[Functional analysis of the human T-type calcium channel alpha 1H subunit gene in cellular proliferation].
An increase in the functional expression of the T-type calcium (Ca) channel previously has been proposed to be associated with vascular smooth muscle cell proliferation although direct evidence for the channel causing these effects remains to be demonstrated. In this study, we provide evidence that stable over-expression of the alpha 1H subunit of the T-type Ca channel (CACNA1H) in HEK-293 cells confers a significant growth advantage. Over-expression of the alpha 1H subunit of the T-channel was confirmed in stable transfects by RT-PCR analysis using specific primer pairs and also by electrophysiology. Growth curve assays showed population doubling time for alpha 1H stable transfects was 14.0 +/- 0.4 h, whereas control cultures had a population doubling time of 22.1 +/- 1.1 h (n = 3, P < 0.05). In addition, total cell numbers significantly increased in stable transfects at all time points investigated from 48-120 h after plating (5 x 10(3) cells/well) compared with control cultures. Consistent with these findings flow cytometry showed that 53.9% of control cells were in G1, 34.5% in S and 11.6% in G2/M whereas alpha 1H transfectants displayed 45.6% of cells in G1, 44.6% in S and 9.8% in G2/M. Finally, the Western blotting results verified that the levels of protein expression of CDK2, cyclin A and cyclin E were relatively high in alpha 1H transfectants compared to control cultures. Our results demonstrate that the T-type Ca channel provides a growth advantage to HEK-293 cells when stably expressed and that it probably exerts these effects via control of the G1/S cell cycle mechanism.
Wang
Yue-Qun
YQ
College of Life Science, Hunan Normal University, Changsha 410081, China.
Brooks
Gavin
G
Zhu
Chuan-Bing
CB
Yuan
Wu-Zhou
WZ
Li
Yong-Qing
YQ
Wu
Xiu-Shan
XS
D015153:Blotting, Western; D042846:CDC2-CDC28 Kinases; D020747:Calcium Channels, T-Type / Q000235:genetics / Q000502:physiology*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D019925:Cyclin A / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D010957:Plasmids / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D020747:Calcium Channels, T-Type; D019925:Cyclin A; D019927:Cyclin E; D021122:Protein Subunits; D012333:RNA, Messenger; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases