Cellular characteristics of primary and immortal canine embryonic fibroblast cells.
Using normal canine embryonic fibroblasts (CaEF) that were shown to be senescent at passages 7th-9th, we established two spontaneously immortalized CaEF cell lines (designated CGFR-Ca-1 and -2) from normal senescent CaEF cells, and an immortal CaEF cell line by exogenous introduction of a catalytic telomerase subunit (designated CGFR-Ca-3). Immortal CGFR- Ca-1, -2 and -3 cell lines grew faster than primary CaEF counterpart in the presence of either 0.1% or 10% FBS. Cell cycle analysis demonstrated that all three immortal CaEF cell lines contained a significantly high proportion of S-phase cells compared to primary CaEF cells. CGFR-Ca-1 and -3 cell lines showed a loss of p53 mRNA and protein expression leading to inactivation of p53 regulatory function, while the CGFR-Ca-2 cell line was found to have the inactive mutant p53. Unlike the CGFR-Ca-3 cell line that down-regulated p16INK4a mRNA due to its promoter methylation but had an intact p16INK4a regulatory function, CGFR-Ca-1 and -2 cell lines expressed p16INK4a mRNA but had a functionally inactive p16INK4a regulatory pathway as judged by the lack of obvious differences in cell growth and phenotype when reconstituted with wild-type p16INK4a. All CGFR-Ca-1, -2 and -3 cell lines were shown to be untransformed but immortal as determined by anchorage-dependent assay, while these cell lines were fully transformed when overexpressed oncogenic H-rasG12V. Taken together, similar to the nature of murine embryo fibroblasts, the present study suggests that normal primary CaEF cells have relatively short in vitro lifespans and should be spontaneously immortalized at high frequency.
You
Seungkwon
S
The Laboratory of Cell Growth and Function Regulation, Korea University, Seoul, Korea. bioseung@korea.ac.kr
Moon
Jai-Hee
JH
Kim
Tae-Kyung
TK
Kim
Sung-Chan
SC
Kim
Jai-Woo
JW
Yoon
Du-Hak
DH
Kwak
Sungwook
S
Hong
Ki-Chang
KC
Choi
Yun-Jaie
YJ
Kim
Hyunggee
H
D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic; D016922:Cellular Senescence / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics; D004285:Dogs; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D015870:Gene Expression; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D019098:Telomerase / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D019098:Telomerase; D018631:ras Proteins