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Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 2002 Jun;93(6):667–676. doi: 10.1111/j.1349-7006.2002.tb01305.x

Establishment and Characterization of a New Lung Cancer Cell Line (MI‐4) Producing High Levels of Granulocyte Colony Stimulating Factor

Ryoji Harada 1, Yoshiki Uemura 1,✉, Makoto Kobayashi 1, Adriana Zamecnikova 1, Hideshi Nakata 1, Takahiro Taguchi 2, Mutsuo Furihata 3, Yuji Otsuki 3, Hirokuni Taguchi 1
PMCID: PMC5927050  PMID: 12079515

Abstract

We established a human lung cancer cell line, MI‐4 from the pleural effusion of a 69‐year‐old male with advanced large cell undifferentiated carcinoma of the lung complicated by leukocytosis. The culture supernatant of MI‐4 contained high levels of granulocyte colony stimulating factor (G‐CSF). The intracellular localization of the G‐CSF was identified by immunocytochemistry. Reverse transcription‐polymerase chain reaction (RT‐PCR) revealed G‐CSF mRNA expression in this cell line. The cell line was successfully transplanted into nude mice. The transplanted nude mice also showed leukocytosis with a high serum G‐CSF level. Southern blot analysis did not show amplification or rearrangement of the G‐CSF gene in MI‐4 cells. Spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) analyses revealed that this cell line has an additional chromosome 17 attached to a segment of chromosome 10 besides two intact chromosomes 17, and that each of these three chromosomes 17 has a G‐CSF gene on chromosome 17q. Inflammatory cytokines, tumor necrosis factor (TNF)‐α and interleukin (IL)‐1β, significantly enhanced G‐CSF expression at both the protein and mRNA levels in MI‐4. However, these cytokines did not stimulate the growth of MI‐4 cells, regardless of abundant G‐CSF production. TNF‐α rather suppressed it, in a dose‐dependent manner. Exogenous recombinant human G‐CSF and anti‐G‐CSF antibody did not promote or inhibit the growth of MI‐4 cells at any concentration examined. In addition, RT‐PCR analysis did not show G‐CSF receptor mRNA expression. These results suggest that this cell line does not have an autocrine growth loop for G‐CSF. This cell line should be very useful for understanding the biological activity of G‐CSF in G‐CSF‐overproducing lung cancer.

Keywords: Lung cancer, G‐CSF, Cell line, MI‐4

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REFERENCES

  • 1. ) Asano , S. , Urabe , A. , Okabe , T. , Sato , N. and Kondo , Y.Demonstration of granulopoietic factor(s) in the plasma of nude mice transplanted with a human lung cancer and in the tumor tissue . Blood , 49 , 845 – 852 ( 1977. ). [PubMed] [Google Scholar]
  • 2. ) Okabe , T. , Sato , N. , Kondo , Y. , Asano , S. , Ohsawa , N. , Kosaka , K. and Ueyama , Y.Establishment and characterization of a human cancer cell line that produces human colony‐stimulating factor . Cancer Res. , 38 , 3910 – 3917 ( 1978. ). [PubMed] [Google Scholar]
  • 3. ) Ueyama , Y. and Tamaoki , N.Leukocytosis in nude mice into which human tumors are transplanted—in vivo screening system for hematopoietic growth factors in human tumors . In “ Hematopoietic Growth Factors: Molecular Biology to Clinical Applications of rG‐CSF ,” ed. Quesen‐berry P. J. , Asano S. and Saito S. , pp. 27 – 53 ( 1991. ). Excerpta Medica; , Tokyo . [Google Scholar]
  • 4. ) Okabe , T. , Nomura , H. and Ohsawa , N.Establishment and characterization of a human colony‐stimulating factor‐producing cell line from a squamous cell carcinoma of the thyroid gland . J. Natl. Cancer Inst. , 69 , 1235 – 1243 ( 1982. ). [PubMed] [Google Scholar]
  • 5. ) Baba , M. , Kaito , M. , Hasegawa , H. , Nakayabu , M. , Yoshida , Y. , Uda , Y. , Shima , T. , Suzuki , S. and Shimizu , N.Establishment and characterization of a new human thyroid anaplastic cancer cell line that produces human colony‐stimulating factor . Mie Med. J. , 41 , 155 ( 1991. ). [Google Scholar]
  • 6. ) Higaki , I. , Hirohashi , K. , Fukushima , S. , Wanibuchi , H. , Seike , N. , Yamane , T. , Kubo , S. , Tanaka , H. , Shuto , T. , Yamamoto , T. and Kinoshita , H.Renal pelvic carcinoma producing granulocyte colony‐stimulating factor: report of a case . Surg. Today , 31 , 266 – 268 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 7. ) Akatsuka , A. , Shimamura , K. , Katoh , Y. , Takekoshi , S. , Nakamura , M. , Nomura , H. , Hasegawa , M. , Ueyama , Y. and Tamaoki , N.Electron microscopic identification of the intracellular secretion pathway of human G‐CSF in a human tumor cell line: a comparative study with a Chinese hamster ovary cell line (IA1‐7) transfected with human G‐CSF cDNA . Exp. Hematol. , 19 , 768 – 772 ( 1991. ). [PubMed] [Google Scholar]
  • 8. ) Ito , N. , Matsuda , T. , Kakehi , Y. , Takeuchi , E. , Takahashi , T. and Yoshida , O.Bladder cancer producing granulocyte colony‐stimulating factor . N. Engl. J. Med. , 323 , 1709 – 1710 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Takeda , T. , Ichiyanagi , A. , Sano , K. , Yoshida , J. , Tsutsumi , Y. and Miyaji , T.A case of gallbladder cancer producing granulocyte‐colony‐stimulating factor . Gastroenterol. Jpn. , 25 , 762 – 767 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Baba , M. , Hasegawa , H. , Nakayabu , M. , Shimizu , N. , Suzuki , S. , Kamada , N. and Tani , K.Establishment and characteristics of a gastric cancer cell line (HuGC‐OOHIRA) producing high levels of G‐CSF, GM‐CSF, and IL‐6: the presence of autocrine growth control by G‐CSF . Am. J. Hematol. , 49 , 207 – 215 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Tohyama , K. , Yoshida , Y. , Kubo , A. , Sudo , T. , Moriyama , M. , Sato , H. and Uchino , H.Detection of granulocyte colony‐stimulating factor produced by a newly established human hepatoma cell line using a simple bioassay system . Jpn. J. Cancer Res. , 80 , 335 – 340 ( 1989. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. ) Lilly , M. B. , Devlin , P. E. , Devlin , J. J. and Rado , T. A.Production of granulocyte colony‐stimulating factor by a human melanoma cell line . Exp. Hematol. , 15 , 966 – 971 ( 1987. ). [PubMed] [Google Scholar]
  • 13. ) Fahey , R. J.Unusual leukocyte responses in primary carcinoma of the lung . Cancer , 4 , 930 ( 1951. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Shijubo , N. , Inoue , Y. , Hirasawa , M. , Igarashi , T. , Mori , M. , Matsuura , A. , Uede , T. and Suzuki , A.Granulocyte colony‐stimulating factor‐producing large cell undifferentiated carcinoma of the lung . Intern. Med. , 31 , 277 – 280 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Teramachi , M. , Miyamoto , N. , Yamamoto , Y. , Sasaka , T. , Nakamura , T. and Kitamura , F.A case of large cell carcinoma of the lung which is suspected of producing granulocyte colony‐stimulating factor . J. Jpn. Respir. Soc. , 30 , 1327 – 1332 ( 1992. ). [PubMed] [Google Scholar]
  • 16. ) Asahi , Y. , Kubonishi , I. , Imamura , J. , Kamioka , M. , Matsushita , H. , Furihata , M. , Ohtsuki , Y. and Miyoshi , I.Establishment of a clonal cell line producing granulocyte colony‐stimulating factor and parathyroid hormone‐related protein from a lung cancer patient with leukocytosis and hypercalcemia . Jpn. J. Cancer Res. , 87 , 451 – 458 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. ) Watanabe , M. , Ono , K. , Ozeki , Y. , Tanaka , S. , Aida , S. and Okuno , Y.Production of granulocyte‐macrophage colony‐stimulating factor in a patient with metastatic chest wall large cell carcinoma . Jpn. J. Clin. Oncol. , 28 , 559 – 562 ( 1998. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Takeuchi , R. , Kasagi , K. , Ohta , H. and Konishi , J.Diffuse bony uptake of thallium‐201‐chloride in the granulocyte colony‐stimulating factor‐producing lung carcinoma . J. Nucl. Med. , 39 , 241 – 243 ( 1998. ). [PubMed] [Google Scholar]
  • 19. ) Kasuga , I. , Yonemaru , M. , Minemura , K. , Utsumi , K. , Torii , Y. , Ichinose , Y. , Toyama , K. and Ebihara , Y.A case of large cell carcinoma of the lung producing granulocyte colony‐stimulating factor . J. Jpn. Respir. Soc. , 32 , 73 – 77 ( 1994. ). [PubMed] [Google Scholar]
  • 20. ) Shibuya , T.Establishment of a G‐CSF and GM‐CSF producing cell line from human large cell carcinoma of the lung . Em. J. Haematol. , 43 , 182 – 183 ( 1989. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Suzuki , A. , Takahashi , T. , Okuno , Y. , Nakamura , K. , Tashiro , H. , Fukumoto , M. , Konaka , Y. and Imura , H.Analysis of abnormal expression of g‐csf gene in a novel tumor cell line (KHC 287) elaborating G‐CSF, IL‐1 and IL‐6 with co‐amplification of c‐myc and c‐ki‐ras . Int. J. Cancer , 48 , 428 – 433 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Inoue , M. , Minami , M. , Fujii , Y. , Matsuda , H. , Shirakura , R. and Kido , T.Granulocyte colony‐stimulating factor and interleukin‐6‐producing lung cancer cell line, LCAM . J. Surg. Oncol. , 64 , 347 – 350 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 23. ) Schrock , E. , Mnoir , S. , Veldman , B. , Schoell , B. and Wienberg , J.Multicolor spectral karyotyping of human chromosomes . Science , 273 , 494 – 497 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 24. ) Simmers , R. N. , Smith , J. , Shannon , M. F. , Wong , G. , Lopez , A. F. , Baker , E. , Sutherland , G. R. and Vadas , M. A.Localization of the human G‐CSF gene to the region of a breakpoint in the translocation typical of acute promyelocytic leukemia . Hum. Genet. , 78 , 134 – 136 ( 1988. ). [DOI] [PubMed] [Google Scholar]
  • 25. ) Simmers , R. N. , Webber , L. M. , Shannon , M. F. , Garson , O. M. , Wong , G. , Vadas , M. A. and Sutherland , G. R.Localization of the G‐CSF gene on chromosome 17 proximal to the breakpoint in the t(15;17) in acute promyelocytic leukemia . Blood , 70 , 330 – 332 ( 1987. ). [PubMed] [Google Scholar]
  • 26. ) Kinoshita , T. , Imamura , J. , Nagai , H. and Shimotohno , K.Quantification of gene expression over a wide range by the polymerase chain reaction . Anal. Biochem. , 206 , 231 – 235 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 27. ) Alt , F. W. , Kellems , R. E. , Bertino , J. R. and Schimke , R. T.Selective multiplication of dihydrofolate reductase genes in methotrexate‐resistant variants of cultured murine cells . J. Biol. Chem. , 5 , 1357 – 1370 ( 1978. ). [PubMed] [Google Scholar]
  • 28. ) Schwab , M. , Alitalo , K. , Varmus , H. E. , Bishop , J. M. and George , D. Acellular oncogene (c‐Ki‐ras) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells . Nature , 303 , 497 – 501 ( 1983. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Bishop , J. M.The molecular genetics of cancer . Science , 235 , 305 – 311 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) Ymer , S. , Tucker , W. Q. , Sanderson , C. J. , Hapel , A. J. , Campbell , H. D. and Young , I. G.Constitutive synthesis of interleukin‐3 by leukaemia cell line WEHI‐3B is due to retroviral insertion near the gene . Nature , 317 , 255 – 258 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 31. ) Leibovitz , A. , Wymer , J. , Massey , K. , Thompson , F. H. and Nelson , M. A.Establishment of a new lung sarcoma cell line from a human lung carcinosarcoma . Cancer Lett. , 82 , 145 – 152 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 32. ) Virmani , A. K. , Tonk , V. S. and Gazdar , A. F.Comparison between fluorescence in situ hybridization and classical cytogenetics in human tumors . Anticancer Res. , 18 , 1351 – 1356 ( 1998. ). [PubMed] [Google Scholar]
  • 33. ) Metcalf , D.The roles of stem cell self‐renewal and autocrine growth factor production in the biology of myeloid leukemia . Cancer Res. , 49 , 2305 – 2311 ( 1989. ). [PubMed] [Google Scholar]
  • 34. ) Tachibana , M. , Miyakawa , A. , Tazaki , H. , Nakamura , K. , Kubo , A. , Hata , J. , Nishi , T. and Amano , Y.Autocrine growth of transitional cell carcinoma of the bladder induced by granulocyte‐colony stimulating factor . Cancer Res. , 55 , 3438 – 3443 ( 1995. ). [PubMed] [Google Scholar]
  • 35. ) Kyo , S. , Kanaya , T. , Takakura , M. and Inoue , M.A case of cervical cancer with aggressive tumor growth: possible autocrine growth stimulation by G‐CSF and Il‐6 . Gynecol. Oncol. , 78 , 383 – 387 ( 2000. ). [DOI] [PubMed] [Google Scholar]
  • 36. ) Kikuchi , T. , Nakahara , S. and Abe , T.Granulocyte colony‐stimulating factor (G‐CSF) production by astrocytoma cells and its effect on tumor growth . J. Neurooncol. , 27 , 31 – 38 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 37. ) Pietsch , T. , Mempel , K. , Menzel , T. , Ockler , R. and Welte , K.Medulloblastoma cells constitutively produce granulocyte colony‐stimulating factor . Klin. Padiatr. , 202 , 235 – 239 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 38. ) Caldwell , J. and Emerson , S. G.IL‐1 alpha and TNF alpha act synergistically to stimulate production of myeloid colony‐stimulating factors by cultured human bone marrow stromal cells and cloned stromal cell strains . J. Cell. Physiol. , 159 , 221 – 228 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 39. ) Uemura , Y. , Nakata , H. , Kobayashi , M. , Harada , R. , Asahi , Y. and Taguchi , H.Regulation of granulocyte colony‐stimulating factor and parathyroid hormone‐related protein production in lung carcinoma cell line OKa‐C‐1 . Jpn. J. Cancer Res. , 91 , 911 – 917 ( 2000. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40. ) Gerharz , C. D. , Reinecke , P. , Schneider , E. M. , Schmits , M. and Gabbert , H. E.Secretion of GM‐CSF and M‐CSF by human renal cell carcinomas of different histologic types . Urology , 58 , 821 – 827 ( 2001. ). [DOI] [PubMed] [Google Scholar]
  • 41. ) Kimura , H. , Yamaguchi , Y. , Sun , L. , Iwagami , S. and Sugita , K.Establishment of large cell lung cancer cell lines secreting hematopoietic factors inducing leukocytosis and thrombocytosis . Jpn. J. Clin. Oncol. , 22 , 313 – 319 ( 1992. ). [PubMed] [Google Scholar]
  • 42. ) Quentmeier , H. , Zaborski , M. and Drexler , H. G.The human bladder carcinoma cell line 5637 constitutively secretes functional cytokines . Leuk. Res. , 21 , 343 – 350 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 43. ) Nakamura , M. , Oshika , Y. , Abe , Y. , Ozeki , Y. , Katoh , Y. , Yamazaki , H. , Kijima , H. , Ueyama , Y. , Ogata , T. and Tamaoki , N.Gene expression of granulocyte colony stimulating factor (G‐CSF) in non‐small cell lung cancer . Anti-cancer Res. , 17 , 573 – 576 ( 1997. ). [PubMed] [Google Scholar]

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