Skip to main content
NCBI home page
Japanese Journal of Cancer Research : Gann logoLink to Japanese Journal of Cancer Research : Gann
. 1990 Jan;81(1):91–99. doi: 10.1111/j.1349-7006.1990.tb02512.x

Kinetics of Internalization and Cytotoxicity of Transferrin‐Neocarzinostatin Conjugate in Human Leukemia Cell Line, K562

Yutaka Kohgo 1,✉, Hitoshi Kondo 1, Junji Kato 1, Katsunori Sasaki 1, Nobuyasu Tsushima 1, Takuji Nishisato 1, Michiaki Hirayama 1, Koshi Fujikawa 1, Naoaki Shintani 1, Yoshihiro Mogi 1, Yoshiro Niitsu 1
PMCID: PMC5917957  PMID: 2139019

ABSTRACT

Human serum transferrin was conjugated with an anticancer‐active polypeptide, neocarzinostatin, by using N‐succinimidy1‐3‐(2‐pyridyldithio)propionate. The conjugate consisted of 1.8 mol of neocarzinostatin per 1 mol of transferrin on average and retained cytotoxic activity against human tumor cells. This conjugate was capable of binding to the transferrin receptor of human myelogenous leukemia K562 cells and was internalized by endocytosis. The LD50 values of the conjugate and neocarzinostatin alone in the presence of excess native bovine transferrin were 0.20 μ/ml and 1.80 μ/ml, respectively, suggesting that the effect of the conjugate was greater than that of neocarzinostatin alone. A pulse‐chase experiment using 125I‐labeled conjugate revealed that 25% of the internalized conjugate was degraded in lysosomes and the rest was recycled back to the cell surface without degradation. About 75% of this conjugate recycled back to the cell surface in 18.3 min (3.4 min for receptor binding and 14.9 min for recycling to the cell surface through the acidosomes), while the rest was delivered from the cell surface to the lysosome in 19.6 min. This phenomenon was confirmed by chasing the radioactivity in subcellular fractions separated by Percoll density gradient centrifugation. Therefore, it was concluded that this conjugate is internalized specifically by transferrin receptors and is at least partly transferred to and accumulated in lysosomal compartments, resulting in the inhibition of cellular DNA synthesis.

Keywords: Transferrin, Transferrin receptor, Neocarzinostatin, Targetting, Conjugate

Full Text

The Full Text of this article is available as a PDF (492.1 KB).

REFERENCES

  • 1. ) Aisen , P. and Listowsky , I.Iron transport and storage proteins . Ann. Rev. Biochem. , 49 , 357 – 383 ( 1980. ). [DOI] [PubMed] [Google Scholar]
  • 2. ) Trowbridge , I. S. and Omary , M. B.Human cell surface glycoprotein related to cell proliferation is the receptor for transferrin . Proc. Natl. Acad. Sci. USA , 78 , 3039 – 3043 ( 1981. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. ) Kohgo , Y. , Niitsu , Y. , Nishisato , T. , Kato , J. , Sasaki , K. , Tsushima , N. , Hirayama , M. , Kondo , H. and Urushizaki , I.Externalization of transferrin receptor in established human cell lines . Cell Biol. Int. Rep. , 11 , 871 – 879 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 4. ) Kohgo , Y. , Niitsu , Y. , Nishisato , T. , Urushizaki , Y. , Kondo , H. , Fukushima , M. , Tsushima , N. and Urushizaki , I.Transferrin receptors of tumor cells — potential tools for diagnosis and treatment of malignancies . In “ Proteins of Iron Storage and Transport ,” ed. Spic G. , Momtruil J. , Chrichton R. R. and Mazurier J. pp. 155 – 169 ( 1985. ). Elsevier/North‐Holland Biomedical Press; , Amsterdam . [Google Scholar]
  • 5. ) Hopkins , C. R. and Trowbridge , I. S.Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells . J. Cell Biol. , 97 , 508 – 521 ( 1983. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. ) Hamilton , T. A. , Wada , H. G. and Sussman , H. H.Identification of transferrin receptor on the surface of human cultured cells . Proc. Natl. Acad. Sci. USA , 76 , 6406 – 6410 ( 1979. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. ) Niitsu , Y. , Kohgo , Y. , Nishisato , T. , Kondo , H. , Kato , J. , Urushizaki , Y. and Urushizaki , I.Transferrin receptor in human cancerous tissues . Tohoku J. Exp. Med. , 153 , 239 – 243 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Trowbridge , I. S. and Domings , D. L.Anti‐transferrin receptor monoclonal antibody and toxin‐antibody conjugates after growth of human tumor cells . Nature , 294 , 171 – 173 ( 1981. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Yeh , C. G. and Faulk , W. P.Killing of human tumor cells in culture with adriamycin conjugates of human transferrin . Clin. Immunol. Immunopathol. , 32 , 1 – 11 ( 1984. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Raso , V. and Basala , M.A highly cytotoxic human transferrin‐ricin A chain conjugate used to select receptor modified cells . J. Biol. Chem. , 259 , 1143 – 1149 ( 1984. ). [PubMed] [Google Scholar]
  • 11. ) Aisen , P. , Liebman , A. and Reich , H. A.Studies on the binding of iron to transferrin and conalbumin . J. Biol. Chem. , 241 , 1666 – 1671 ( 1966. ). [PubMed] [Google Scholar]
  • 12. ) Fukuda , K.Study of targeting chemotherapy against gastrointestinal cancer: preparation of anticancer drug‐monoclonal conjugate and investigation about its biological activities . Akita J. Med. , 12 , 451 – 468 ( 1985. ) ( in Japanese ). [Google Scholar]
  • 13. ) Klausner , R. D. , van Rensworde , J. , Ashwell , G. , Kemph , C. , Schechter , A. N. , Dean , A. and Bridges , K. R.Receptor‐mediated endocytosis of transferrin in K562 cells . J. Biol. Chem. , 258 , 4715 – 4724 ( 1983. ). [PubMed] [Google Scholar]
  • 14. ) Niemann , R. and Buddecke , E.Acceptor‐specific glucuronyl transfer catalyzed by β‐glucuronidase . Biochim. Biophys. Acta , 567 , 196 – 206 ( 1979. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Ciechanover , A. , Schwartz , A. L. , Dautry‐Varsat , A. and Lodish , H. F.Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line . J. Biol. Chem. , 258 , 9681 – 9689 ( 1983. ). [PubMed] [Google Scholar]
  • 16. ) Torpe , P. E. and Ross , W. C. J.The preparation and cytotoxic properties of antibody‐toxin conjugates . Immunol. Rev. , 62 , 121 – 158 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Jansen , F. K. , Blythman , H. E. , Carriere , D. , Casellas , P. , Gros , P. , Laurent , J. L. , Paolucci , F. , Pau , B. , Poncelet , P. , Recher , H. , Vedal , H. and Voisin , G. A.Immunotoxins; hybrid molecules combining high specificity and potent toxicity . Immunol. Rev. , 62 , 185 – 216 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Cumber , A. J. , Forrster , J. A. , Foxwell , B. M. J. , Ross , W. C. , Thorpe , P. E. , Faulstich , H. , Fiume , L. , Domingo , D. L. , Trowbridge , I. S. and Shier , W. T.Drug conjugates . Methods Enzymol. , 112 , 207 – 247 ( 1985. ). 3900635 [Google Scholar]
  • 19. ) Vitetta , E. S. , Krplick , K. A. and Uhr , J. W.Neoplastic B cells as targets for antibody‐ricin A chain immunotoxins . Immunol. Rev. , 62 , 159 – 183 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Raso , V. , Ritz , J. , Basala , M , and Schlossman , S. F.Monoclonal antibody‐ricin A chain conjugate selecting cytotoxic for cells bearing the common acute lymphoblastic leukemia antigen . Cancer Res. , 42 , 457 – 464 ( 1982. ). [PubMed] [Google Scholar]
  • 21. ) Shimizu , N. , Shimizu , Y. and Miskimins , W. K.EGF‐ricin A conjugate, kinetic profiles of cytotoxic effect and resistant cell variants . Cell Struct. Funct. , 9 , 203 – 212 ( 1984. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Miskimins , W. K. and Shimizu , N.Synthesis of a cytotoxic insulin crosslinked to diphtheria toxin fragment A capable of recognizing insulin receptors . Biochem. Biophys. Res. Commun. , 91 , 143 – 151 ( 1979. ). [DOI] [PubMed] [Google Scholar]
  • 23. ) Simpson , D. L. , Cawley , D. B. and Herschman , H. R.Killing of cultured hepatocytes by conjugates of asialofetuin and EGF linked to the A chains of ricin or diphtheria toxin . Cell , 29 , 469 – 473 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 24. ) Gibson , B. W. , Herlihy , W. C. , Anantha Samy , T. S. , Hahm , K‐S. , Maeda , H. , Meienhofer , J. and Biemann , K.A revised primary structure for neocarzinostatin based on fast atom bombardment and gas chromatographic‐mass spectrometry . J. Biol. Chem. , 259 , 10801 – 10806 ( 1984. ). [PubMed] [Google Scholar]
  • 25. ) Ashwell , G. and Harford , J.Carbohydrate specific receptors of liver . Ann. Rev. Biochem. , 51 , 531 – 554 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 26. ) King , A. C. , Hernaez‐Davis , L. and Cuatrecasas , P.Lysomotropic amines cause intracellular accumulation of receptors for epidermal growth factor . Proc. Natl. Acad. Sci. USA , 77 , 3283 – 3287 ( 1980. ). [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Japanese Journal of Cancer Research : Gann are provided here courtesy of Wiley

RESOURCES