20. Increase in Spermidine / spermine N¹-acetyltransferase mRNA as a Delayed Response of HeLa S3 Cells Following X-ray lrradiation

S. Ichimura, M. Nenoi, and K. Hamana* (*Gunma Univ.)

Keywords: X-ray irradiation, delayed cell death, polyamine, SSAT

In a broad range of biological organisms, including animals, plants, and microorganisms, polyamines mediate multiple essential functions such as DNA replication and protein biosynthesis. The cellular polyamine levels are regulated to maintain the optimal condition for the physiological state through biosynthetic, catabolic and transport pathways. Regulation of polyamines is also thought to be integral to the cellular response to toxic stresses. In fact, the polyamine catabolic enzyme spermidine/ spermine N¹-acetyltransferase (SSAT) is activated by various anti-proliferative agents. For example N¹, N¹¹ diethylnorspermtne, whrch us cytotoxic to cells and a potential chemotherapeutic polyamine analog for cancers, markedly enhances the cellular level of SSAT, while natural polyamine spermtne IS far less effective at increasing the SSAT levels. Our previous result LhxL SSAT mRNA accumulated at the plateau phase in HeLa cells suggests that SSAT mediates the growth arrest of these cells. In order to examine the contribution of SSAT to growth arrest following X-ray irradiation, we measured the intracellular levels of DNA, SSAT mRNA, ODC mRNA, 18S rRNA and polyamines at several days postirradiation.

Exponentially growing asynchronous HeLa S3 cells were irradiated by 2, 5 and 10 Gy X-rays and the proceeding growth was monitored according to DNA content. The growth was not inhibited up to 24 h after irradiation of even 10Gy in either series. The cell cycle of exponentially growing HeLa cells would be arrested after the first or second replication following X-irradiation. Total RNAs of cells in a dish were purified simultaneously with the DNA preparation at various post-irradiation times. 'The Northern blots of an aliquot of total RNA were hybridized with oligonucleotide probes for SSAT and ODC mRNAs and for 18S rRNA. SSAT mRNA increased after irradiation and reached a maxtmum amount at day 3. It is noteworthy that the increase in SSAT mRNA by X-ray treatment appeared suddenly at the beginning of growth inhibition. On the other hand, the ODC mRNA level decreased as the growth proceeded in both irradiated and non-irradiated cells, and reached the minimum level at day 2. The level of 18S rRNA, which would correlate with the translational activity, was maximal at day 1 and decreased gradually to a stationary state in control cells. The fact that X-ray irradiation had only a slight effect on the rRNA level suggests that the growth inhibition induced by X-ray irradiation was not due to the depression in translational activity

The content of individual polyamines among the cells adhering to each dish was also measured. As expected, the levels of N¹-acetylspermidine increased in parallel with the enhancement of SSAT mRNA. The relatively lower level of acetylated polyamines would be due to rapid gradation by polyamine oxidase to reduced polyamines.