Neplanocin A. Actions on S-adenosylhomocysteine hydrolase and on hormone synthesis by GH4C1 cells.

G Wolfson,J Chisholm,A H Tashjian,S Fish,R H Abeles

doi:10.1016/s0021-9258(17)38527-7

Abstract

We have investigated the biochemical actions of Neplanocin A (Nepl A), a carbocyclic adenosine analog, on purified calf liver S-adenosylhomocysteine hydrolase and in the GH4C1 strain of functional rat pituitary cells. Addition of 1 mol of Nepl A/2 mol of S-adenosylhomocysteine hydrolase subunit led to rapid and complete inactivation. Concomitant with inactivation, half of the enzyme-bound NAD was reduced and adenine was released stoichiometrically from Nepl A. In GH4C1 cells Nepl A caused a dose-dependent rapid (within 5 min) and irreversible inactivation of S-adenosylhomocysteine hydrolase and concomitant increase in intracellular S-adenosylhomocysteine. In cells treated with Nepl A for 4-5 days, methylation of DNA cytosine was depressed approximately 50%, and the level of cytoplasmic prolactin mRNA was elevated 2-fold. While acute (30 min) release of prolactin from intracellular stores was unaffected, Nepl A acted in a dose- and time-dependent manner to increase the production of both prolactin and growth hormone, the two hormones synthesized and secreted by GH4C1 cells. The lowest effective dose was 0.12 microM, the concentration required to decrease S-adenosylhomocysteine hydrolase activity by 50%. By 4-7 days the production of both hormones in Nepl A-treated cells was increased 2-3 times above control. The action on hormone production persisted for at least 7 days after removal of Nepl A from the culture medium. We conclude that Nepl A inhibits S-adenosylhomocysteine hydrolase, raises cellular S-adenosylhomocysteine, decreases bulk DNA methylation, and increases hormone synthesis in GH4C1 cells.

Highlights

EXPERIMENTAL PROCEDURESNepl A was a gift from Dr Elslager (Warner-Lambert Company, Ann Arbor, MI)
We have investigated thebiochemical actions of Ne- dependent transmethylases [2]
Transmethylation reactions are involved in a wide variety to thatproposed for 2’-deoxyadenosine [18].Nepl A has been of biological processes such as modification of DNA, RNA, protein, and phospholipid [1].Most transmethylases utilize S-adenosyl-L-methionine as a methyl donor converting it to AdoHcy’which, in turn, is a potent inhibitor of AdoMetreported to have cytotoxic activity against murine L5178Y cells and human colon carcinoma HT-29 cells in culture [14, 19], growth inhibitory activity on certain plant pathogenic fungi [14],and to prolong the life of mice bearing L1210 leukemia cells [11,15]

Summary

EXPERIMENTAL PROCEDURES

Nepl A was a gift from Dr Elslager (Warner-Lambert Company, Ann Arbor, MI). Fresh stock solutionsin M e 8 0 (Fisher) were prepared every 4-5 months and stored at -20"C. The following compounds were purchased from Sigma: HTL, cystine, Cys, Met, 5AzaCyd. Fresh stock solutions in distilled Hz0 were prepared for each experiment. 5-AzaCyd stock was prepared in phosphate-buffered saline (pH 7.6). [6-3H]dCyd (850 mCi/mmol) was from Moravek Biochemicals (Brea, CA) Fresh stock solutions in distilled Hz0 were prepared for each experiment. 5-AzaCyd stock was prepared in phosphate-buffered saline (pH 7.6). [6-3H]dCyd (850 mCi/mmol) was from Moravek Biochemicals (Brea, CA)

Methods

RESULTS

R5 days

DISCUSSION