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Regulated Expression of the Prodynorphin Gene in the R2C Leydig Tumor Cell Line^*

CYNTHIA T. McMURRAY, LAKSHMI DEVI, LISA CALAVETTA and JAMES O. DOUGLASS

Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University Portland, Oregon 97201
Department of Pharmacology, New York University Medical Center (L.D.) New York, New York 10016

Address all correspondence and requests for reprints to: Dr. James O. Douglass, Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, 3181 Sam Jackson Park Road, Portland, Oregon 97201.

Abstract

We report here that prodynorphin mRNA and prodynorphin-derived peptides are synthesized in the R2C rat Leydig tumor cell line. The size of the prodynorphin transcript found in these cells (-2200 nucleotides) is identical to that found in the intact testis. R2C cells also contain proteolytically processed prodynorphin-derived peptides. In R2C cells, the endogenous prodynorphin gene and cellular levels of prodynophinderived peptides are positively regulated by cAMP analogs, while phorbol esters exert a slight negative regulation of the prodynorphin mRNA. Using gene transfer techniques, we have identified a 210-basepair fragment of the rat prodynorphin gene which initiates the transcription of the bacterial reporter molecule, chloramphenicol acetyl transferase. The chimeric fusion gene, when transfected into R2C cells, exhibited the same positive response to cAMP analogs as the endogenous gene. The results suggest that a cAMP regulatory element resides within the cloned rat prodynorphin fragment, and that the element is functionally active in R2C cells. (Endocrinology 124: 49–59, 1989)

Footnotes

^* This work was supported by a research award from the McKnight Foundation and Grant DA-04154 (to J.D.) from the NIDA.

Supported by Postdoctoral Fellowship PF-2997 from the American Cancer Society.

Received July 29, 1988.

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