Adenovirus vectors differentially modulate proliferation of pituitary lactotrophs in primary culture in a mitogen and infection time-dependent manner

Abstract

Adenoviruses are powerful, widely utilized vectors for gene transfer. Limitations to their application, however, have not been well described. We used rat pituitary lactotrophs in primary culture as a model for studying how adenovirus vector infection modulates mitogen-induced proliferation and the activities of mitogen signaling pathways. Infection with adenovirus vectors expressing b-galactosidase (bgal) raised basal proliferative levels and blocked fetal bovine serum (FBS)-induced proliferation of lactotrophs, but did not influence the changes in proliferation induced by forskolin, IGF-I, and bromocriptine. The bgalexpressing adenoviruses did not alter the inhibitory action of 17b-estradiol (E 2 ) in the presence of IGF-I; however, they blocked the stimulatory action of E 2 in the presence of dextrancoated charcoal-striped serum or forskolin. An adenovirus expressing no protein failed to block FBS-induced proliferation, but was effective in modulating basal proliferative levels and the stimulatory actions of E 2 . The increased basal proliferative level and the blockade of FBS-induced proliferation were transient, and lost 5 days after infection while the blockade of the stimulatory action of E 2 in the presence of forskolin persisted. Adenovirus infection raised basal protein levels of the phosphorylated forms of cAMP response element-binding protein (pCREB) and ERK1/2 and increased the proportion of pCREB-immunoreactive lactotrophs. Adenoviruses also altered estrogen-induced responses in mRNA expression of several estrogen-responsive genes in a gene-specific manner. The results demonstrate that an adenovirus vector differentially interferes with lactotroph proliferation in response to various mitogens. Our results suggest that the effects of the adenovirus that are independent of the genes transferred must be considered when performing adenoviral gene transfer in the primary cultures of normal cells.

Ca2+-induced PARP-1 activation and ANF expression are coupled events in cardiomyocytes

“…The relative quantities of c-fos per sample were calculated in relation to each of two reference genes in rat S14 (hepatic lipogenic S14 protein [31]; 5 -TCCTCTCCGTATGC-AGCCATGT-3 and 3 -ACCCTTCCTTCGAGTGCTGTCA-5 ), ARBP (acidic ribosomal phosphoprotein [32]; 5 -CTGACTAC-ACCTTCCCACTG-3 and 3 -TCCGACTCTTCCTTTGCTTC-5 ) and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 3 -CTGGAAAGCTGTGGCGTGATGG-5 and 5 -TCCTCAG-TGTAGCCCAGGATGC-3 ) [33] using the C t formula as described previously [34]. RNA was prepared (RNeasy Mini Kit, Qiagen) 15, 30, or 60 min after treatment with 1 μM AngII (angiotensin II) for 10 min at 37 • C. RNA samples were treated with DNAse-I (Ambion #1906) to avoid the presence of DNA residues.…”

“…The presented autoradiograms identify Ca 2+ -dependent [32 P]polyADP-ribosylation of immunolabelled PARP-1 (MCA1522, Serotec). After the cells were thoroughly washed, their nuclei were isolated and exposed to increasing Ca 2+ concentrations in the presence of [ 32 P]NAD (1000 Ci/mmol; 1 μCi/sample).…”

<i>Rasd1</i> is an estrogen-responsive immediate early gene and modulates expression of late genes in rat anterior pituitary cells

“…Expression levels of mRNAs for estrogen-responsive genes were determined by qRT-PCR as described previously [23]. Total pituitary RNA was isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol and treated with RNase-free DNase I (Qiagen).…”

Activation of G protein-coupled estrogen receptor 1 mimics, but does not mediate, the anti-proliferative action of estradiol on pituitary lactotrophs in primary culture

“…Immunoblotting was performed as described previously [29]. Pituitary cells were lysed with ice-cold lysis buffer composed of 20 mM Tris, pH 7.6, 137 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 2 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (aprotinin, bestatin, leupeptin, and pepstatin A) (Santa Cruz Biotechnology, Santa Cruz, CA), and a whole-cell extract of soluble proteins was obtained by centrifugation at 17,500 g for 15 min at 4 °C.…”