Effect of phorbol esters on activity of Ha-ras gene promoter in HeLa cells.
Abstract
AIM: To study the role of phorbol esters (PMA) in the activity of promoter of
Ha-ras in HeLa cells.
METHODS: After treatment with PMA, the growth rate of HeLa cells was measured by
MTT assay and the expression of Ha-ras gene was detected by reversed
transcriptase polymerase chain reaction. The regulation fragment of Ha-ras was
constructed into the plasmid enhanced yellow fluorescent protein (EYFP) which did
not contain promoter element. The recombinant plasmid pRasEYFP was transient
transfected to HeLa cells. Using the EYFP gene as reporter, the activity of
promoter of Ha-ras after treatment of PMA was assayed.
RESULTS: The growth rate of PMA-treated HeLa cells was markedly reduced compared
with untreated cells. The expression of Ha-ras gene was obviously decreased and
the activity of promoter of Ha-ras was decreased by 34.0 %and 26.7 % respectively
in HeLa cells after treated with PMA (100 microg/L) for 48 h and 72 h. In the
meanwhile the activity of protein kinase C (PKC) was decreased by 76.3 % and 73.2
% compared with the control cells, respectively.
CONCLUSION: PMA play an important role in regulation of the activity of promoter
of Ha-ras in HeLa cells. The molecular mechanism may be through the PKC pathway.
Keywords:
Ha-ras in HeLa cells.
METHODS: After treatment with PMA, the growth rate of HeLa cells was measured by
MTT assay and the expression of Ha-ras gene was detected by reversed
transcriptase polymerase chain reaction. The regulation fragment of Ha-ras was
constructed into the plasmid enhanced yellow fluorescent protein (EYFP) which did
not contain promoter element. The recombinant plasmid pRasEYFP was transient
transfected to HeLa cells. Using the EYFP gene as reporter, the activity of
promoter of Ha-ras after treatment of PMA was assayed.
RESULTS: The growth rate of PMA-treated HeLa cells was markedly reduced compared
with untreated cells. The expression of Ha-ras gene was obviously decreased and
the activity of promoter of Ha-ras was decreased by 34.0 %and 26.7 % respectively
in HeLa cells after treated with PMA (100 microg/L) for 48 h and 72 h. In the
meanwhile the activity of protein kinase C (PKC) was decreased by 76.3 % and 73.2
% compared with the control cells, respectively.
CONCLUSION: PMA play an important role in regulation of the activity of promoter
of Ha-ras in HeLa cells. The molecular mechanism may be through the PKC pathway.