POLYAMINE AND POLYAMINE ANALOG REGULATION OF SPERMIDINE SPERMINE N1-ACETYLTRANSFERASE IN MALME-3M HUMAN-MELANOMA CELLS

In MALME-3M human melanoma cells the polyamine analog N1,N-12-bis(ethy l)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the po lyamine catabolizing enzyme, spermidine/spermine N1-acetyl-transferase (SSAT) by more than 200-fold. In the present study increases in SSAT activity in MALME-3M cells treated with 10 muM BESPM were found to be accompanied by a substantial (up to 45-fold) accumulation of SSAT mRNA . By Northern blot analysis three RNA transcripts were found to hybrid ize with the coding region of human SSAT cDNA: a minor high molecular weight (approximately 3.5 kilobases) species designated form A and two lower molecular weight species designated forms B and C (approximatel y 1.5 and approximately 1.3 kilobases, respectively). Form A increased uniformly during BESPM treatment and was most obvious in nuclear RNA preparations. On the basis of size similarity to the transcribing regi on of the gene and hybridization with the coding region of SSAT cDNA a nd its prevalence in nuclear mRNA preparations, form A is thought to r epresent precursor SSAT RNA. Form C is present in control cells and in creases steadily during treatment, whereas form B increases transientl y during early treatment (1-3 h). By RNase H digestion assay, form B w as found to have a 200-base pair longer poly(A) tract and as such may represent a precursor to form C. Accumulation of SSAT mRNA was found t o be a result of increased gene transcription and stabilization of SSA T mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the tr anscription rate of the SSAT gene. As indicated by actinomycin D studi es, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-f old at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effec tive than the analog at increasing enzyme activity. Lowering intracell ular polyamine pools with inhibitors of biosynthesis decreased basal S SAT mRNA levels by at least 70% indicating, that the gene can be down- regulated as well as up-regulated by polyamines. These findings indica te that SSAT represents a unique example of gene expression being posi tively influenced at the RNA level by polyamines and their analogs.