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Characterization of Cultured Bovine Adrenocortical Cells and Derived Clonal Lines: Regulation of Steroidogenesis and Culture Life Span^*

MICHAEL H. SIMONIAN, PETER J. HORNSBY, CHARLES R. ILL, MICHAEL J. O'HARE and GORDON N. GILL

Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine La Jolla, California 92093; and the Ludwig Institute for Cancer Research Royal Marsden Hospital (M.J.O'H.), Surrey, England

Address requests for reprints to: Dr. Gordon N. Gill, Department of Medicine, M-013, University of California, San Diego, La Jolla, California92093.

Abstract

Steroid production by cultured bovine adrenocortical cells and derived clones has been characterized by high pressure liquid chromatography. Although bovine adrenocortical cell cultures produce 17-hydroxy-⁴,3-ketosteroids characteristic of the zona fasciculata-reticularis, the ability to llβ-hydroxylate steroids is rapidly lost in culture. Thus, in response to steroidogenic factors, later passage cultures and clonal lines produce progesterone, 17a-hydroxyprogesterone (17-hydroxypregn- 4-ene-3,20-dione), 20a-dihydroprogesterone (20-hydroxypregn- 4-en-3-one), 17-hydroxy-20-dihydroprogesterone (17a,20-dihydroxypregn-4-en-3-one), and 11-deoxycortisol (17,21-dihydroxypregn-4-ene-3,20-dione). Although mass cultures respond to ACTH throughout their life span of 55–65 generations in culture, clonal lines uniformly lack responsiveness to ACTH. Steroidogenic activity, which is low in mass cultures and clones in the absence of steroidogenic factors, is progressively increased in response to angiotensin II, cholera toxin, and monobutyryl cAMP. In stimulated cells, low density lipoprotein is the principal source of cholesterol for steroidogenesis.

Fibroblast growth factor (FGF) is required for proliferation of both mass cultures and clonal lines throughout the finite life span in culture. Removal of FGF from later passage cells results in a rapid increase in generation time. In the continuous presence of FGF, the life span of the clonal line ACl is the same as that of uncloned cultures. FGF also has a weak steroidogenic effect, with dose-response characteristics identical to those for FGFstimulated growth

The clonal line ACl, which has lost responsiveness to ACTH, thus retains regulation of steroidogenesis and growth by FGF, angiotensin II, cholera toxin, prostaglandin E_t, and monobutyryl cAMP similar to that of mass uncloned cultures. These mass cultures and cloned lines represent well defined homogeneous populations of endocrine cells suitable for study of growth and differentiated function throughout a finite life span. (Endocrinology 105: 99, 1979)

Footnotes

^* This work was supported by NIH Research Grant AM-13149 from the NIAMDD and NIH Research Grant AG-00936 from the National Institute on Aging.

Bank of America–Giannini Foundation Fellow.

Received October 31, 1978.