chromosome numbers considered normal (21/22) for this cell line.Multiple chromatid exchanges were also observed after carbaryl treatment. The lack of single exchange events indicates that the effect may be systemic. However, additional experiments are required before this hypothesis can be confirmed or discarded. Considering the results obtained and the possible importance of gene dosage for tumor promotion, it is suggested that carbaryl should be tested for tumor promotion in vivo.     

Sister-chromatid exchanges and thioguanine resistance in V79 Chinese hamster cells after treatment with the aneuploidy-inducing agent carbaryl +/- S9 mix

Carbaryl induced sister-chromatid exchanges (SCEs) but no thioguanine resistance in V79 Chinese hamster cells. Addition of S9 from Aroclor-pretreated rats, or glutathione, reduced the toxic effects of carbaryl. Glutathione or S9 mix reduced the effect of carbaryl on SCE. However, the latter result indicates that carbaryl's effect may be enhanced at a certain compound/S9 ratio. Since treatment with microsomes alone, but not S9 mix, was clastogenic it cannot be excluded that this enhancement of SCE was due to perturbations in the S9 mix by carbaryl rather than to formation of some particular SCE-inducing metabolite from the compound. The effects of carbaryl on chromosomes and chromosomal distribution are comparable to those sometimes reported for TPA. This, in conjunction with the weak indications on carcinogenic activity of carbaryl, makes it of interest that the compound be tested for promotion or co-carcinogenicity in vivo.     

Timing of initiation of chromosome replication in individual Escherichia coli cells.

The synchrony of initiation of chromosome replication at multiple origins within individual Escherichia coli cells was studied by a novel method. Initiation of replication was inhibited with rifampicin or chloramphenicol and after completion of ongoing rounds of replication the numbers of fully replicated chromosomes in individual cells were measured by flow cytometry. In rapidly growing cultures, with parallel replication of several chromosomes, cells will end up with 2n (n = 1, 2, 3) chromosomes if initiation occurs simultaneously at all origins. A culture with asynchronous initiation may in addition contain cells with irregular numbers (not equal to 2n) of chromosomes. The frequency of cells with irregular numbers of chromosomes is a measure of the degree of asynchrony of initiation. After inhibition of initiation and run-out of replication in rapidly growing B/r A and K-12 cultures, a small fraction of the cells (2-7%) contained 3, 5, 6 or 7 chromosomes. From these measurements it was calculated that initiation at four origins in a single cell occurred within a small fraction, 0.1, of the doubling time (tau). A dnaA(Ts) mutant strain grown at permissive temperature exhibited a very large fraction of cells with irregular numbers of chromosomes after drug treatment demonstrating virtually random timing of initiation. A similar pattern of chromosome number per cell was found after treatment of a recA strain.     

Schedule dependent variation in human lymphocyte sensitivity to bleomycin and repair of chromosomal aberrations in G2.

Bleomycin (BLM) induced chromosomal damage in G2 phase and its repair kinetics in normal human lymphocytes were studied following different treatment schedules. As a first step, a dose-response curve was obtained (concentrations of 5-50 micrograms/ml). For repair kinetics studies, blood samples were treated with BLM at a concentration of 20 micrograms/ml. Continuous treatment produced equal numbers of breaks per cell (br/c) when the cells were treated 3, 4 or 5 h before fixation. If the treatment time was extended to 6 h, the level of br/c was increased 2-fold (p < 0.001) as a result of an increased number of cells with more than 3 br/c. The curves obtained after pulse treatment showed maximal chromosome damage at time 3 (45 min BLM treatment, followed by 2 h repair in drug free medium). When the time after treatment was extended to 4 h (treatment time 5), a 50% reduction in chromosome damage was measured. It was found out that at treatment points 3, 4 and 5 the differences in breaks per cell at the different schedules applied were statistically highly significant. If caffeine (CAF) was added, the continuous treatment, BLM+CAF, induced a statistically significant increase in the frequency of br/c at every treatment point, but the shape of the curve illustrating the kinetics of chromosomal damage remained unchanged. Moreover, the addition of CAF at continuous BLM treatment brings the level of br/c close to that measured at the pulse BLM treatment except for treatment time 3. When applied in a combination with BLM, CAF considerably modified the kinetics of chromosome damage for a pulse (BLM alone) treatment. The possible reasons for the changes in the level of br/c as well as a tentative scheme for assessment of chromosome damage repair capacity after BLM treatment are discussed.     

Regualtion of the immune response to tumor antigens. I. Immunosuppressor cells in tumor-bearing hosts.

A/Jax mice were rendered immune to the syngeneic and transplantable methylcholanthrene-induced Sarcoma 1509a by the surgical removal of the tumor 7 days after implantation; subsequent injection i.v. transfer of 10(7) to 10(8) washed thymus or spleen cells of tumor-bearing animals (TBA) to immune animals significantly inhibited the rejection of the tumor; this suppressive effect was entirely abolished by the treatment of these lymphocytes with anti-theta serum or anti-thymocyte serum (ATS) and complement before adoptive transfer. On the other hand, an equal number of thymus or spleen cells of normal animals or of animals bearing an unrelated tumor had no suppressive effect. Treatment of normal syngeneic animals with ATS after tumor cell inoculation or splenectomy of TBA resulted in the suppression of the tumor growth. The serum of TBA had no effect on tumor growth in immune syngeneic mice. Together these results suggest that TBA possess immunosuppressor T cells regulating negatively their immune response to the tumor.     

NK cells rapidly remove B16F10 tumor cells in a perforin and interferon-gamma independent manner in vivo

Natural killer (NK) cells have been shown critical in reducing tumor lung metastasis in various murine cancer models. Effector molecules such as perforin and IFN-γ may play important roles in inhibition of metastasis. However, most of these conclusions were based on experiments that involved quantitation of metastatic colonies several weeks after tumor challenge. The roles of NK cells and their effector molecules (perforin and IFN-γ) in the initial immune responses against tumor metastasis in lungs are still unknown. By using the B16F10 melanoma tumor model combined with confocal microscopy, we observed an increase in numbers of B16F10 cells in NK-depleted mice at 60 min post tumor inoculation, but this effect was independent of perforin or IFN-γ. In addition, NK cell numbers in lungs after tumor injection rapidly increased suggesting a redistribution of NK cells in the lungs. However, NK cells were not found in contact with tumor cells until day 6 or later. Our data indicate that during early responses against B16F10 cells, NK cells use another mechanism(s) besides perforin and IFN-γ to prevent tumor metastasis.     

Enhancement of varicella-zoster virus replication in cultured human embryonic lung cells treated with the pesticide carbaryl.

In studies designed to determine the factors responsible for control of herpesvirus replicaton in an infected cell, we examined the interaction of varicella-zoster (VZ) virus-infected human embryonic lung cells with the pesticide carbaryl. The replication of the cell-associated VZ virus was enhanced 2- to 13-fold as compared to control cultures in Sevin 4 Oil-treated cultures and in cultures treated with the pesticide's active ingredient, carbaryl. The replication of VZ virus in cultures teated with the base oil plus inert ingredients found in the pesticide formulation was not enhanced. Possible differences in cytotoxicity induced by Seven 4 Oil, pure carbaryl, or the base oil preparation were ruled out since treated and control cultures were shown to have similar numbers of viable cells when measured by trypan blue exclusion tests or by the ability of treated cells to form foci. A dose response study showed a decrease in viral enhancement in cells treated with decreasing carbaryl concentrations.     

Unique uncoupler-stimulation pattern of mitochondrial ATPase activity of tumor cells, brain, and fetal liver

Mitochondria were prepared from various kinds of normal tissues and tumor cells of mice, and their ATPase activities were measured in the presence of an uncoupler. The ATPase activities of all mitochondria were stimulated by the uncoupler when it was added to the mitochondrial suspension just before or after addition of ATP. However, when mitochondria were preincubated with the uncoupler for four minutes or more before the addition of ATP, its stimulating effect on mitochondrial ATPase activities was greatly reduced in all tumor cells tested, but not in normal adult liver. Reduction of the stimulating effect of the uncoupler by preincubation with it was also observed with mitochondrial ATPase of brain and fetal liver. Thus this pattern of change in the effect of uncoupler on preincubation may be common to tumor mitochondria, but it is not specific to tumor mitochondria. The pattern of uncoupler stimulation observed in fetal liver changed rapidly to that of adult liver immediately after birth. Thus the difference between the two uncoupler stimulation patterns is probably not due to a difference in molecular species of mitochondrial ATPase.     

Chromosomal variability of ginseng cells transformed with plant oncogene rolC

Chromosome numbers were was studied in ginseng cell line 1c transformed with Agrobacterium rhizogenes strain A4, which carried plasmid pRiA4, and with A. tumefaciens strain GV3101, which carried vector pPCV002-35S rolC. As compared with the nontransformed cell line 1c, tumor cell cultures 1c-A4 and 1c-rolC and the tissues of rolC teratoma (excluding leaves) displayed higher polyploidy and aneuploidy. The 1c-A4 and 1c-rolC hairy-root cultures also had aneuploid and polyploid cells, but the chromosome variation was lower than in tumor cells or the initial culture 1c. Generally, an increase of chromosome variation in cultivated cells was the main effect of the integration of several oncogenes, which were in the A. rhizogenes A4 T-DNA, or of the individual rolC gene in the ginseng genome. Another effect consisted in stabilization of the chromosome number in some differentiated transgenic tissues. Possible reasons for this effect are discussed.     

Mevalonate deprivation leads to aneuploidy in Chinese hamster ovary cells

After exposure to compactin, the competitive inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 22% of CHO-K1 cells contained abnormally high numbers of chromosomes. In two populations of cells selected for compactin resistance 31 and 33% of the cells contain more than 22 chromosomes. Some cell lines isolated from these populations have the wild type chromosome number of 20-21, while others have a broad distribution of chromosome number, often with a mean around 36-40. Finally, Chinese hamster ovary cells that are mutant for 3-hydroxy-3-methylglutaryl-CoA reductase and therefore auxotrophic for mevalonate were starved for that compound. This treatment also increased the number of cells containing extra chromosomes. These results indicate that interruption of the cellular supply of mevalonate results in abnormal chromosome number.     

Immunotherapy of spontaneous mammary tumors in mongrel dogs with autologous tumor cells and neuraminidase

Summary The effect produced on tumor progression by the injection of either VCN-treated tumor cells or tumor cells mixed with VCN to dogs with spontaneous mammary tumors was investigated. Dogs of different breeds and ages with at least two palpable spontaneous mammary tumors were selected. One tumor was left in the animal for further clinical examination, whereas the other tumor(s) was (were) excised for histologic diagnosis and for preparation of a single-cell suspension. Autologous M-cells were treated with VCN, subsequently extensively washed and injected SC into the neck of the dog on the day of operation and on the next day or different numbers of autologous M tumor cells (10⁵, 10⁶, 10⁷, 5×10⁷, 10⁸) were mixed with different amounts of VCN (0, 0.65, 6.5, 65 mU), and these various mixtures were injected ID at different sites to each dog on the day of operation. This procedure has been called chessboard vaccination (Seiler and Sedlacek, 1978). Altogether 79 dogs were blindly distributed into six groups in three consecutive studies. The results show that the therapeutic effect of the injection of VCN-treated autologous tumor cells depends on the number of tumor cells injected: injection of 2×10⁷ tumor cells repeatedly induced regression of the residual tumor mass (Studies I, II, and III) in most dogs and prevention of metastasis (Study I), while the application of 1×10⁸ tumor cells caused enhanced tumor proliferation in all and early metastasis in most of the dogs (Study I). The injection of 2×10⁶ tumor cells induced only a transient regression, with subsequent progression of the residual mammary tumor (Study II). Repetition of the injection of 2×10⁶ tumor cells three times every 4 weeks did not improve this effect (Study II). The chessboard vaccination proved to be at least as effective as the injection of 2×10⁷ VCN-treated tumor cells (Study III), although 1×10⁸ or more tumor cells had been injected; this number of cells caused tumor enhancement when the cells were treated with VCN only and injected SC (Study I). Moreover, the DTH reaction after ID injection of autologous tumor cells could be increased by the addition of VCN: low numbers of tumor cells and high amounts of VCN or high numbers of tumor cells and low amounts of VCN caused the most pronounced skin response. The relevance of these data to overcoming the risk of tumor enhancement after injection of an inadequate number of VCN-treated tumor cells and the possible diagnostic and therapeutic relevance of the DTH response after chessboard vaccination will be discussed.The abbreviations used in this work are: VCN, vibrio cholerae neuraminidase; M, mitomycin-treated; M-VCN, treated with mitomycin and VCN; DTH, delayed-type hypersensitivity; PBS, phosphate-buffered saline     

Isolation and characterization of variant clones of Chinese hamster cells after treatment with irradiated 5-iodouridine.

Variant clones were isolated from cultured Chinese hamster Don cells after treatment with irradiated 5-iodouridine. The following characters of a primary variant clone, C-11 and a secondary variant clone, C-24 were compared with those of the original clone C-1: colony-forming activity, growth rate in the presence of irradiated and unirradiated 5-iodouridine, distribution of chromosome numbers and cell cohesion. The variant clones C-11 and C-24 were partially resistant to unirradiated 5-iodouridine at lower concentration and C-24 cells were slightly resistant to short-term treatment with irradiated 5-iodouridine. Unlike clines C-1 and C-11 the variant clone C-24 showed no lag phase on growth in 5-iodouridine medium. The modal numbers of the chromosomes of all three clones were 22, like that of normal Chinese hamster diploid cells. Of the three clones, the variant C-24 cells showed the least mutual cohesion and the original C-1 cells showed the most. The possibility that an alteration in cellular membrane might be related to an increase in the resistance to radiosensitizing agents are discussed.     

Chromosomal aneuploidy in African Wildcat somatic cells and cloned embryos

In the present study, we compared the incidence of aneuploidy in in vitro fertilized domestic cat embryos (DSH-IVF) with that of African Wildcat (AWC) cloned embryos reconstructed with AWC fibroblast donor cells from different passages (AWC-NT). Fibroblast cells were cultured to passages 1 (P1), 3 (P3), 4 (P4), and 9 (P9), after which cells at each passage were karyotyped and serum-starved before being frozen for nuclear transfer. AWC-NT embryos were produced by fusion of a single AWC somatic cell at P1, P3, P4, or P9 to enucleated domestic cat cytoplast derived from in vitro matured (IVU) oocytes. DSH-IVF embryos were produced after IVU oocytes were fertilized in vitro with domestic cat spermatozoa. To determine chromosome numbers, embryos (2-4-cell) or fibroblast cells were cultured in medium containing 0.28 microg/mL of Colcemid for 22-24 h or 15-24 h, respectively. Subsequently, embryos and cells were placed in hypotonic solution, fixed, and stained for analysis of chromosome spreads by bright field microscopy. Chromosomal abnormalities in AWC fibroblast cells increased progressively during culture in vitro: P1 (43%), P3 (46%), P4 (62%), and P9 (59%). In fibroblast cells, hypoploidy (94/202, 46%) was the major chromosomal abnormality, and it occurred more frequently than hyperploidy (14/202, 7%; p < 0.05). While the percentage of hyperploid cells remained stable during all passages, the proportion of hypoploidy in fibroblast cells increased significantly after P4. The overall incidence of chromosomal abnormalities in AWC-NT embryos at P1 (45%), P3 (60%), and P4 (50%) was similar to that of the fibroblast cells from which they were derived; however, the incidence was higher for embryos reconstructed with donor fibroblasts at P9 (89%). Hypoploidy was the most common chromosomal abnormality observed in either AWC-NT or DSH-IVF embryos. AWCNT embryos reconstructed with donor cells at early passages (P1, P3, and P4) had similar frequencies of chromosomal diploidy, as did DSH-IVF embryos. Accordingly, based on the present results, for NT we are currently using cat donor cells at early passages, when the percentage of cells with chromosomal abnormalities is low. It is recommended that the chromosomal stability of each cell line be analyzed before use as NT donor cells to reduce the incidence of chromosomal anomalies in reconstructed embryos and to possibly produce a subsequent increase in cloning efficiency.     

The effect of factors released from the tumor-transformed cells on DNA synthesis, mitosis, and cellular enlargement in 3T3 fibroblasts

Quiescent serum-starved 3T3 cells can be stimulated to initiate DNA synthesis after addition of conditioned media from spontaneously tumor-transformed 3T3 cells (3T6-cells) or from SV-40-transformed 3T3 cells (SV-3T3 cells). The conditioned media were found to stimulate both the chromosome cycle (i.e., DNA synthesis and cell division) and the growth cycle (i.e., cellular enlargement). Furthermore, addition of conditioned media to quiescent 3T3 cells increased the activity of HMG CoA reductase--an enzyme previously proposed to exercise some control on cell proliferation in 3T3 cells (Larsson and Zetterberg: J. Cell. Physiol. 129:99-102, 1986. The increased activity of HMG CoA reductase after treatment with tumor cell conditioned media was correlated to the stimulatory effects on DNA synthesis. By treating 3T3 cells stimulated to resume proliferation by addition of conditioned media with mevinolin (a competitive inhibitor of HMG CoA reductase) the activity of HMG CoA reductase as well as the DNA synthesis and cell division were efficiently inhibited. In contrast, HMG CoA activity was not coupled to the cellular enlargement. Therefore, it is proposed that one set of factors present in tumor cell conditioned media preferentially stimulates the chromosome cycle by increasing the HMG-CoA reductase activity, whereas another set of factors is responsible for growth in cell size. Both types of factors are required for balanced growth.     

Induction of aneuploidy by nickel sulfate in V79 Chinese hamster cells

The ability of nickel sulfate (NiSO(4)) to induce chromosome aneuploidy was investigated in vitro using the V79 Chinese hamster cell line. V79 cells were treated with 100-400 microM NiSO(4) for 24h, and monitored up to 72 h following treatment with a chromosome aberration assay, a micronuclei assay using antikinetochore antibodies (CREST assay) and an anaphase/telophase assay.Aneuploid cells were induced in a significant fraction of the cell population 24-48 h following treatment with nickel sulfate. The majority of these cells were hyperdiploid. In addition, nickel sulfate caused increased frequency of cells with kinetochore-positive micronuclei as well as kinetochore-negative micronuclei. Abnormal chromosome segregation such as lagging chromosomes, chromosome bridges and asymmetric segregation were also observed in more than 50% of anaphase or telophase cells following treatment with NiSO(4). The incidences of these abnormalities were dose-dependent in general, although the effects were prominent in a sublethal dose.These results indicate that NiSO(4) has the ability to induce aneuploidy in V79 cells. In addition, the results in anaphase/telophase assay suggest that the compound may have an effect on spindle apparatus, which could result in aneuploidy following abnormal chromosome segregation.     

Effects of GTP on cyclic AMP concentrations in intact Ehrlich ascites tumor cells.

Experiments were conducted to test the hypothesis that intracellular concentrations of guanosine triphosphate (GTP) regulate the activity and hormonal sensitivity of adenylate cyclase in intact cells. By appropriate treatments, the GTP concentrations of Ehrlich ascites tumor cells could be varied between 28% and 680% of control values. Cyclic AMP concentrations were measured before and after addition of epinephrine in cells containing this range of GTP concentratins. Basal cyclic AMP concentrations were unaffected by changes in GTP concentrations. In cells containing lowered concentrations of GTP, the cyclic AMP concentration following addition of epinephrine was half that in control cells. Elevation of GTP concentrations above normal and had no effect on cyclic AMP concentrations following epinephrine treatment.     

Cytological and cytogenetical studies on brain tumors. V. Preferential loss of sex chromosomes in human meningiomas.

Twelve out of 88 cytogenetically examined meningiomas of female patients showed, in addition to the typical loss of a chromosome 22, a loss of 1 or more chromosomes of group C. Among them 8 tumors had less than 8% cells with Barr-body-like particles, whereas in one tumor 12% and in 3 others over 20% Barr bodies were found, which, based on control studies, were classified as sex-chromatin negative, partly positive, and positive, respectively. In one case the loss of an X chromosome was verified by Giemsa banding. In 6 out of 24 meningiomas of male origin, the chromosomal morphology and association pattern strongly indicated that besides the loss of a chromosome 22, the Y chromosome was also missing. Moreover, the loss of the male sex chromosome could be ascertained in 4 tumors by the conspicuous absence of Y fluorescence in interphase nuclei and in metaphase plates after fluorescence staining. The findings are discussed in connection with the gonosomal loss in other human tumors and in old age.     

Rapid interphase and metaphase assessment of specific chromosomal changes in neuroectodermal tumor cells by in situ hybridization with chemically modified DNA probes

Repeated DNAs from the constitutive heterochromatin of human chromosomes 1 and 18 were used as probes in nonradioactive in situ hybridization experiments to define specific numerical and structural chromosome aberrations in three human glioma cell lines and one neuroblastoma cell line. The number of spots detected in interphase nuclei of these tumor cell lines and in normal diploid nuclei correlated well with metaphase counts of chromosomes specifically labeled by in situ hybridization. Rapid and reliable assessments of aneuploid chromosome numbers in tumor lines in double hybridization experiments were achieved, and rare cells with bizarre phenotype and chromosome constitution could be evaluated in a given tumor cell population. Even with suboptimal or rare chromosome spreads specific chromosome aberrations were delineated. As more extensive probe sets become available this approach will become increasingly powerful for uncovering various genetic alterations and their progression in tumor cells.     

Inhibition of in vitro lymphoproliferative responses by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infectious agent.

Small numbers of X-irradiated 13762 cells added as third-party cells to mitogen response assays or mixed lymphocyte cultures caused a significant reduction in viability of the cocultivated lymphocytes, and completely inhibited the expected lymphoproliferative responses. Results showed that the factor(s) responsible for the inhibitory effect was preserved after ultrasonic disruption of the tumor cells, could be sedimented by ultracentrifugation, and was sensitive to treatment with ultraviolet light. Further, cytopathic effects could be serially propagated using cell-free supernatants obtained from sonicated 13762 tumor cells. The results suggest that the 13762 adenocarcinoma line, as carried in vivo in this laboratory, harbors an infectious particle which can affect the proliferative responses of lymphocytes in vitro.     

Ascitic and solid Ehrlich tumor inhibition by Chenopodium ambrosioides L. treatment

The leaves of Chenopodium ambrosioides L. [Chenopodiaceae] ('mastruz') have been indicated for the treatment of several diseases, among which the cancer. There are no results focusing the effect of C. ambrosioides treatment on tumor development in vivo. The aim of this study was to investigate the effect of treatment with C. ambrosioides on Ehrlich tumor development. Swiss mice were treated by intraperitoneal route (i.p.) with hydroalcoholic extract from leaves of C. ambrosioides (5 mg/kg) or with PBS (control group) 48 h before or 48 h later the Ehrlich tumor implantation. The tumor cells were implanted on the left footpad (solid tumor) or in the peritoneal cavity (ascitic tumor). To determine the solid tumor growth, footpad was measured each 2 days until the fourteenth day, when the feet were weighed. Ascitic tumor development was evaluated after 8 days of tumor implantation by quantification of the ascitic fluid volume and tumor cell number. The i.p. administration of C. ambrosioides extract before or after the tumor implantation significantly inhibited the solid and ascitic Ehrlich tumor forms. This inhibition was observed in ascitic tumor cell number, in the ascitic volume, in the tumor-bearing foot size and foot weight when compared to control mice. The treatments also increased the survival of tumor-bearing mice. In conclusion, C. ambrosioides has a potent anti-tumoral effect which was evident with a small dose and even when the treatment was given two days after the tumor implantation. This effect is probably related with anti-oxidant properties of C. ambrosioides.