Estrogen Induction of Telomerase Activity through Regulation of the Mitogen-Activated Protein Kinase (MAPK) Dependent Pathway in Human Endometrial Cancer Cells

Given that prolonged exposure to estrogen and increased telomerase activity are associated with endometrial carcinogenesis, our objective was to evaluate the interaction between the MAPK pathway and estrogen induction of telomerase activity in endometrial cancer cells. Estradiol (E2) induced telomerase activity and hTERT mRNA expression in the estrogen receptor (ER)-α positive, Ishikawa endometrial cancer cell line. UO126, a highly selective inhibitor of MEK1/MEK2, inhibited telomerase activity and hTERT mRNA expression induced by E2. Similar results were also found after transfection with ERK 1/2-specific siRNA. Treatment with E2 resulted in rapid phosphorylation of p44/42 MAPK and increased MAPK activity which was abolished by UO126. The hTERT promoter contains two estrogen response elements (EREs), and luciferase assays demonstrate that these EREs are activated by E2. Exposure to UO126 or ERK 1/2-specific siRNA in combination with E2 counteracted the stimulatory effect of E2 on luciferase activity from these EREs. These findings suggest that E2-induction of telomerase activity is mediated via the MAPK pathway in human endometrial cancer cells

Inhibition of cyclin-dependent kinases by purine analogues

While testing purines related to the non-specific protein kinase inhibitors M-dimethyl-aminopurine and N6-(A'-isopenteny1)adenine as potential inhibitors of the p34'd'z/cyclin B kinase, we discovered a compound with high specificity, 2-(2-hydroxyethylamino)-6-benzylamino-9-meth-ylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non-competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34"dk4/~y~lin D1, p4Wdk6/cyclin D3, CAMP-dependent and cGMP-dependent ki-nases, eight protein kinase C isoforms, calmodulin-dependent kinase 11, myosin light-chain kinase, mitogen-activated S6 kinase, casein kinase 2, double-stranded EWA-activated protein kinase, AMP-stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell-cycle regulating p34'dc2/cyclin B, p33cdk2/~y~lin A and p33cdk2/~y~lin E kinases, the brain ~ 3 3 ' ~ ~ ' / p35 kinase and the ERKlMAP-kinase (and its starfish homologue ~44"'~~) are substantially inhibited by olomoucine (I & values are 7, 7, 7, 3 and 25 pM, respectively). The cdk4/cyclin D1 and cdk6/ cyclin D3 kinases are not significantly sensitive to olomoucine (I & values greater than 1 mM an