Caveolin-1 sensitizes rat pituitary adenoma GH3 cells to bromocriptine induced apoptosis.

BACKGROUND: Prolactinoma is the most frequent pituitary tumor in humans. The dopamine D2 receptor agonist bromocriptine has been widely used clinically to treat human breast tumor and prolactinoma through inhibition of hyperprolactinemia and induction of tumor cell apoptosis, respectively, but the molecular mechanism of bromocriptine induction of pituitary tumor apoptosis remains unclear. Caveolin-1 is a membrane-anchored protein enriched on caveolae, inverted flask-shaped invaginations on plasma membranes where signal transduction molecules are concentrated. Currently, caveolin-1 is thought to be a negative regulator of cellular proliferation and an enhancer of apoptosis by blocking signal transduction between cell surface membrane receptors and intracellular signaling protein cascades. Rat pituitary adenoma GH3 cells, which express endogenous caveolin-1, exhibit increased apoptosis and shrinkage after exposure to bromocriptine. Hence, the GH3 cell line is an ideal model for studying the molecular action of bromocriptine on prolactinoma. RESULTS: The expression of endogenous caveolin-1 in GH3 cells was elevated after bromocriptine treatment. Transiently expressed mouse recombinant caveolin-1 induced apoptosis in GH3 cells by enhancing the activity of caspase 8. Significantly, caveolin-1 induction of GH3 cell apoptosis was sensitized by the administration of bromocriptine. Phosphorylation of caveolin-1 at tyrosine 14 was enhanced after bromocriptine treatment, suggesting that bromocriptine-induced phosphorylation of caveolin-1 may contribute to sensitization of apoptosis in GH3 cells exposed to bromocriptine. CONCLUSION: Our results reveal that caveolin-1 increases sensitivity for apoptosis induction in pituitary adenoma GH3 cells and may contribute to tumor shrinkage after clinical bromocriptine treatment.

Proliferin enhances microvilli formation and cell growth of neuroblastoma cells.

Proliferins (also termed mitogen-regulated proteins; MRP/PLFs) belong to the prolactin gene family. Mrp/Plfs are involved in angiogenesis of the uterus and placenta and maximally expressed during midgestation and decline through the remainder of the gestation period in mouse placenta. The tissue expressions of Mrp/Plfs are mainly documented in placenta, hair follicles of skin and in wound healing. In this report, we demonstrate that Plf1, Plf1 minus exon3, Plf2 and Mrp3 but not Mrp4 are expressed in mouse whole brain by diagnostic RT-PCR and Western blotting. The expression levels of Mrp/Plf mRNAs in mouse brains were low during the neonatal period, but higher in embryonic and adult stages, indicating Mrp/Plfs expression profiles are different in mouse brain and placenta. Interestingly, endogenous Mrp/Plfs were detected using immunostaining both in mouse brain sections and the neuroblastoma cell line, Neuro-2a cells. The function of PLF1 was explored by expressing exogenous PLF1 in Neuro-2a cells. This resulted in increased microvilli. Neuro-2a cells with stable expression of PLF1 had increased proliferation compared with normal and stable expressing EGFP cells when cell reached saturation density. Together these data, strongly suggest that MRP/PLFs mediate microvilli formation and contribute to cell proliferation of neuroblastoma cells.

In vitro and ex vivo green fluorescent protein expression in alveolar mammary epithelial cells and mammary glands driven by the distal 5'-regulative sequence and intron 1 of the goat beta-casein gene.

The 5'-regulative sequence and intron 1 of the goat beta-casein gene from -4044 to +2123 bp was cloned and fused with the reporter gene of green fluorescent protein (GFP) to create a plasmid termed pGB562/GFP. To detect GFP expression, pGB562/GFP was transfected in vitro via liposomes into the mammary epithelial cell line NMuMG. Cells could not express GFP unless the transfected NMuMG cells lined up to create functional alveoli. These functional cells were cultured with lactogenic hormones, including insulin, dexamethasone and prolactin, and were grown on a layer of the extracellular matrix Matrigel. Green fluorescent protein expression levels in NMuMG cells were 25-, 55- and 42-fold those in the control group at 24, 48, and 72 h after pGB562/GFP transfection respectively. In addition, pGB562/GFP was transfected ex vivo by electroporation into mammary gland fragments and cells were then cultured in vitro with a supplement of lactogenic hormones. Strong GFP expression localized in fragments of the mammary gland was observed 24 h after gene transfer. The novel strategy of ex vivo gene transfer into mammary tissue using GFP as a reporter gene to detect the function of a tissue-specific promoter is efficient and convenient. The data obtained herein reveal that the 5'-regulative sequence and intron 1 of the 6.2 kb goat beta-casein gene can enhance the efficiency of transgene expression. Thus, the GB562 sequence may act as a good promoter and effectively elevate the production of exogenous protein in mammary glands.

Serum progesterone changes in luteal cyclicity and duration of estrous cycle in Formosan sika deer (Cervus nippon taiouanus) hinds.

A study was conducted to investigate the serum progesterone (SP(4)) profiles and duration of estrous cycles in the farmed Formosan sika deer (FSD; Cervus nippon taiouanus) during the major breeding season. Five parous, open and non-milking hinds were allotted to collect peripheral blood samples twice weekly for P(4) measurement by radioimmunoassay beginning at the initiation of the rutting season indicated by rutting behaviors of the sexually mature stags. The hinds were polyestrous as proved by cyclic changes of SP4 levels. After the presumptive estrus shown by the lowest concentration of SP(4) (0.20+/-0.01 ng/ml), this ovarian hormone markedly elevated on day 7 of the cycle (1.67+/-0.11 ng/ml), reached plateau (3.15+/-0.16 ng/ml, P<0.01) during days 11 to 18, and then declined to the basal levels in the subsequent estrus. It is concluded that mean duration of the estrous cycle in FSD during the major rutting season is 19.3 days with a range of 17 to 21 days, and that the patterns of circulating progesterone profiles during the estrous cycles of the FSD are similar to those of other deer species so far investigated.