Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines.

BACKGROUND: Estrogen receptor-alpha (ERalpha) is essential for mammary gland development and is a major oncogene in breast cancer. Since ERalpha is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERalpha mediated cell proliferation. In the paracrine model, ERalpha-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERalpha does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERalpha in some primary breast tumors. METHODS: Colocalization of ERalpha with Ki-67 in ERalpha-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1) was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERalpha was determined by co-immunofluorescent staining of ERalpha and the major cyclins (D, E, A, B), and by flow cytometry analysis of ERalphahigh cells. To further confirm the autocrine action of ERalpha, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERalpha and cell cycle progression. RESULTS: Colocalization of ERalpha with Ki-67 was present in all three ERalpha-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERalpha is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha. CONCLUSION: Our data indicate that ERalpha can mediate estrogen-induced cell proliferation in an autocrine mode in ERalpha-positive breast cancer cell lines. All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

Effects of androgen on embryo implantation in the mouse delayed-implantation model.

OBJECTIVE: To examine the effects of androgen on implantation and decidualization in the mouse delayed-implantation model. DESIGN: Experimental animal study. SETTING: University research laboratory. ANIMAL(S): Sexually mature female mice (Kunming White strain). INTERVENTION(S): Delayed and activated implantation; pseudopregnancy; embryo transfer (ET); E(2) assay; inhibitor. MAIN OUTCOME MEASURE(S): Effects of androgen on embryo implantation were determined by treating the mice under delayed implantation with different doses of testosterone propionate (TP); the effects of androgen on the expression of implantation-related genes were examined by in situ hybridization. RESULT(S): Delayed implantation could be initiated by TP. Dihydrotestosterone was also able to initiate implantation in the delayed-implantation model. The implantation window could be maintained for at least 48 hours by 5 mg TP per mouse. Prostaglandin endoperoxide synthase 2 (Ptgs2) and microsomal prostaglandin E synthase (mPtges) were aberrantly expressed in mouse uterus at implantation sites after delayed implantation was activated by high doses of TP. CONCLUSION(S): A low dose of TP led to a delay in embryo implantation, but a high dose caused aberrant expression of both Ptgs2 and mPtges at the implantation site. It is possible that high doses of TP may disturb peri-implantation development or may be involved in early pregnancy loss by disturbing the uterine prostaglandin system.

Rat ovulation, implantation and decidualization are severely compromised by COX-2 inhibitors.

Although Cyclooxygenase-2 (COX-2) is essential for mouse ovulation, fertilization, implantation and decidualization, the regulation and function of COX-2 in rat reproduction are still unknown. This study was designed to examine the action of COX-2 in rat ovulation, implantation and decidualization by using two specific inhibitors of COX-2 (nimesulide and niflumic acid). Compared to control, either nimesulide or niflumic acid significantly inhibited the ovulation in the superovulated rats. Although nimesulide had no obvious effects on the number of implantation sites and the vascular permeability, the expression of PPARdelta, HB-EGF and vimentin proteins was down-regulated in the nimesulide-treated groups. COX-1 protein was upregulated by nimesulide treatment. Nimesulide also had an inhibitory effect on decidualization during early pregnancy and under artificial decidualization. Moreover, nimesulide caused the increase of the gestation period and the reduction of litter size and birth weight compared to controls. Based on our data, rat implantation and decidualization were delayed by nimesulide treatment, resulting in the reduction of litter size and birth weight and the prolongation of gestational length, suggesting that COX-2 plays an important role in implantation and decidualization.

Cyclooxygenases and prostaglandin E synthases in preimplantation mouse embryos.

Prostaglandin E2 (PGE2) is shown to be essential for female reproduction. Cyclooxygenase (COX) is a rate-limiting enzyme in prostaglandin synthesis from arachidonic acid and exists in two isoforms: COX-1 and COX-2. Prostaglandin E synthase (PGES) is a terminal prostanoid synthase and can catalyse the isomerization of the COX product PGH2 to PGE2, including microsomal PGES-1 (mPGES-1), cytosolic PGES (cPGES) and mPGES-2. This study examined the protein expression of COX-1, COX-2, mPGES-1, cPGES and mPGES-2 in preimplantation mouse embryos by immunohistochemistry. Embryos at different stages collected from oviducts or uteri were transferred into a flushed oviduct of non-pregnant mice. The oviducts containing embryos were paraffin-embedded and processed for immunostaining. COX-1 immunostaining was at a basal level in zygotes and a low level at the 2-cell stage, reaching a high level from the 4-cell to blastocyst stage. COX-2 immunostaining was at a low level at the zygote stage and was maintained at a high level from the 2-cell to blastocyst stages. A low level of mPGES-1 immunostaining was observed from the zygote to 8-cell stages. The signal for mPGES-1 immunostaining became stronger at the morula stage and was strongly seen at the blastocyst stage. cPGES immunostaining was strongly observed in zygotes, 2-cell and 8-cell embryos. There was a slight decrease in cPGES immunostaining at the 4-cell, morula and blastocyst stages. mPGES-2 immunostaining was at a low level from the zygote to morula stages and at a high level at the blastocyst stage. We found that the COX-1, COX-2, mPGES-1, cPGES and mPGES-2 protein signals were all at a high level at the blastocyst stage. PGE2 produced during the preimplantation development may play roles during embryo transport and implantation.