FM3A Mutant

Experimental Cell Research

Volume 171, Issue 1, July 1987, Pages 24-36

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase α activity☆

Author links open overlay panelToshihikoEkiMasa-AtsuYamada

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https://doi.org/10.1016/0014-4827(87)90248-5Get rights and content

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One spontaneous and four N-methyl-N′-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase α activity and cannot grow at 39 °C, were isolated and characterized with respect to the thermolability of their DNA polymerase α activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase α activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 °C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 °C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 °C. At 39 °C, the rate for the wild-type increased considerably compared to that at 33 °C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 °C.Objectives: The aim of this study was to examine the cytotoxic effects of Carisolv on mouse mammary carcinoma cell line (FM3A) at different application times.

Methods: The FM3A cell line obtained from the European Collection of Animal Cell Cultures was used in the cell culture assays. Exponentially growing cells were seeded in 5×105 cells/well in 5 ml of RPMI 1640 medium supplemented with 10(%) fetal calf serum and antibiotics in each well of a six-well plate. Carisolv gel was applied onto the cell culture medium for 1, 5 and 20 min and incubated for 24 h at 37 °C in a humidified atmosphere of 5(%) carbon dioxide (CO2). After 24 h incubation, the cells were collected by trypsinisation and counted with a hemocytometer. The cytotoxicity of the Carisolv was determined by evaluation of cell growth and viability in comparison to untreated controls (cell growth=100%). For cell viability, the trypane blue exclusion assay was used. Dunnett's t-test was used for statistical analysis.

Results: Cell growth was significantly reduced after 20 min application of Carisolv in comparison to the control (p<0.001) and 1 min treatment groups (p<0.05). No significant differences were found in cell viability between the study groups.

Conclusions: It can be concluded that prolonged application of Carisolv did not affect cell viability, but had a reducing effect on cell growth in the FM3A cell line.Biochemical and Biophysical Research Communications

Volume 225, Issue 3, 23 August 1996, Pages 924-931

Regular Article

Promotion of Heat-Induced Apoptosis in FM3A Cells by Protease Inhibitors

Author links open overlay panelWei-GuoZhua1ShigetoshiAntokua

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https://doi.org/10.1006/bbrc.1996.1273Get rights and content

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Although it has been shown that proteases may play a positive role in causing apoptosis of some cells, we report here that, on the contrary, protease inhibitors can promote heat-induced apoptosis in FM3A cells. Cysteine protease inhibitor, trans-Epoxy-succinyl-l-leucylamido-(4-guanidino)butane (E-64, 100 μg/ml) and aspartate protease inhibitor, pepstatin-A (100 μg/ml) were used to test hyperthermic effect on FM3A cells and showed remarkable cytotoxicity when they were present in cell suspension during heating at 44 °C. The cytotoxicity was due to promotion of heat-induced apoptosis as judged by DNA agarose electrophoresis. Furthermore, using flow cytometric analysis, we observed a decrease in the G0/G1phase cell and an increase in the S phase cell as well as increased apoptosis after heat shock. E-64 and pepstatin-A exhibited a promotive effect on the changes of cell cycle induced by heat. The data presented suggest that the enhancement of hyperthermic cell killing by protease inhibitors may be related to promotion of heat-induced apoptosis and changes of cell cycle.We have shown previously that induction of HSP70 synthesis in murine FM3A and its mutant ts85 cells by heat shock is somehow modulated by culture temperature. In this study, we further examined activation of heat shock transcription factor (HSF) and induction kinetics of HSP70 synthesis and HSP70 mRNA in FM3A and ts85 cells maintained at 37°C (37°C-cells) and 33°C (33°C-cells). Upon exposure to heat shock, HSF was activated to a high level in 37°C-FM3A cells, whereas HSF was activated only to a low level in the 33°C-cells. The induction of HSP70 mRNA and HSP70 synthesis occurred successively in the 37°C-cells but not in the 33°C-cells. On the other hand, in both 37 and 33°C-ts85 cells, activation of HSF, induction of HSP70 mRNA, and HSP70 synthesis occurred successively. Characteristically, protein synthesis in both 33°C-FM3A and ts85 cells was significantly lower than in the respective 37°C-cells, but constitutive HSP73 levels were similar among both the 37 and 33°C-cells. Furthermore, inhibition of protein synthesis of FM3A cells did not influence the activation of HSF, but accelerated inactivation of the activated HSF. We discuss the possible inhibition mechanisms of activation of HSF in 33°C-FM3A cells, regarding the function of HSP70 in both protein synthesis and repression of HSF.Biochemical and Biophysical Research Communications

Volume 243, Issue 2, 13 February 1998, Pages 550-554

Regular Article

A Mutant Strain of Mouse FM3A Cells Defective in Apoptotic DNA Fragmentation

Author links open overlay panelYukikaYamauchiaKoOkumurabc

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https://doi.org/10.1006/bbrc.1998.8121Get rights and content

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A mutant strain of mouse FM3A cells was defective in apoptotic DNA fragmentation. Upon DNA damage by UV-radiation, the strain did not form DNA fragments, though it accumulated a large amount of p53 protein as did wild-type cells. No DNA fragmentation was observed when the strain was subjected to other kinds of DNA damaging agents such as X ray-radiation, or addition of etoposide to the culture medium. Other well-known biological stimulations inducing apoptosis, serum depletion or addition of TNF-α to the culture medium, also failed to cause DNA fragmentation in the cells. Using this apoptosis-deficient strain, we characterized another apoptotic reaction, export of phosphatidylserine to the cell surface. This molecule was exported onto the surface in both wild-type and the mutant cells when cells were treated with TNF-α and cycloheximide. Thus, the export of phosphatidylserine is suggested to be independent of the pathway for apoptotic DNA fragmentation.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 129, Issue 3, December 1984, Pages 389-395

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

Author links open overlay panelMasaoHyodo∗KenshiSuzuki∗

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https://doi.org/10.1016/0027-5107(84)90094-0Get rights and content

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A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process.

To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 94, Issue 1, May 1982, Pages 199-211

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

Author links open overlay panelMasaoHyodoaMuneoOhkidoc

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https://doi.org/10.1016/0027-5107(82)90182-8Get rights and content

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To investigate the response of cells to one type of DNA damage — namely DNA crosslinks — cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 μg/ml plus 5 kJ/m2 or 1.0 μg/ml plus 1–2.5 kJ/m2)_halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [3H]thymidine into cellular DNA immediately after the treatment. Then, after 8 h of incubation, the incorporation of [3H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [3]Hthymidine were correlated with the halt and resumption in the cell-cycle process.

Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.Biochimica et Biophysica Acta (BBA) - General Subjects

Volume 884, Issue 2, 19 November 1986, Pages 304-310

Purification and characterization of adenine phosphoribosyltransferase from mouse mammary carcinoma FM3A cells in culture

Author links open overlay panelGensakuOkadaaHidekiKoyamab

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https://doi.org/10.1016/0304-4165(86)90178-9Get rights and content

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Adenine phosphoribosyltransferase has been purified to apparent homogeneity from mouse mammary tumor FM3A cells. The purified enzyme, with a specific activity of 20.6·106 units/g protein at 30°C, was homogeneous as judged by polyacrylamide gel electrophoresis and Ouchterlony double immunodiffusion analysis. The native enzyme had a molecular weight of 44000 and a subunit composition of 23000. Apparent Km values for adenine and 5-phosphoriboxyl-1-pyrophosphate (PRib-PP) were 6.6 μM and 1.2 μM, respectively. Free Mg2+ was an essential activator with a half-maximal effect at 0.4 mM. AMP was an inhibitor, competitive with PRib-PP, and the Ki value was estimated to be 24 μM. The enzyme activity was not significant affected by 2,6-diaminopurine, 4-carbamoylimidazolium 5-olate, 8-azaadenine, and 2-fluoro-6-aminopurine. An antibody against the purified mouse adenine phosphoribosyltransferase was raised in a rabbit. The enzyme derived from either mouse, Chinese hamster, or human cells was completely neutralized and precipitated by this antibody, indicating that these enzymes share a common antigenis determinant.Biochemical and Biophysical Research Communications

Volume 140, Issue 1, 15 October 1986, Pages 414-418

Heat production as a cell cycle monitoring parameter

Author links open overlay panelM.Yamamura1T.Miyamae2

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https://doi.org/10.1016/0006-291X(86)91106-XGet rights and content

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A microcalorimetric method was applied to define the cell cycle by measuring heat production of mouse breast cancer cell line, FM3A. FM3A cells, were synchronised at the Go phase and produced 13.0 μ Watts(W) per 1×106 cells. Although the number of cells in fresh medium remained unchanged during the following 24 hour, a dramatic increase of heat production was observed and maximum heat (46.2μW) was produced by the cells at 24 hours when the cell cycle was presumably at the G2 phase. At 26 hours, although cell number increased, heat production decreased. Since the cells were not treated in any manner, this microcalorimetric method of measuring the cell cycle by monitoring heat production can be a very useful tool.The rate of DNA-chain elongation was studied in mouse FM3A cells after treatment with 8-methoxypsoralen plus near-ultraviolet radiation using the minimal doses (1 μg/ml 8-methoxypsoralen plus 1–2.5 kJ/m2 of near-ultraviolet radiation) which inhibited cell-cycle progression or DNA replication. A rapid decrease in incorporation of [3H]thymidine and recovery to some extent during incubation after treatment have been reported (Hyodo, M., Fujita, H., Suzuki, K., Yoshino, K., Matsuo, I. and Ohkido, M. (1982) Mutat. Res. 94, 199–211). The results of the present study showed that the rate was not changed suggesting that the decrease in [3H]thymidine incorporation was not due to the rate of DNA-chain elongation, but was due to change in the frequency of initiation of replication. Formation of DNA crosslinks was then studied by the sedimentation of pre-labeled DNA in an alkaline sucrose gradient. The results showed that, at these doses of 8-methoxypsoralen plus near-ultraviolet radiation, approx. 2–7 crosslinks were formed per 109 Da. It was also suggested that some of the DNA crosslinks might be repaired during the prolonged incubation, but unrepaired crosslinks were still present after 24 h incubation.Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 350, Issue 1, 19 February 1996, Pages 143-152

Research paper

Natural antimutagenic agents

Author links open overlay panelLester A.MitscheraDelbert M.Shankelb

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https://doi.org/10.1016/0027-5107(95)00099-2Get rights and content

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Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the author's laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles. The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P. plicata. The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system. The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.FEBS Letters

Volume 185, Issue 1, 3 June 1985, Pages 95-100

Research letters

Murine mammary FM3A carcinoma cells transformed with the herpes simplex virus type 1 thymidine kinase gene are highly sensitive to the growth-inhibitory properties of (E)-5-(2-bromovinyl)-2'-deoxyuridine and related compounds

Author links open overlay panelJ.BalzariniaT.Senoab

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https://doi.org/10.1016/0014-5793(85)80747-XGet rights and content

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Murine mammary carcinoma (FM3A TK−/HSV-1 TK+) cells, which are thymidine kinase (TK)-deficient but have been transformed with the herpes simplex virus type 1 (HSV-1) TK gene are inhibited in their growth by (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU) and (E)-5-(2-bromovinyl)-2'-deoxycytidine (BVDC) at 0.5, 0.5 and 0.8 ngml, respectively; i.e., a concentration 5000 to 20000-fold lower than that required to inhibit the growth of the corresponding wild-type FM3A/0 cells. Hence, transformation of tumor cells with the HSV-1 TK gene makes them particularly sensitive to the cytostatic action ofBVDU and related compounds.

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