無蛋白培養液にて増殖可能な線維肉腫株の分泌因子による自己増殖抑制

無蛋白培養液にて増殖可能なマウス線維肉腫 Gc-4 PF細胞に conditioned medium 濃縮液 (CM) を添加して培養すると, 今まで多く報告されている結果とは異なり, 細胞が障害され増殖が抑制されることをdye exclusion法と3- (4, 5-dimethylthiazo1-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) 法にて確認した.増殖の抑制効果は, 培養開始時の細胞数が多いほど早期に認められたので分泌因子によるものと考えられた.Hoechst33342にて染色した細胞をフローサイトメトリーで検索したところ, CMの...

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Published in北関東医学 Vol. 45; no. 6; pp. 503 - 513
Main Author 斯波, 俊祐
Format Journal Article
LanguageEnglish
Japanese
Published 北関東医学会 01.11.1995
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ISSN0023-1908
1883-6135
DOI10.2974/kmj1951.45.503

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Abstract 無蛋白培養液にて増殖可能なマウス線維肉腫 Gc-4 PF細胞に conditioned medium 濃縮液 (CM) を添加して培養すると, 今まで多く報告されている結果とは異なり, 細胞が障害され増殖が抑制されることをdye exclusion法と3- (4, 5-dimethylthiazo1-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) 法にて確認した.増殖の抑制効果は, 培養開始時の細胞数が多いほど早期に認められたので分泌因子によるものと考えられた.Hoechst33342にて染色した細胞をフローサイトメトリーで検索したところ, CMの添加により小型高染色細胞が有意に増加し, アポトーシスを誘発することが示唆された.しかしながら, DNAの断片化は, 高分子量のレベルでむしろ抑制されていた.細胞周期を調べると, CMの添加によりS期の細胞が約10%減り, G2/M期の細胞が増加したが, phase特異的な細胞周期の静止状態は明らかではなかった.以上の結果より, 蛋白非依存性培養株である線維肉腫細胞は, 自己の死を導く因子を分泌していることが示唆された.さらにその死滅過程は, クロマチンの変化には依存しないタイプのアポトーシスの関与が考えられた.
AbstractList 無蛋白培養液にて増殖可能なマウス線維肉腫 Gc-4 PF細胞に conditioned medium 濃縮液 (CM) を添加して培養すると, 今まで多く報告されている結果とは異なり, 細胞が障害され増殖が抑制されることをdye exclusion法と3- (4, 5-dimethylthiazo1-2-yl) -2, 5-diphenyl tetrazolium bromide (MTT) 法にて確認した.増殖の抑制効果は, 培養開始時の細胞数が多いほど早期に認められたので分泌因子によるものと考えられた.Hoechst33342にて染色した細胞をフローサイトメトリーで検索したところ, CMの添加により小型高染色細胞が有意に増加し, アポトーシスを誘発することが示唆された.しかしながら, DNAの断片化は, 高分子量のレベルでむしろ抑制されていた.細胞周期を調べると, CMの添加によりS期の細胞が約10%減り, G2/M期の細胞が増加したが, phase特異的な細胞周期の静止状態は明らかではなかった.以上の結果より, 蛋白非依存性培養株である線維肉腫細胞は, 自己の死を導く因子を分泌していることが示唆された.さらにその死滅過程は, クロマチンの変化には依存しないタイプのアポトーシスの関与が考えられた.
Author 斯波, 俊祐
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2) Sporn MB, Roberts AB : Autocrine growth factors and cancer. Nature 313 : 745-747, 1985.
11) Sun X-M, Snowden RT, Skilleter DN, et al : A flow-cytomeric method for the separation and quantitation of normal and apoptotic thymocytes. Anal. Biochem. 204 : 351-356, 1992.
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7) Watanabe H, Shinozaki T, Raz A, et al : Expression of aurocrine motility factor receptor in serum- and protein-independent fibrosarcoma cells : Implication for autonomy in tumor-cell motility and metastasis. Int. J. Cancer 53 : 689-695, 1993.
21) Reed JC : Bcl-2 and regulation of programmed cell death. J. Cell Biol. 124 : 1-6, 1994.
12) Tomei LD, Shapiro JP, Cope FO : Apoptosis in C3H/10T1/2 mouse embryonic cells : Evidence for internucleosomal DNA modification in the absence of double-strand cleavage. Proc. Natl. Acad. Sci. USA 90 : 853-857, 1993.
22) Fesus L, Davies PJA, Piacentini M : Apoptosis : molecular mechanism in programmed cell death. European J. Cell Biol. 56 : 170-177, 1991.
30) Yanagihara K, Tsumuraya M : Transforming growth factor β1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 52 : 4042-4045, 1992.
23) Vaux DL : Toward an understanding of the molecular mechanism of physiological cell death. Proc. Natl. Acad. Sci. USA. 90 : 786-789, 1993.
18) Walker PR, Smith C, Youdale T, et al : Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res. 51 : 1078-1085, 1991.
16) Wyllie AH : Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284 : 555-556, 1980.
10) Hardin JA, Sherr DH, DeMaria MA, et al : A simple fluorescence method for surface antigen phenotyping of lymphocytes under-going DNA fragmentation. J. Immunol. Methods 154 : 99-107, 1992.
3) Watanabe H, Chigira M, Arita S : Murine fibrosarcoma clone established in defined medium. In vitro Cell Dev. Biol. 27A : 267-269, 1991.
29) Dulic V, Kaufmann WK, Wilson SJ, et al : p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76 : 1013-1023, 1994.
25) Lockshin RA, Zakeri Z : Programmed cell death and apotosis. In Tomei LD, Cope FO (eds) : Apoptosis : The molecular basis of cell death. Cold Spring Harbor Laboratory Press, New York, 1991, pp47-60.
17) Brown DG, Sun X-M, Cohen GM : Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J. Biol. Chem. 268 : 3037-3039, 1993.
13) Watanabe H, Kanbe K, Shinozaki T, et al : Apoptosis of a fibrosarcoma induced by protein-free culture involves DNA cleavage to large fragments but not internucleosomal fragmentation. Int. J. Cancer : in press.
15) Areds MJ, Morris RG, Wyllie AH : Apoptosis The role of the endonuclease. Am. J. Pathol. 136 : 593-608, 1990.
4) Watanabe H, Chigira M : The role of endogenous proteins in the protein-free maintenance of three distinct tumor cell lines in vitro. Int. J. Oncol. 1 : 481-487, 1992.
5) 有田 覚, 千木良正機, 渡辺秀臣ら : 転移性肉腫Gunma Clone-4 の基礎的研究.日整会誌57 : 1847-1857, 1983.
31) Lin J-K, Chou C-K : In vitro apoptosis in human hepatoma cell line induced by transforming growth factor β1. Cancer Res. 52 : 385-388, 1992.
26) Osthoff KS, Walczak H, Droge W, et al : Cell nucleus and DNA fragmentation are not required for apoptosis. J. Cell Biol. 127 : 15-20, 1994.
9) Tada H, Shiho O, Kurosima K, et al : An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93 : 157-165, 1986.
8) Mosmann T : Rapid colorimetric assay for cellular growth and survival : Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 : 55-63, 1983.
6) Watanabe H, Kanbe K, Chigira M : Differential purification of autocrine motility factor derived from a murine protein-free fibrosarcoma. Clin. Exp. Metastasis 2 : 155-163, 1994.
14) Schwartz DC, Cantor CR : Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37 : 67-75, 1984.
27) Murray AW : Creative blocks : cell-cycle checkpoints and feedback controls. Nature 359 : 599-604, 1992.
19) Chigira M, Noda K, Watanabe H : Autonomy in tumor cell proliferation. Med. Hypoth. 32 : 249-254, 1990.
References_xml – reference: 25) Lockshin RA, Zakeri Z : Programmed cell death and apotosis. In Tomei LD, Cope FO (eds) : Apoptosis : The molecular basis of cell death. Cold Spring Harbor Laboratory Press, New York, 1991, pp47-60.
– reference: 24) Wyllie AH, Kerr JFR, Currie AR : Cell death : the significance of apotosis. Int. Review of Cytol. 68 : 251-306, 1980.
– reference: 22) Fesus L, Davies PJA, Piacentini M : Apoptosis : molecular mechanism in programmed cell death. European J. Cell Biol. 56 : 170-177, 1991.
– reference: 10) Hardin JA, Sherr DH, DeMaria MA, et al : A simple fluorescence method for surface antigen phenotyping of lymphocytes under-going DNA fragmentation. J. Immunol. Methods 154 : 99-107, 1992.
– reference: 29) Dulic V, Kaufmann WK, Wilson SJ, et al : p53-dependent inhibition of cyclin-dependent kinase activities in human fibroblasts during radiation-induced G1 arrest. Cell 76 : 1013-1023, 1994.
– reference: 7) Watanabe H, Shinozaki T, Raz A, et al : Expression of aurocrine motility factor receptor in serum- and protein-independent fibrosarcoma cells : Implication for autonomy in tumor-cell motility and metastasis. Int. J. Cancer 53 : 689-695, 1993.
– reference: 18) Walker PR, Smith C, Youdale T, et al : Topoisomerase II-reactive chemotherapeutic drugs induce apoptosis in thymocytes. Cancer Res. 51 : 1078-1085, 1991.
– reference: 1) Barnes D, Sato G : Methods for growth of cultured cells in serum-free medium. Anal. Biochem. 102 : 255-270, 1980.
– reference: 30) Yanagihara K, Tsumuraya M : Transforming growth factor β1 induces apoptotic cell death in cultured human gastric carcinoma cells. Cancer Res. 52 : 4042-4045, 1992.
– reference: 4) Watanabe H, Chigira M : The role of endogenous proteins in the protein-free maintenance of three distinct tumor cell lines in vitro. Int. J. Oncol. 1 : 481-487, 1992.
– reference: 16) Wyllie AH : Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284 : 555-556, 1980.
– reference: 20) Thompson CB : Apoptosis in the pathogenesis and treatment of disease. Science 267 : 1456-1462, 1995.
– reference: 9) Tada H, Shiho O, Kurosima K, et al : An improved colorimetric assay for interleukin 2. J. Immunol. Methods 93 : 157-165, 1986.
– reference: 26) Osthoff KS, Walczak H, Droge W, et al : Cell nucleus and DNA fragmentation are not required for apoptosis. J. Cell Biol. 127 : 15-20, 1994.
– reference: 31) Lin J-K, Chou C-K : In vitro apoptosis in human hepatoma cell line induced by transforming growth factor β1. Cancer Res. 52 : 385-388, 1992.
– reference: 19) Chigira M, Noda K, Watanabe H : Autonomy in tumor cell proliferation. Med. Hypoth. 32 : 249-254, 1990.
– reference: 8) Mosmann T : Rapid colorimetric assay for cellular growth and survival : Application to proliferation and cytotoxicity assays. J. Immunol. Methods 65 : 55-63, 1983.
– reference: 5) 有田 覚, 千木良正機, 渡辺秀臣ら : 転移性肉腫Gunma Clone-4 の基礎的研究.日整会誌57 : 1847-1857, 1983.
– reference: 13) Watanabe H, Kanbe K, Shinozaki T, et al : Apoptosis of a fibrosarcoma induced by protein-free culture involves DNA cleavage to large fragments but not internucleosomal fragmentation. Int. J. Cancer : in press.
– reference: 15) Areds MJ, Morris RG, Wyllie AH : Apoptosis The role of the endonuclease. Am. J. Pathol. 136 : 593-608, 1990.
– reference: 11) Sun X-M, Snowden RT, Skilleter DN, et al : A flow-cytomeric method for the separation and quantitation of normal and apoptotic thymocytes. Anal. Biochem. 204 : 351-356, 1992.
– reference: 23) Vaux DL : Toward an understanding of the molecular mechanism of physiological cell death. Proc. Natl. Acad. Sci. USA. 90 : 786-789, 1993.
– reference: 2) Sporn MB, Roberts AB : Autocrine growth factors and cancer. Nature 313 : 745-747, 1985.
– reference: 17) Brown DG, Sun X-M, Cohen GM : Dexamethasone-induced apoptosis involves cleavage of DNA to large fragments prior to internucleosomal fragmentation. J. Biol. Chem. 268 : 3037-3039, 1993.
– reference: 21) Reed JC : Bcl-2 and regulation of programmed cell death. J. Cell Biol. 124 : 1-6, 1994.
– reference: 28) Mercer WE, Shields MT, Amin M, et al : Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53. Proc. Natl. Acad. Sci. USA. 87 : 6166-6170, 1990.
– reference: 27) Murray AW : Creative blocks : cell-cycle checkpoints and feedback controls. Nature 359 : 599-604, 1992.
– reference: 6) Watanabe H, Kanbe K, Chigira M : Differential purification of autocrine motility factor derived from a murine protein-free fibrosarcoma. Clin. Exp. Metastasis 2 : 155-163, 1994.
– reference: 14) Schwartz DC, Cantor CR : Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis. Cell 37 : 67-75, 1984.
– reference: 3) Watanabe H, Chigira M, Arita S : Murine fibrosarcoma clone established in defined medium. In vitro Cell Dev. Biol. 27A : 267-269, 1991.
– reference: 12) Tomei LD, Shapiro JP, Cope FO : Apoptosis in C3H/10T1/2 mouse embryonic cells : Evidence for internucleosomal DNA modification in the absence of double-strand cleavage. Proc. Natl. Acad. Sci. USA 90 : 853-857, 1993.
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SubjectTerms アポトーシス
フローサイトメトリー
内因性増殖抑制因子
無蛋白細胞培養
線維肉腫
Title 無蛋白培養液にて増殖可能な線維肉腫株の分泌因子による自己増殖抑制
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