Expression of Transgenes in Normal and Neoplastic Anterior Pituitary Cells Using Recombinant Adenoviruses: Long Term Expression, Cell Cycle Dependency, and Effects on Hormone Secretion*

Abstract

Adenovirus vectors have recently been used to transfer genes into a variety of cell types, including neurons, glial cells, Schwann cells, and epithelial cells. To evaluate the efficiency of gene transfer into pituitary cells using viral vectors, we used replication-deficient recombinant adenovirus vectors (RAds) encoding beta-galactosidase driven by various viral promoters. We tested the ability of RAds to infect and express beta-galactosidase within the different identified cell populations of the anterior pituitary anterior pituitary gland and also in tumor cells of anterior pituitary origin, i.e. GH3 and AtT20 cells. Our results demonstrate that transgenes encoded by RAds are expressed within all cell types of the adenohypophysis in vitro and also within AtT20 and GH3 endocrine tumor cells. Our long term expression studies indicate that long term expression with low cytotoxicity can be achieved, but that the longevity of transgene expression from RAds depends on the proliferative status of the target cells. Slowly dividing cells (endocrine population) express transgenes for longer than actively dividing cells (tumor cells and nonendocrine anterior pituitary cells). The ability of anterior pituitary cells to secrete ACTH or LH through the regulated secretory pathway decreased after infection with RAds at high multiplicity of infection (> or = 20 plaque-forming units/target cell), whereas cell viability was not affected. We also demonstrate that a higher percentage of cells expressed the transgene beta-galactosidase when we infected actively dividing GH3 cells compared with the infection of growth-arrested GH3 cells. This could reflect differential virus entry or differential activity of the individual promoters during different stages of the cell cycle. This work demonstrates that high efficiency gene transfer into all pituitary cell types can be achieved with RAds, and that this system can be exploited to characterize and experimentally manipulate pituitary-specific gene expression. The higher efficiency of infection and transgene expression in actively dividing cells compared to that in their growth-arrested counterparts could also be exploited for the treatment of pituitary adenomas that do not respond to classical treatment strategies, using suicide or cytotoxic gene therapy.

Involvement of cAMP response element-binding protein in the regulation of cell proliferation and the prolactin promoter of lactotrophs in primary culture

“…To express a dominant-negative form of CREB and reporter genes we used adenovirus vectors, which have proved efficient for expressing proteins in normal pituitary cells in primary culture (3,30). To express a dominant-negative form of CREB and reporter genes we used adenovirus vectors, which have proved efficient for expressing proteins in normal pituitary cells in primary culture (3,30).…”

Generation of a Recombinant Herpes Simplex Virus Type 1 Expressing the Rat Corticotropin- Releasing Hormone Precursor: Endoproteolytic Processing, Intracellular Targeting and Biological Activity

“…Primary rat AP culture and biological activity studies were performed as described previously [7]. Briefly, cells were pre-incubated in DMEM containing 0.25% BSA for 2 h before the addition of conditioned medium from infected cells for 3 h. ACTH released into the culture medium was measured using a specific radioimmunoassay (RIA; NIDDK; National Hormone and Pituitary Program, Rockville, Md., USA) as described previously [8]. Immunoreactive CRH released into the culture medium and stored intracellularly was measured as described previously [7, 8].…”

“…Briefly, cells were pre-incubated in DMEM containing 0.25% BSA for 2 h before the addition of conditioned medium from infected cells for 3 h. ACTH released into the culture medium was measured using a specific radioimmunoassay (RIA; NIDDK; National Hormone and Pituitary Program, Rockville, Md., USA) as described previously [8]. Immunoreactive CRH released into the culture medium and stored intracellularly was measured as described previously [7, 8]. ACTH released from AP cells exposed to medium from HSV1/ ts K-infected cells was considered as basal release, and ACTH released from AP cells exposed to medium from ts K/CRH10-infected cells was considered as stimulated release.…”

Gene therapy for the treatment of pituitary tumors

“…Initial studies with adenoviral and herpes-derived vectors demonstrated that these two vector systems could be used to efficiently transfer different types of genes into normal rat anterior pituitary (AP) cells in primary culture as well as in the corticotropic AtT 20 and somatomammotropic GH 3 tumor cell lines [34, 35]. Interestingly, neoplastic AP cells were found to be more susceptible to viral vector-mediated transduction than were normal AP cells.…”