Purification of Host DNA Synthesis-Suppressing Factor (DSF) Produced by Infection with Measles Virus

Yamamoto, Tomoko; Minagawa, Tomonori; Iida, Hiroo

doi:10.1111/j.1348-0421.1976.tb01018.x

Cited by 2 publications

(5 citation statements)

References 15 publications

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“…Mouse tumor cells (Meth-A) derived from ascitic tumor induced in a BALB/c mouse by 20-methylcholanthrene was cultivated in suspension in RPMI-1640 medium supplemented with 5% fetal calf serum. As previously described by Yamamoto et al (8), Meth-A cells were more sensitive to DSF than HeLa or NC-37 cells. Therefore, Meth-A cells were used in the present experiments.…”

Section: Methodssupporting

confidence: 50%

“…The preparation and purification of DSF were described in previous papers (5,8), and a pure preparation (302 unit/mg of 133…”

Section: Methodsmentioning

confidence: 99%

“…The preparation and purification of DSF were described in previous papers (5,8) dine incorporated into the total cellular materials (a), acid insoluble fraction (b), and acid-soluble fraction (c) were counted. As shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

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Thymidine Metabolism in Cells Treated with DNA‐Suppressing Factor (DSF)

Fujii

Minagawa

Kato

et al. 1978

Microbiology and Immunology

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Inhibition of thymidine incorporation into DNA in cells treated with DNA-suppressing factor (DSF) has been studied. After 16 hr treatment with DSF, transport of labeled thymidine across the cell membrane was not inhibited, since equilibrium of labeled thymidine with the acid-soluble pool occurred at the same rate and the radioactivity was at the same level as in untreated cells. The values of Vmax and Km in the kinetics of transport of exogenous thymidine were not changed by DSF. Phosphorylation of labeled thymidine to deoxythymidine triphosphate (dTTP) was not inhibited by DSF. After a chase of labeled thymidine, radioactivity of the acid-soluble fraction in DSF-treated cells decreased more rapidly but that of the acid-insoluble fraction remained at a lower level than in untreated cells. It was assumed that DSF might block the entry of dTTP into DNA.As described in our previous paper (6), DNA-suppressing factor (DSF) inhibited cellular DNA synthesis only when added to cells in the early G1-phase, and did not affect the stability of double-stranded DNA or the chain-elongation of singlestranded DNA in HeLa cells. We discussed the mechanisms of the inhibition of DNA synthesis by DSF and suggested the following possibilities: 1) DSF might inhibit transport of exogenous thymidine across the cell membrane, 2) DSF might inhibit phosphorylation of thymidine, or 3) DSF might inhibit the initiation of DNA synthesis. Recently, Hand(3) reported that two mechanisms underlay the inhibition of DNA synthesis in L-929 cells by Newcastle disease virus (NDV) ; NDV inhibited DNA synthesis directly and, in addition, decreased thymidine transport. The present paper describes the metabolism of exogenous thymidine in DSF-treated cells. MATERIALS AND METHODSCell culture. Mouse tumor cells (Meth-A) derived from ascitic tumor induced in a BALB/c mouse by 20-methylcholanthrene was cultivated in suspension in RPMI-1640 medium supplemented with 5% fetal calf serum. As previously described by Yamamoto et al (8), Meth-A cells were more sensitive to DSF than HeLa or NC-37 cells. Therefore, Meth-A cells were used in the present experiments. Meth-A cells were kindly provided by Dr. Kobayashi, Cancer Institute, Hokkaido University.Host DNA-suppressing factor (DSF). The preparation and purification of DSF were described in previous papers (5,8), and a pure preparation (302 unit/mg of 133

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Section: Methodssupporting

confidence: 50%

“…The preparation and purification of DSF were described in previous papers (5,8), and a pure preparation (302 unit/mg of 133…”

Section: Methodsmentioning

confidence: 99%

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Thymidine Metabolism in Cells Treated with DNA‐Suppressing Factor (DSF)

Fujii

Minagawa

Kato

et al. 1978

Microbiology and Immunology

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“…On the next day, the culture fluid was replaced with serum-free MEM, and the cultures were further incubated for 24 to 48 hr when the culture fluid was harvested. The harvested fluid was centrifuged at 5,000 rpm for 10 min, and DSF contained in the supernatant was purified by the methods previously described (22 0.1 N NaOH, 0.9 M NaC1, and 0.001 M EDTA and centrifuged as described above.…”

Section: Methodsmentioning

confidence: 99%

“…Recently we succeeded in purification of the DSF. The molecular weight of purified DSF was about 55,000 and the activity of DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin (22).…”

mentioning

confidence: 99%

The Effect of the DNA‐Suppressing Factor (DSF) on Host DNA Synthesis in Synchronized Cell Cultures

Minagawa

Fujii

Yamamoto

et al. 1977

Microbiology and Immunology

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Purified host DNA-suppressing factor (DSF) produced into culture fluid of HeLa C-9 cells infected with measles virus inhibited cellular DNA synthesis in HeLa cells. When purified DSF was added into cultures of synchronous HeLa cells at the early G1-phase, cellular DNA synthesis was irreversibly inhibited. However, DSF did not affect the stability of native double-stranded DNA nor the chain-elongation of single-stranded DNA in cells of the S-phase.Many RNA viruses are known to suppress host macromolecular synthesis (1,3,4,15,24). Among them, reovirus type 3 (2, 6, 16), measles virus (8, 18, 25) and pancreatic necrosis virus (7) inhibit only cellular DNA synthesis, while leaving unaffected the synthesis of protein and RNA. In our previous paper (9), it was reported that the host DNA-suppressing factor (DSF) which was produced into culture fluid after infection with measles virus inhibited cellular DNA synthesis. Recently we succeeded in purification of the DSF. The molecular weight of purified DSF was about 55,000 and the activity of DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin (22).In the suppression of DNA synthesis by reovirus and pancreatic necrosis virus, a factor similar to DSF has not yet been proved. However, Cox and Shaw (2) showed that the block of DNA synthesis by reovirus appeared to be due to an event(s) during the early G1-phase of the cell cycle. In addition, Lothrop and Nicholson (7) reported that DNA synthesis in the early synthetic phase was relatively insensitive to the inhibition by pancreatic necrosis virus. Thus it is of interest to determine how the DSF reacts at the specific stages of the cell cycle. The present study was undertaken to determine the steps of the suppression of cellular DNA synthesis in HeLa cells by the DSF. MATERIALS AND METHODSCell culture. HeLa C-9 cells which had been cloned from HeLa cells were cultivated in Eagle's minimum essential medium (MEM) supplemented with 5% calf serum. 639

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Purification of Host DNA Synthesis-Suppressing Factor (DSF) Produced by Infection with Measles Virus

Cited by 2 publications

References 15 publications

Thymidine Metabolism in Cells Treated with DNA‐Suppressing Factor (DSF)

Thymidine Metabolism in Cells Treated with DNA‐Suppressing Factor (DSF)

The Effect of the DNA‐Suppressing Factor (DSF) on Host DNA Synthesis in Synchronized Cell Cultures

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