hTERT phosphorylation by PKC is essential for telomerase holoprotein integrity and enzyme activity in head neck cancer cells

Abstract

Telomerase activity is suppressed in normal somatic tissues but is activated in most cancer cells. We have previously found that all six telomerase subunit proteins, including hTERT and hsp90 are needed for full enzyme activity. Telomerase activity has been reported to be upregulated by protein kinase C (PKC), but the mechanism is not clear. In this study, we examined how PKC regulates telomerase activity in head and neck cancer cells. PKC inhibitor, bisindolylmaleimide I (BIS), inhibited telomerase activity but had no effect on the expressions of telomerase core subunits. RNA interference (RNAi) and in vitro phosphorylation studies revealed that PKC isoforms alpha, beta, delta, epsilon, zeta specifically involved in telomerase regulation, and the phosphorylation target was on hTERT. Treatment with the hsp-90 inhibitor novobiocin dissociated hsp90 and hTERT as revealed by immunoprecipitation and immunoblot analysis and reduced telomerase activity. Treatment with the PKC activator SC-10 restored the association of hsp90 and hTERT and reactivate telomerase, suggesting that hTERT phosphorylation by PKC is essential for telomerase holoenzyme integrity and function. Analysis on clinical normal and tumour tissues reveal that the expressions of PKC alpha, beta, delta, epsilon, zeta were higher in the tumour tissues, correlated with telomerase activity. Disruption of PKC phosphorylation by BIS significantly increased chemosensitivity to cisplatin. In conclusion, PKC isoenzymes alpha, beta, delta, epsilon, zeta regulate telomerase activity in head and neck cancer cells by phosphorylating hTERT. This phosphorylation is essential for telomerase holoenzyme assembly, leading to telomerase activation and oncogenesis. Manipulation of telomerase activity by PKC inhibitors is worth exploring as an adjuvant therapeutic approach.

Figure 1

Effects of PKC inhibitor, BIS, on telomerase activity and the telomerase subunit expressions. OEC-M1 cells were treated with 40 μM (A, B, D) or various amounts (B) of BIS for 48 h. (A) Effect of BIS on PKC isoenzyme expressions. Eight PKC isoenzymes (α, βI, βII, γ, δ, ε, ζ, η) were determined by immunoblot, as indicated at the left of the figure. (B) Effect of BIS on PKC phosphorylation activity. PKC activity was determined by examining the amount of PKC-phosphosubstrate using pan-antibody by immunoblot analysis. C: Control, without drug treatment; BIS: BIS-treated cells. (C) Effect of BIS on telomerase activity. Telomerase activity was determined by TRAP-EIA as described in the materials and methods. (D) Effect of BIS on the expressions of telomerase subunits. Telomerase subunits, hTERT, hsp90, TEP1 and p23 were determined by RT–PCR as indicated on the left of the figure. Actin expression was determined as an internal control.

Figure 2

Effects on telomerase activity by specific PKC isoenzymes. (A) OEC-M1 cells were transfected with specific PKC-RNAi plasmids for 48 h. Immunoblot analysis to determine the protein expression levels of each PKC isoenzyme. Actin gene expression was measured as an internal control. Actin expression remained consistent in every transfected sample. In this figure, the results of PKC α-RNAi transfection are shown as representative of all the RNAi experiments. C: control cells without plasmid transfection, V: cells transfected with vector, RNAi: cells transfected with specific PKC-RNAi plasmid as indicated on the left of the figure. (B) Telomerase activity was determined after transfection with specific PKC-RNAi plasmids as indicated on the bottom of the figure. (C) OEC-M1 cells were treated with 40 μM BIS for 48 h. Cell lysates were subjected to in vitro phosphorylation by specific PKC isoenzymes as indicated, followed by determination of telomerase activity. ^*Statistical significance using Student's t-test (P<0.05 of two-sided test).

Figure 3

In vitro phosphorylation study for the target molecule hTERT. Nuclear proteins were phosphorylated using [γ-P³²]-ATP by specific PKC isoenzymes as indicated on the top of the figure. Protein samples were immunoprecipitated by hTERT antibody. Samples were then subjected to autoradiography or immunoblot analysis. Proteins without phosphorylation (lanes 1 and 2) or immunoprecipitated with pre-immune goat IgG (IgG) (lane 9) were used as negative controls. An immunoblot of hTERT for each sample served as an internal control. Autoradiography data for immunoprecipitated samples demonstrate that hTERT molecules were phosphorylated by the PKC isoenzymes.

Figure 4

Effect of PKC phosphorylation on telomerase holoprotein integrity and the enzyme activity. (A) After treatment with 10 μM SC-10, PKC activity was determined by examining the amount of PKC-phosphosubstrates using pan-antibody immunoblot analysis. (B) Association study of hTERT-hsp90 and the influence of PKC phosphorylation. Lanes 1 and 2: Nuclear proteins from OEC-M1 cells with or without PKC-RNAi plasmid transfection were extracted and subjected to immunoprecipitation and immunoblot. Lanes 3 to 4: OEC-M1 cells were treated with 300 μM novobiocin for 24 h to disrupt the hsp-hTERT association. Cells were harvested or continuously cultured with 10 μM SC-10 for an additional 24 h (novobiocin+SC-10). Nuclear proteins were subjected to immunoprecipitation by hsp90 followed by immunoblot by hTERT or hsp90 (as control). (C) Alterations of telomerase activity after hTERT-hsp90 disruption and reassociation. Lanes 1 and 2: Cellular proteins from OEC-M1 cells with or without PKC-RNAi plasmid transfection were extracted for determination of telomerase activity by TRAP-EIA. Lanes 3 to 4: OEC-M1 cells were treated with 300 μM novobiocin for 24 h to disrupt the hsp-hTERT association. Cells were harvested or continuously cultured with 10 μM SC-10 for an additional 24 h. Cellular proteins were extracted for determination of telomerase activity.

Figure 5

Relative levels of telomerase activity and the expressions of PKC isoenzymes in normal and tumour tissues. Four pairs of normal (N) and tumour (T) tissues from head and neck cancer patients were examined. Each sample is indicated at the top of the figure. (A) The protein expression was determined by immunoblot analysis and is indicated at the left of the figure. Actin protein expression was used as an internal control. Telomerase activity in each sample was determined by TRAP-EIA and was normalised with that in the OEC-M1 cell lines. Relative levels of telomerase activity (%TS) are indicated at the bottom of the figure. (B) Average of telomerase activity and PKC isoenzyme expression in tumour and normal tissues. After quantitation of the immunoblot densities in each sample, the levels of PKC isoenzymes were normalised with their respective actin level to calculate the relative expression. Average of telomerase activity in each sample was also determined as indicated. ^*Statistical significance using student t-test (P<0.05 of two-sided test).

Figure 6

Increased chemosensitivity to cisplatin-induced cell death after inhibition of telomerase activity BIS-induced dephosphorylation of PKC. OEC-M1 cells were treated with 40 μM BIS for 48 h, followed by administration of various amounts of cisplatin (0, 3 or 10 μg ml⁻¹) for an additional 12 h. Cell viability was determined using trypan blue staining.