Transformation and immortalization of Leydig cells from the Sprague-Dawley rat by an early genetic region of simian virus 40 DNA.
Two transformed cell lines of rat Leydig cells were established by transfection of primary cells with the transforming region of simian virus (SV40) DNA. Normal adult Leydig cells are non-proliferating cells and cease to grow after the first trypsinization for cell culturing. The cell lines, NWL2 and NWL15, continued to proliferate and subsequently needed subculturing every 2 weeks (split ratio 1:2). No crisis was observed after 35 passages for 18 months. Nile red staining showed the presence of lipid droplets in both normal and transformed cells, although the transformed cells had 2-3-fold higher amounts than the normal cells. The integration of T-antigen DNA has taken place in at least 2 and 1 sites in NWL2 and NWL15, respectively. Both cell lines expressed T-antigen mRNA. The cell lines expressed luteinizing hormone receptor (LH-R) (a Leydig cell-specific gene), insulin-like growth factor-I, insulin-like growth factor-I receptor (IGF-I-R) and insulin-like growth factor binding protein-2 (IGFBP-2) genes. The amounts of transcripts of LH-R were lower in the transformed cells as compared to the normal cells. The IGF-I-R mRNA levels were comparable to those of the normal Leydig cells. NWL2 and NWL15 cells also expressed IGF-I mRNA although to a lesser extent than the normal Leydig cells. IGFBP-2 mRNA levels were much higher in both the transformed cell lines than in the normal Leydig cells. The transformed cells were evaluated for the expression of P450scc, which catalyzes the conversion of cholesterol to pregnenolone.(ABSTRACT TRUNCATED AT 250 WORDS)
Nagpal
M L
ML
Medical Service, W.J.B. Dorn Veterans Hospital, Columbia, South Carolina.
Wang
D
D
Calkins
J H
JH
Lin
T
T
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics; D002455:Cell Division; D002461:Cell Line, Transformed / Q000737:chemistry / Q000166:cytology; D002472:Cell Transformation, Viral; D004279:DNA, Viral / Q000032:analysis; D015870:Gene Expression; D018971:Insulin-Like Growth Factor Binding Protein 2; D007334:Insulin-Like Growth Factor I / Q000235:genetics; D007985:Leydig Cells / Q000737:chemistry / Q000166:cytology*; D008297:Male; D012333:RNA, Messenger / Q000032:analysis; D012367:RNA, Viral / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011974:Receptors, LH / Q000235:genetics; D017451:Receptors, Somatomedin / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics*; D013002:Somatomedins / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D002352:Carrier Proteins; D004279:DNA, Viral; D018971:Insulin-Like Growth Factor Binding Protein 2; D012333:RNA, Messenger; D012367:RNA, Viral; D011974:Receptors, LH; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I