Abstract
In MALME-3M human melanoma cells the polyamine analog N1,N12-bis(ethyl)spermine (BESPM) suppresses the key polyamine biosynthetic enzymes, ornithine and S-adenosylmethionine decarboxylase, and increases the polyamine 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 microM 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 hybridize 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 (approximately 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 region of the gene and hybridization with the coding region of SSAT cDNA and its prevalence in nuclear mRNA preparations, form A is thought to represent precursor SSAT RNA. Form C is present in control cells and increases steadily during treatment, whereas form B increases transiently during early treatment (1-3 h). By RNase H digestion assay, form B was 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 to be a result of increased gene transcription and stabilization of SSAT mRNA. Nuclear run-on studies indicated a 2-4-fold increase in the transcription rate of the SSAT gene. As indicated by actinomycin D studies, the SSAT mRNA half-life increased with BESPM treatment from 17 to 64 h. The natural polyamine, spermine, also increased SSAT mRNA (5.5-fold at 24 h) and behaved similarly to BESPM in inducing the appearance of the same three transcript forms. The polyamine was much less effective than the analog at increasing enzyme activity. Lowering intracellular polyamine pools with inhibitors of biosynthesis decreased basal SSAT mRNA levels by at least 70% indicating, that the gene can be down-regulated as well as up-regulated by polyamines. These findings indicate that SSAT represents a unique example of gene expression being positively influenced at the RNA level by polyamines and their analogs.
Highlights
Base pair longer poly(A) tract andas such may represent a precursor to form C
The cDNA, carcinoma cell lines (15,17,18).More recently we reported which consisted of a 324-bp PstI-PstI fragment of the SSAT coding that impressiveantitumor activityby BESPM and two related region extending from the first to the last exon, was 32P-labeledby homologs against MALME-3M human melanoma xenografts correlated with the ability of the homologs to induce SSAT in vitro (19.20)
In the present study we report that SSAT induction by the polyamine analog BESPM is associated with the appearance ofnew SSAT transcripts and an increase in steady-state levels of SSAT mRNA whichappears to be at least partially caused by SSAT mRNA stabilization and anincrease in the SSAT gene transcription rate
Summary
The increase in hybridization with the coding regionof SSAT cDNA and the RNA in response to BESPM treatment did not occur at the fact that itwas most prevalent in nuclear RNApreparations same rate or to thesame extent as SSAT activity (Fig. 2,right (seebelow),the highest molecular weighttranscript (-3.5 kb; panel). The latter began a t basal levels of 70 pmol/min/mg form A) was presumed to represent precursor SSAT RNA. Decreases labeled 324-bp PstI-PstI fragment of SSAT cDNA coding region (10, 11).Densitometric values at thebottom indicate the fold increase of the lower SSAT mRNA band relative to control This blot is representative of others obtainedin a total of three experiments. A 48-h treatment with the inhibitors did not fully deplete polyamine pools (Table I), the modest deranged from & 0.5 to 2.0
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