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. 2003 Jan;41(1):11–21. doi: 10.1023/A:1024296220592

Characterisation of BHK-21 cells engineered to secrete human insulin

Patrick Gammell 1, Lorraine O'Driscoll 1, Martin Clynes 1
PMCID: PMC3449758  PMID: 19002958

Abstract

Autoimmune destruction of β cells in the pancreas leads to type I, or insulin dependent diabetes mellitus (IDDM), through the loss of endogenous insulin production capacity. This paper describes an attempt to generate ‘artificial’β cells using the fibroblast cell line BHK21. Stable transfectants expressing the human preproinsulin (PPI) gene were isolated and characterised. The resulting clone selected for further analysis (BHK-PPI-C16) was capable of secreting 0.12 pmol proinsulin/hr/105 cells and maintained a steady cellular proinsulin content of 0.36 ± 0.04 pmol l−1. There was no processing of the proinsulin to mature insulin. The cells were unresponsive to glucose but there was increased proinsulin secretion in the presence of agents that stimulated formation of intracellular cAMP. Transfection of cDNAs for the key elements of the glucose sensing apparatus (GLUT2 and glucokinase) led to a subphysiological stimulation of secretion when glucokinase was transfected alone while there was a complete loss of insulin secretion when both components were overexpressed. The deleterious effect on proinsulin secretion observed upon co-expression of the glucose sensing genes may have implications for applications requiring multigene expression in BHK21 cells.

Keywords: BHK21, Cell Therapy, Diabetes, Glucokinase (GCK), Glucose Sensing, Glucose transporter type 2 (GLUT2), Human Insulin Gene, Insulin secretion, Multigene expression

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References

  1. Aebischer P., Pochon N.A., Heyd B., Deglon N., Joseph J.M., Zurn A.D., et al. Gene therapy for amyotrophic lateral sclerosis (ALS) using a polymer encapsulated xenogenic cell line engineered to secrete hCNTF. Hum Gene Ther. 1996;7:851–860. doi: 10.1089/hum.1996.7.7-851. [DOI] [PubMed] [Google Scholar]
  2. Davies E.L., Shennan K.I.J., Docherty K., Bailey C.J. Expression of GLUT2 in insulin secreting AtT20 pituitary cells. J Molec Endocrinol. 1998;20:75–82. doi: 10.1677/jme.0.0200075. [DOI] [PubMed] [Google Scholar]
  3. Falqui L., Martinenghi S., Severini G.M., Corbella P., Taglietti M.V., Arcelloni C., et al. Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin. Hum Gene Ther. 1999;10:1753–1762. doi: 10.1089/10430349950017437. [DOI] [PubMed] [Google Scholar]
  4. Halban P.A., Irminger J.C. Sorting and processing of secretory proteins. Biochem J. 1994;299:1–18. doi: 10.1042/bj2990001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hughes S.D., Quaade C., Milburn J.L., Cassidy L., Newgard C.B. Expression of normal and novel glucokinase mRNA's in anterior pituitary and islet cells. J Biol Chem. 1991;266:4521–4530. [PubMed] [Google Scholar]
  6. Hughes S.D., Johnson J.H., Quaade C., Newgard C.B. Engineering of glucose stimulated insulin secretion and biosynthesis in non-islet cells. Proc Natl Acad Sci USA. 1992;89:688–692. doi: 10.1073/pnas.89.2.688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kawakami Y., Yamaoka T., Yamashita K., Itakura M., Nakcuchi H. Somatic gene therapy for diabetes with an immunological safety system for complete removal of transplanted cells. Diabetes. 1992;41:956–960. doi: 10.2337/diab.41.8.956. [DOI] [PubMed] [Google Scholar]
  8. Kelly R.B. Pathways of protein secretion in eukaryotes. Science. 1985;230:25–32. doi: 10.1126/science.2994224. [DOI] [PubMed] [Google Scholar]
  9. Kintsurashvili E., Zhou D., Wheeler M.B., Vacek I., Sum A.M. Genetic engineering of glucose stimulated insulin secretion in Chinese hamster ovary cells. Art Cells Blood Subs and Immob Biotech. 1998;26:329–340. doi: 10.3109/10731199809117675. [DOI] [PubMed] [Google Scholar]
  10. Law E., Gilvarry U., Lynch V., Gregory B., Grant G., Clynes M. Cytogenetic comparison of two poorly differentiated human lung squamous cell carcinoma lines. Cancer Genet Cytogenet. 1992;59:111–118. doi: 10.1016/0165-4608(92)90204-l. [DOI] [PubMed] [Google Scholar]
  11. Lee S.W., Eldin G. Expression of tetracycline resistance in pBR322 derivatives reduces the reproductive fitness of plasmid containing Escherichia coli. Gene. 1985;39:173–180. doi: 10.1016/0378-1119(85)90311-7. [DOI] [PubMed] [Google Scholar]
  12. Lu D., Tamemoto H., Saito I., Takeuchi T. Regulatable production of insulin from primary-cultures hepatocytes: insulin production is up-regulated by glucagon and cAMP and down regulated by insulin. Gene Ther. 1998;5:888–895. doi: 10.1038/sj.gt.3300677. [DOI] [PubMed] [Google Scholar]
  13. Merten O.W., Kallel H., Manuguerra J.C., Tardy-Panit M., Crainic R., Delpeyroux F., et al. The new medium MDSS2N, free from any animal protein supports cell growth and production of various viruses. Cytotechnology. 1999;30:191–201. doi: 10.1023/A:1008021317639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Motoyoshi S., Shirotani T., Araki E., Sakai K., Kaneko K., Motoshima H., et al. Cellular characterisation of pituitary adenoma cell line (AtT20 cell) transfected with insulin glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose. Diabetologia. 1998;41:1492–1501. doi: 10.1007/s001250051096. [DOI] [PubMed] [Google Scholar]
  15. O'Driscoll L., Gammell P., Clynes M. Engineering vero cells to secrete human insulin. In vitro cell dev biol anim. 2002;38:146–153. doi: 10.1290/1071-2690(2002)038<0146:EVCTSH>2.0.CO;2. [DOI] [PubMed] [Google Scholar]
  16. Selden R.F., Skoskiewicz M.J., Russel P.S., Goodman H.M. Regulation of insulin gene expression. implications for gene therapy. New Engl J Med. 1987;317:1067–1076. doi: 10.1056/NEJM198710223171706. [DOI] [PubMed] [Google Scholar]
  17. Simpson A.M., Tuch B.E., Swan M.A., Tu J., Marshall G.M. Functional expression of the human insulin gene in a human hepatoma cell line (Hep G2) Gene Ther. 1995;2:223–231. [PubMed] [Google Scholar]
  18. Stewart C., Taylor N.A., Green I.C., Docherty K., Bailey C.J. Insulin-releasing pituitary cells as a model for somatic cell gene therapy in diabetes mellitus. J Endocrinol. 1994;142:339–343. doi: 10.1677/joe.0.1420339. [DOI] [PubMed] [Google Scholar]
  19. Taniguchi H., Fukao K., Nakcuchi H. Constant delivery of proinsulin by encapsulation of transfected cells. J Surgical Res. 1997;70:41–45. doi: 10.1006/jsre.1997.5085. [DOI] [PubMed] [Google Scholar]
  20. Yallop C.A., Svedsen I. The effects of G418 on the growth and metabolism of recombinant mammalian cell lines. Cytotechnology. 2001;35:101–114. doi: 10.1023/A:1017550902771. [DOI] [PMC free article] [PubMed] [Google Scholar]

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