Characterization of GH3 cells overexpressing basic fibroblast growth factor (FGF-2).
Basic fibroblast growth factor (FGF-2) is not only a potent mitogen for various cells but also a multifunctional factor with angiogenic and chemotactic activity, and the capacity to induce the synthesis of various proteinases and to modulate endocrine function. To clarify the role played by FGF-2 in the progression of pituitary tumor, we fused rat FGF-2 cDNA to the promoter SR alpha, consisting of the early promoter of SV40 and HTLV(I)-LTR, and we cotransfected GH3 cells with pSV2-neo by an electroporation method. After selection by G418, we obtained 7 neomycin-resistant clones. Southern blot analysis of genomic DNA revealed the presence of transfected rat FGF-2 cDNA in 4 of the 7 clones. To measure FGF-2 molecules, we established a new immuno-fluorometric assay system, using 3 monoclonal antibodies against different portions of human FGF-2. This assay had a minimum sensitivity of 10 pg/ml and cross-reacted neither with acidic fibroblast growth factor (FGF-1) nor insulin-like growth factor 1 (IGF-1), even at a concentration of 100 ng/ml. Although FGF-2 was undetectable in the culture medium of any of the clones, the cell homogenate contained a significant amount of FGF-2 (7.2 ng/mg protein) in 1 of the 4 FGF-2-transfected clones (GH3FGF(+)), whereas FGF-2 was not detected (< 5.2 pg/mg protein) in the cell homogenates of either the parent GH3 cells or the control cells transfected with pSV2-neo alone (GH3FGF(-)), GH3FGF(+) grew as adherent cells and formed epithelial sheets with a growth rate similar to that of control cells. The amount of prolactin(PRL) released by TRH was greater in GH3FGF(+) than that in GH3 or GH3FGF(-). On the other hand, the sensitivity to SRIF was increased in GH3FGF(+) compared with that in other clones. The findings of these in vitro studies indicate that FGF-2, if it is expressed in pituitary tumor cells, plays little if any role in cell growth but may modulate certain cell functions such as responsiveness to hormones.
Wada
S
S
Department of Medicine, Kobe University School of Medicine, Japan.
Kaji
H
H
Yoshimura
K
K
Chihara
K
K
D000236:Adenoma; D000818:Animals; D015139:Blotting, Southern; D002455:Cell Division / Q000502:physiology; D018076:DNA, Complementary; D004273:DNA, Neoplasm / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D016222:Fibroblast Growth Factor 2 / Q000032:analysis / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000502:physiology; D013006:Growth Hormone / Q000096:biosynthesis; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D006801:Humans; D010911:Pituitary Neoplasms; D010957:Plasmids; D011388:Prolactin / Q000096:biosynthesis; D012334:RNA, Neoplasm / Q000032:analysis; D051381:Rats; D013973:Thyrotropin-Releasing Hormone / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000166:cytology / Q000378:metabolism
D018076:DNA, Complementary; D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D016222:Fibroblast Growth Factor 2; D013973:Thyrotropin-Releasing Hormone; D011388:Prolactin; D013006:Growth Hormone; D013007:Growth Hormone-Releasing Hormone