Effects of MLT and atRA on ER and TGFp mRNA Levels

Previous work by our laboratory has demonstrated that MLT can down-regulate the expression of the ER in human breast cancer cells (18), while others have shown that RA also suppresses ER gene expression (59). It has also been shown that both MLT (72) and RA (72) can alter the expression of estrogen-regulated genes, such as TGFP, which are involved in breast tumor cell proliferation. We, therefore, examined the steady state levels of mRNA encoding the ER and TGF-P1 in MCF-7 cells by Northern blot analysis following 48h of treatment with either MLT or atRA alone, or with the sequential regimen of MLT and atRA. Figure 3 shows that both atRA and MLT alone significantly decreased the steady state level of ER mRNA by 62% and 79%, respectively (p < 0.01 vs control). However, the sequential regimen of MLT and atRA reduced ER mRNA expression to almost undetectable levels (p < 0.001 vs MLT or atRA alone). In addition, both atRA and MLT alone enhanced the steady state level of TGF-P1 mRNA by 40% and 53%, respectively; while the sequential regimen produced a super-induction ofTGF-P1 mRNA levels (91% increase over control, and 65% and 52% increase over atRA or melatonin treatment, respectively). These results suggest that the sequential treatment of MCF-7 cells with MLT followed by atRA results in an additive or synergistic induction ofTGF-P1 mRNA expression.

'36B4

Figure 3. Effects of treatment with MLT or atRA alone, versus the sequential regimen of MLT and atRA on steady state ER and TGF-131 mRNA levels in MCF-7 cells cultured in medium supplemented with 5% CS-FBS. MCF-7 cells were incubated with ethanol diluent (control), 10-9M MLT (Mel), 10-9 M atRA fatRA), or a regimen of MLT followed 24 h later by atRA (M + R). For each time point, 50 ^g of total RNA was fractionated on denaturing 1 % agarose gels and blotted. Northern blots were probed with [32P]-labeled human ER and human TGF- P1 cDNAs. The 36B4 cDNA was used to monitor RNA loading. The autoradiograph of this Northern blot analysis is representative of three independent experiments. ER and TGF- 1 mRNA expression was quantified by scanning densitometry and normalized to 36B4 mRNA.

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