Correlation of dyskerin expression with active proliferation independent of telomerase

Abstract

Background: Dyskerin, which is an important component of the telomerase complex and is needed for normal telomerase activity, is frequently overexpressed in neoplasia. Dyskerin also plays an essential role in ribosome biogenesis. Because protein synthesis increases during tumorigenesis, this led us to hypothesize that dyskerin expression would be upregulated independently of the cell immortalization mechanism.

Methods: Dyskerin and telomerase reverse transcriptase (TERT) expression were examined in oral squamous cell carcinomas (OSCC) and patient-matched controls, as well as in a panel of telomerase-positive and telomerase-negative cells. Antisense inhibition of TERT was used to test the effects of downregulation of telomerase on dyskerin expression.

Results: Dyskerin was frequently overexpressed in OSCC and in immortalized and transformed keratinocytes relative to primary cells, independently of TERT and telomerase activity. Instead, dyskerin expression strongly correlated with cell proliferation rates.

Conclusions: The role of dyskerin in tumorigenesis does not correlate with its function within the telomerase complex.

Figure 1. Upregulation of dyskerin in immortalized oral keratinocytes is not dependent upon TERT

A, OKF6-TERT2 cells were transfected with either 10 μM TERT antisense DNA oligonucleotide (ANTI) or a control, non-specific oligonucleotide (CTRL). Twenty-four hours later, relative telomerase activity levels were assessed by quantitative RT-PCR and normalized to telomerase levels in the untransfected OKF6-TERT2 cells. B, TERT / β-actin and DKC1 / β-actin expression was normalized to levels of the respective transcripts in the OKF6-TERT2 cells. The solid bars represent the mean values of four transfections conducted in one representative experiment; error bars denote the standard deviations. C, A qualitative TRAP assay was used to measure telomerase activity in exponentially-growing OKF6-TERT2 and OKF6-Δp53D1 cells. The characteristic laddering effect was observed in the OKF6-TERT2 and other telomerase-expressing cells, including UM-SCC1 oral cancer cells and a control cell pellet included with the kit, but not in the ALT-immortalized OKF6-Δp53D1 cells. The internal control served as a control for the PCR reaction. D, DKC1 mRNA expression was assessed by quantitative RT-PCR in OKF6, OKF6-Δp53D1 and OKF6-TERT2 cells. E, Dyskerin protein levels were quantitated relative to β-actin using NIH Image J.

Figure 2. Dyskerin expression correlates with the rate of cell proliferation

A, Fold increase in cell proliferation rates were determined by normalizing the relative WST-1 absorbance for each of the respective cell lines to those measured on the first day after plating. Error bars denote standard deviation from triplicate wells for each time point. Experiments were repeated twice with similar results. B, Cells were harvested in log growth phase (three days after initial plating) and total cell lysates were subjected to Western blot. Dyskerin was increased in the TERT-immortalized cells relative to their parental cells, and even more so in the transformed cells. For comparison purposes, dyskerin expression was also examined in the transformed squamous epithelial cell lines, SCC-10B and SCC-22B. C, Dyskerin expression was normalized to actin, and then compared to the fold increase in WST-1 absorbance three days after initial plating. A linear regression analysis showed strong correlation (r² = 0.927, p < 0.001) between relative dyskerin levels and cell proliferation. D, Dyskerin expression was reduced in contact-inhibited OKF6 and OKF6-TERT2 cells (100%) relative to the exponentially growing cells (50%-60% confluent). Except where indicated, all protein lysates were obtained from actively proliferating cells. E, Cell cycle distribution of OKF6-TERT2 cells at 60% and 100% confluency. OKF6 cells showed a similar profile (not shown).

Figure 3. DKC1 and TERT mRNA expression do not correlate in patient-derived OSCC

A, Box plot illustrating DKC1 upregulation in OSCC (N=49) relative to matched normal controls (N=16; p<0.023). Data represent the average relative signals for DKC1 from two independent arrays. B, DKC1 mRNA levels were significantly (p<0.03) increased (*) in 9/13 OSCC (light grey bars) relative to patient-matched normal mucosal controls (white bars). C, TERT mRNA levels were significantly (p<0.001) increased (*) in 11/13 OSCC (dark grey bars) relative to the controls (white bars). D, There was no correlation between the relative fold increase in TERT and DKC1 mRNA expression in the tumors. Using an arbitrary threshold of a 2-fold increase in tumor mRNA expression relative to the matched normal control (horizontal line), 10/11 high TERT expressing tumors surpassed the threshold, while only 4/9 high DKC1 expressing tumors were increased beyond this threshold. DKC1 and TERT mRNA levels were measured relative to TBP) mRNA by quantitative RT-PCR using the 2^−ΔΔCt method. N = normal, T = tumor.

Figure 4. DKC1 and TERT mRNA expression do not correlate in transformed human cell lines

A, A linear regression analysis of the log-transformed expression values revealed no correlation (r² = 0.019, p=0.21) between TERT and DKC1 mRNA expression. B, DKC1 and MYC expression showed significant correlation (r²=0.17, p<0.0001). C, Relative levels of DKC1 and its H/ACA snoRNP binding partner, NHP2, also strongly correlated (r²=0.26, p<0.0001). D, DKC1 and the cell proliferation marker MKI67 also showed correlation (r²=0.11 p=0.0006). E, There was no correlation between DKC1 and CASP3 expression (r²=0.0036, p>0.5); CASP3 was randomly chosen as a factor that does not have any known relationship to DKC1. The raw data and corresponding log transformations for each of the genes are listed in Supplemental Table 2.