Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied by complementary DNA.

Abstract

To investigate the mechanisms by which androgens regulate ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17) in mouse kidney, a cDNA clone encoding OrnDCase mRNA was prepared. Purification of OrnDCase mRNA from kidneys of androgen-treated mice was accomplished by immunoadsorption of renal polysomes to a protein A-Sepharose column and enrichment for poly(A)-containing RNA by oligo(dT)-cellulose. Double-stranded cDNA synthesized from this mRNA was inserted into the Pst I site of plasmid pBR322 by using oligo(dGdC)-tailing and was propagated in Escherichia coli. Plasmids containing cDNA sequences coding for OrnDCase were identified by differential colony hybridization, by radioimmunological detection of OrnDCase-like antigens in bacterial cultures, and by cell-free translation of hybrid-selected mRNA followed by imununoprecipitation with monospecific OrnDCase antiserun%. A restriction endonuclease fragment of the selected plasmid DNA (pODCS4) was labeled by nick-translation and used to study changes in OrnDCase mRNA concentration. After a single dose of testosterone, renal OrnDCase mRNA concentration increased as soon as 6 hr and peaked 24 hr after steroid injection, as measured by RNA blot hybridization. Continuous androgen treatment for 4 days resulted in a 10-to 20-fold increase in OrnDCase mRNA concentration in normal animals, but no induction of this mRNA was detected in mice that have an inherent defect of the androgen receptor (testicular feminization). These results indicate that androgens regulate OrnDCase synthesis in mouse kidney, at least in part, by increasing OrnDCase mRNA accumulation.Ornithine decarboxylase (OrnDCase; L-ornithine carboxylyase, EC 4.1.1.17) catalyzes the conversion of ornithine to putrescine and is the first and apparently rate-limiting enzyme in polyamine biosynthesis. A striking feature of this enzyme is that it has extremely rapid induction kinetics and a high turnover rate (for review, see refs. 1-4). Several studies (1-6) have suggested that regulation of OrnDCase activity occurs through modulation of the amount of the enzyme protein, although other factors such as inhibitors (7-10) or activators (11) of OrnDCase and post-translational enzyme modifications (12, 13) also may be involved. A major reason for difficulties in studying the regulation of this enzyme is that it represents only 0.01-0.05% of the total cellular protein, even when maximally induced. Nonetheless, purification of the enzyme to apparent homogeneity (5,(14)(15)(16)(17) and development of radioimmunoassays (5, 6) have permitted studies on OrnDCase turnover.We reasoned that further understanding of the factors that regulate OrnDCase would be facilitated if a probe were available that would allow direct measurement of OrnDCase mRNA sequences. Kidneys of testosterone-treated mice were chosen as a source for isolation of OrnDCase mRNA because androgen administration increases enzyme protein in this organ to a higher concentration than is known in most tissues (2, 5, 6). In the presen...

Regulation of mouse ornithine decarboxylase activity by cell growth, serum and tetradecanoyl phorbol acetate is governed primarily by sequences within the coding region of the gene

“…Induction of ODC activity and mRNA by TPA appears uniformly reduced in transfectants (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13) fold induction of activity; fig. 3, table 2) as compared to NIH3T3 cells fold induction of activity; fig 3, table 1).…”

“…To better understand the nature of regulation of ODC activity, we and others have cloned mouse ODC genomic DNA and cDNA from cells in which that gene was selectively amplified [9][10][11][12][13]. We showed the cloned ODC gene to be functional by stably transfecting [9,14] ODC-deficient also produce correctly initiated and sized mouse ODC mRNA transcripts and enzymatically active ODC polypeptide [9].…”

Mitogens and protein synthesis inhibitors induce ornithine decarboxylase gene transcription through separate mechanisms in the BC3H1 muscle cell line.

“…1B (Fig. 2A), corresponding to the size of mature ODCase mRNA (1,14,16,18,23 Proliferating, undifferentiated cells contained a relatively high level of ODCase mRNA ( Fig. 2A and B ODCase mRNA induction by protein synthesis inhibition.…”

A novel mechanism of resistance to α‐difluoromethylornithine induced by cycloheximide

“…Since ODC gene amplification is a rather common result of drug resistance, we performed a Southern blot analysis of the DNAs derived from the parental and variant cells using a mouse ODC cDNA clone as a probe [15]. Fig.2 shows that the normal pattern of the multigene ODC family [6, 21,22] is equally distributed in the wild-type and mutant BP-CD5 cells without any relative increase in the intensity of the different bands corresponding to the resistant cells.…”

“…The resulting fragments were fractionated by 0.8% agarose gel electrophoresis, transferred onto nitrocellulose and hybridized as described [14] to the nick-translated mouse ODC-cDNA clone pODC54, kindly provided by Dr O.A. Jgnne [15]. Filters were subjected to autoradiography for 48-72 h.…”