Cellular characteristics of primary and immortal canine embryonic fibroblast cells

Seungkwon You, Jai Hee Moon, Tae Kyung Kim, Sung Chan Kim, Jai Woo Kim, Du Hak Yoon, Sungwook Kwak, Ki Chang Hong, Yun Jaie Choi, Hyunggee Kim

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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 sub-unit (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 p16 INK4a mRNA due to its promoter methylation but had an intact p16 INK4a 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.

Original languageEnglish
Pages (from-to)325-335
Number of pages11
JournalExperimental and Molecular Medicine
Volume36
Issue number4
Publication statusPublished - 2004 Aug 31

Fingerprint

Fibroblasts
Canidae
Cells
Cell Line
Messenger RNA
Telomerase
Methylation
S Phase
Cell growth
Cell Cycle
Embryonic Structures
Assays
Phenotype

Keywords

  • Canine embryonic fibroblast
  • Immortalization
  • p16
  • p53
  • Telomerase

ASJC Scopus subject areas

  • Biochemistry
  • Genetics

Cite this

Cellular characteristics of primary and immortal canine embryonic fibroblast cells. / You, Seungkwon; Moon, Jai Hee; Kim, Tae Kyung; Kim, Sung Chan; Kim, Jai Woo; Yoon, Du Hak; Kwak, Sungwook; Hong, Ki Chang; Choi, Yun Jaie; Kim, Hyunggee.

In: Experimental and Molecular Medicine, Vol. 36, No. 4, 31.08.2004, p. 325-335.

Research output: Contribution to journalArticle

You, S, Moon, JH, Kim, TK, Kim, SC, Kim, JW, Yoon, DH, Kwak, S, Hong, KC, Choi, YJ & Kim, H 2004, 'Cellular characteristics of primary and immortal canine embryonic fibroblast cells', Experimental and Molecular Medicine, vol. 36, no. 4, pp. 325-335.
You, Seungkwon ; Moon, Jai Hee ; Kim, Tae Kyung ; Kim, Sung Chan ; Kim, Jai Woo ; Yoon, Du Hak ; Kwak, Sungwook ; Hong, Ki Chang ; Choi, Yun Jaie ; Kim, Hyunggee. / Cellular characteristics of primary and immortal canine embryonic fibroblast cells. In: Experimental and Molecular Medicine. 2004 ; Vol. 36, No. 4. pp. 325-335.
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AU - Yoon, Du Hak

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AU - Kim, Hyunggee

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AB - 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 sub-unit (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 p16 INK4a mRNA due to its promoter methylation but had an intact p16 INK4a 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.

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