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NCI-Navy Medical Oncology [A. F. G., H. K. O.], Medicine [C. E. M., C. A. S., R. V. L. R.], and Surgery Branches [M. F. B.] Laboratory of Pathology [T. J. T.], National Cancer Institute and Naval Hospital [A. F. G., H. K. O.], and Developmental Endocrinology Branch [G. P. C.], National Institute of Child Health and Development, Bethesda, Maryland 20814; Children's Hospital Medical Center, Oakland, California 94609 [C. H. S.]; and the American Type Culture Collection, Rockville, Maryland 20852 [T. R. C.]
We established a continuous cell line, NCI-H295, from an invasive primary adrenocortical carcinoma. The cell line was established in a fully defined medium (HITES) and later could be adapted for growth in a simple medium supplemented only with selenium, insulin, and transferrin and devoid of serum, steroids, fibroblast growth factor, and a source of exogenous cholesterol. NCI-H295 cells had a relatively long population doubling time and were tumorigenic when inoculated s.c. into athymic nude mice. The cultured cells had ultrastructural features of steroid-secreting cells and contained complex cytogenetic abnormalities including the presence of multiple marker chromosomes. Steroid analyses (radio-immunoassays and mass spectrometry), performed 7 to 9 years after culture initiation, demonstrated secretion of more than 30 steroids characteristic of adrenocortical cells. Total unconjugated steroid secretion in serum-supplemented medium was 2.83 µg/106 cells/24 h and about 4-fold less in serum-free medium. The major pathway of pregnenolone metabolism in NCI-H295 cells is androgen synthesis, with formation of dehydroepiandrosterone, androstenedione, testotesterone, and at least three sulfated androgens, as well as estrogens. In addition, formation of cortisol, corticosterone, aldosterone, and 11ß-hydroxyandrostenidione indicated the presence of 11ß-hydroxylase. Thus, multiple pathways of steroidogenesis are expressed by NCI-H295 cells, including formation of corticosteroids, mineralocorticoids, androgens, and estrogens. Our findings indicate the presence in NCI-H295 cells of all of the major adrenocortical enzyme systems, including 11ß-hydroxylase, desmolase, 21
-hydroxylase, 17-hydroxylase, 18-hydroxylase, lyase, sulfokinase, and aromatase. The NCI-H295 cell line should prove of value in studying the regulation, metabolic pathways, and enzymes involved in steroid formation and secretion. In addition, it may provide insights into the biology and treatment of adrenocortical carcinoma.
1 C. H. S. is supported by the NIH (Grant DK34400). HPLC/MS was carried out on an instrument provided through the NIH Shared Instrument Program (Grant RR03300).
2 To whom requests for reprints should be addressed, at NCI-Navy Medical Oncology Branch, Naval Hospital, Bethesda, MD 20814.
3 Current address: Department of Laboratories, Children's Hospital, Los Angeles, CA 90027.
4 Current address: Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
Received 5/ 1/89.
Revised 4/30/90.
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