Phenotypic conversion of SV40-immortalized human diploid fibroblasts to senescing cells by introduction of an antisense gene for SV40-T antigen.
Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70% as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13 and about 50% of cells were at G1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (pRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total pRB was much higher than that in VA-13. The pRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and pRB or the underphosphorylation of pRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.
Tanaka
Y
Y
Gene Bank, Tsukuba Life Science Center, RIKEN (The Institute of Physical and Chemical Research, Ibaraki, Japan.
Tang
X
X
Hou
D X
DX
Gao
H
H
Kitabayashi
I
I
Gachelin
G
G
Yokoyama
K
K
D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D002453:Cell Cycle; D002472:Cell Transformation, Viral / Q000235:genetics*; D016922:Cellular Senescence; D016373:DNA, Antisense / Q000235:genetics*; D005347:Fibroblasts / Q000473:pathology*; D015967:Gene Expression Regulation, Viral; D006801:Humans; D010641:Phenotype; D010766:Phosphorylation; D016160:Retinoblastoma Protein / Q000096:biosynthesis; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*
D000952:Antigens, Polyomavirus Transforming; D016373:DNA, Antisense; D016160:Retinoblastoma Protein