Regulation of Msx2 gene expression by steroid hormones in human nonmalignant and malignant breast cancer explants cultured in vitro.

Muscle segment homeobox genes, which regulate developmental programs and are expressed in embryonic and adult tissue, play a role in development of some malignancies. There are no reports on the expression of these families of genes in breast cancer tissue. The aim of this study was to compare expression of Msx2 gene in breast cancer of different genotypes as well as in surrounding nonmalignant tissues. Explants obtained during surgery were divided according to their sex steroid receptor status determined by immunocytochemistry. Four explants obtained from malignant and nonmalignant tissue of each individual patient were incubated in a control medium or with the addition of progesterone (10(-7) M) alone, estradiol 17 beta (10(-5) M) or both. The relative level of Msx2 transcripts was evaluated by a semiquantitative RT-PCR and cell proliferation by Alamar Blue test. Results of RT-PCR analysis showed that the relative expression of Msx2 gene depended on the presence of ER/PR receptors both in nonmalignant and malignant tissues Relative amount of Msx2 mRNA was the highest in surrounding nonmalignant ER+/PR- and ER-/PR+ tissue, whereas in ER-/PR- and ER+/PR+ tissue it was 1.4-1.6-fold lower. Tumorigenesis led to about a twofold decrease in the relative amount of Msx2 mRNA except for ER+/PR+ immunophenotype, where no changes were observed. Addition of estradiol or progesterone to the culture of ER-/PR- type tissue explants did not change significantly the relative amount of Msx2 gene mRNA. An opposite effect was observed in ER+/PR- type of tissue. Addition of estradiol alone, or estradiol and progesterone together to tissue culture explants decreased two to three fold the relative amount of Msx2 gene mRNA in both, malignant and surrounding tissues. Progesterone alone had no effect on Msx2 gene expression in this type of tissue. The most complicated regulation was observed in ER+/PR+ type of tissue. Culture of tissue explants supplemented with estradiol significantly increased the relative amount of Msx2 gene mRNA in the surrounding tissue. Progesterone enhanced the stimulatory effect of estradiol in surrounding tissues but not in the malignant tissue. Increased expression of Msx2 correlated with an increased proliferation in ER-/PR- and ER+/PR+ types, but not in ER+/PR- type of tissues. In conclusion, obtained results provide evidence that estrogen affects Msx2 gene expression. Significant changes in the relative amount of Msx2 gene mRNA and lack of canonical ERE element in 5'-upstream sequence of this gene suggest that regulation takes place indirectly probably by protein-protein interaction. The decrease in the relative amount of Msx2 gene mRNA in ER+/PR- type tumor suggests that progesterone also affects Msx2 gene expression by an indirect mechanism(s).

Progesterone but not estradiol 17beta potentiates local GH secretions by hormone-dependent breast cancer explants. An in vitro study.

The aim of this study was to evaluate the effects of treatment of breast cancer explants with tamoxifen (TMX) or RU486 on GH secretory dynamics in the presence of exogenous estrogen (E2), progesterone (P4) or both. Explants obtained during surgery were divided according to their sex steroid hormone receptor status. P4 (10(-7) M) or 17beta-estradiol (10(-5) M) or both were tested in vitro for their ability to induce hGH secretion and cell proliferation. TMX (10(-7) M) was added to E2, RU486 (10(-7) M) to P4, and both were applied to E2 plus P4-supplemented cultures. The stimulatory action of P4 on GH secretion was noted in hormone-dependent (ER+/PR+) but not in hormone-independent explants (ER-/PR-). RU486 did not abolish this effect. The stimulatory action of P4 on GH release was not parallel to the stimulation of cell proliferation. E2 alone was without effect on GH secretion by both types of breast cancer explants. Combined treatment with both steroids stimulated GH secretion and cell proliferation by (ER+/PR+) explants. Both TMX and RU486 reversed this effect on cell proliferation while only RU486 abolished augmentation of GH secretion. In none of the hormone-dependent breast cancers, the combined treatment with E2 and P4 had any effect on GH secretion and cell proliferation. Taken together, these results lead us to the hypothesis that P4 but not E2 potentiates local GH secretion by hormone-dependent breast cancer explants. The fact that RU486 reversed neither GH secretion nor cell proliferation in hormone-dependent explants indicates its non-receptor-mediated mechanism of action.

Comparison of accumulation and altered steroid secretion by placental tissue treated with TCDD and natural mixture of PCDDs-PCDFs.

Explants of human placental tissue harvested immediately after expulsion were used to determine differences between accumulation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated dibenzo-p-dioxin (PCDD)-polychlorinated dibenzo-p-furans (PCDF) environmental mixture, and their influence on placental steroidogenesis. Explants were cultured in vitro for 5 days in media supplemented each day with either TCDD or a mixture of PCDD-PCDF. Media were collected every day for steroid content analysis by radioimmunoassay. At 24 h after the last treatment, the tissue was frozen for further analysis of the content of TCDD or other congeners present in the mixture. Determinations of TCDD and all 17 PCDDs and PCDFs were performed using gas chromatography equipped with DB-5 MS and DB-17 capillary columns. In the control tissue, the amounts of both TCDD and mixture components were close to the limit of detection of the method. In the treated tissue, the TCDD accumulation was 94% of the total exposure to TCDD. The most toxic congeners 2,3,7,8-TCDD, 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 1,2,3,7,8-pentachlorodibenzo-p-furans (PeCDF) and 2,3,4,7,8-PeCDF showed the highest accumulation, which covered >50% of the total toxic equivalents present in this mixture. During the first 3 days of exposure to TCDD there was no effect on the conversion of dehydroepiandrosterone to oestradiol, whereas on days 4 and 5 of exposure, a twofold decrease in oestradiol secretion was observed. However, a small but significant increase in oestradiol secretion was noted at all times of exposure to the PCDD-PCDF mixture. All observed changes in oestradiol secretion were not accompanied by changes in progesterone secretion after exposure to TCDD or the PCDD-PCDF mixture. In conclusion, a high accumulation of TCDD in the placental tissue resulted in a decrease in oestradiol secretion and in vivo this could result in a decrease in blood flow through the placenta. From the mixture, PeCDD and PeCDF in the higher amount accumulated in the placental tissue caused an increase in oestrogen secretion and as a consequence could activate oxytocin secretion from the pituitary and early pregnancy outcome.

Polichlorinated biphenyls (PCB126 and PCB 153) action on proliferation and progesterone secretion by cultured in vitro porcine luteal cells.

SUMMARY: To characterise PCBs action on luteal cell steroidogenesis and cell viability two PCB congeners were selected as model substances. PCB 126 because of its dioxin-like configuration and high toxicity while 153 because it is one of the most commonly detected congeners in breast milk. Luteal cells collected from mature corpora lutea were cultured in M199 medium at 37 degrees C. Control cultures were maintained in that medium alone, while other cultures were supplemented with either PCB 126 (5, 10, 50 and 100 pg/ml) or PCB 153 (5, 10, 50 and 100 ng/ml). After 24 h, 48 h and 72 h of culture media were collected for P4 content analysis. Cell viability was measured using LDH cytotoxicity test. Exposure of luteal cells to all doses of PCB 126 for 24 h had no effect on progesterone secretion while longer, 48 h and 72 h exposure decreased progesterone secretion in a statistically significant manner. Concentration dependent decrease in progesterone secretion by luteal cells was seen after 24 h and 48 h exposure to PCB153 while concentration dependent increase in progesterone secretion was noted after 72 h exposition to this congener. The toxic effect of both congeners was observed only after 72 h exposition to 50 pg/ml and 100 pg/ml in the case of PCB 126 and 50 ng/ml and 100 ng/ml in the case of PCB 153. In conclusion, these results suggest that there are differences in PCB 153 and 126 action in luteal cells. Since information concerning mechanism of PCBs action on luteal cells is scarce, these preliminary experiments are of pioneering character.