Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most human cells. In this work, we examined the participation of protein kinase C (PKC) in the regulation of hTERT expression in human T lymphocytes. Transient expression assays using luciferase reporter plasmids containing hTERT promoter showed that overexpression of PKC h, but not the other PKC isoforms, could activate the promoter activity of hTERT in resting T lymphocytes. Among the PKC h-activated signalings, we presented evidence that the expression of hTERT is mediated through NFjB but not through MEK or c-Jun N-terminal kinase pathways. Analysis of the hTERT promoter occupancy in vivo using chromatin immunoprecipitation assays, however, did not detect an increased binding of NFjB to the hTERT promoter in the activated T cells, although an increased binding of cMyc and Sp1 was detected. Together with the observation that inhibition of NFjB eliminated the induction of cMyc in activated T cells, these results suggest that PKC h-activated NFjB signaling regulates the expression of hTERT via cMyc in human T lymphocytes.

Protein kinase C (PKC) has been implicated to play an essential function in the upregulation of telomerase activity in activated T cells, yet its role in the regulation of telomerase activity remains largely unknown. In this work, we present evidence that PKC activity is required both for the induction of hTERT expression and for the post-transcriptional control of telomerase enzyme activity in T lymphocytes. Of the several PKC isoforms present in lymphocytes, only the level of PKCj j was greatly increased during T cell activation, implicating that PKC-j j may be required for the post-transcriptional control of telomerase enzyme activity in T lymphocytes. ß

Despite the important role of T cell activation in the adaptive immunity, very little is known about the functions of proteins that are differentially expressed in the activated T cells. In this study, we have employed proteomic approach to study the differentially expressed proteins in activated T cells. A total of 25 proteins was characterized that displayed a decreased expression, while a total of 20 proteins was characterized that displayed an increased expression in the activated T cells. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified unexpectedly as one of the up-regulated proteins. Western blot analysis of proteins separated by 2-dimensional gel electrophoresis had identified several modified GAPDHs which were detectable only in the activated T cells, but not in resting T cells. These modified GAPDHs had higher molecular mass and more basic PI, and were present in the nucleus of activated T cells. Promoter occupancy studies by chromatin immunoprecipitation assay revealed that nuclear GAPDH could be detected in the promoter of genes that were up-regulated during T cell activation, but not in the promoter of genes that were not unaffected or down-regulated. Our results suggest that nuclear GAPDH may function as transcriptional regulator in activated T cells.

SummaryTelomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC 50 value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 lM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC 50 values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hematopoietic cancer cells. The potent and selective inhibition of telomerase by U-73122 raises the potential exploitation of this drug and other alkylating agents as telomerase inhibitor.

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