Expression of NES-hTERT in Cancer Cells Delays Cell Cycle Progression and Increases Sensitivity to Genotoxic Stress

Telomerase is a reverse transcriptase associated with cellular immortality through telomere maintenance. This enzyme is activated in 90% of human cancers, and inhibitors of telomerase are currently in clinical trials to counteract tumor growth. Many aspects of telomerase biology have been investigated for therapy, particularly inhibition of the enzyme, but little was done regarding its subcellular shuttling. We have recently shown that mutations in the nuclear export signal of hTERT, the catalytic component of telomerase, led to a mutant (NES-hTERT) that failed to immortalize cells despite nuclear localization and catalytic activity. Expression of NES-hTERT in primary fibroblast resulted in telomere-based premature senescence and mitochondrial dysfunction. Here we show that expression of NES-hTERT in LNCaP, SQ20B and HeLa cells rapidly and significantly decreases their proliferation rate and ability to form colonies in soft agar while not interfering with endogenous telomerase activity. The cancer cells showed increased DNA damage at telomeric and extra-telomeric sites, and became sensitive to ionizing radiation and hydrogen peroxide exposures. Our data show that expression of NES-hTERT efficiently counteracts cancer cell growth in vitro in at least two different ways, and suggest manipulation with the NES of hTERT or its subcellular shuttling as a new strategy for cancer treatment

Expression of _NES-hTERT increases nuclear and mitochondrial DNA damage in skin cancer and prostate cancer cells.

<p>(A) Cells were immunostained with antibodies against γH2AX and against 53BP1. DNA was counterstained with DAPI. Graph shows percentage of cells positive for both γH2AX and 53BP1 foci and the number and size of foci per cell (represented by the different colors according to the graph labeling). Bars are mean ± s.d. (B) ImmunoFISH staining to visualize simultaneously DNA damage foci and telomeres. DAPI was used to counterstain DNA. Graph shows percentage of DNA damage foci localized at telomeres (TIF) per single cell. (C) mtDNA integrity was analyzed by QPCR in three independent experiments. Graph show estimated lesion frequency ± s.e.m.</p

Expression of _NES-hTERT does not alter endogenous levels of telomerase enzymatic activity.

<p>(A) Levels of hTERT RNA were gauged by RT-PCR. GAPDH was amplified and used as loading control. (B) 100 ng of total cell extracts were used to perform the TRAP. Arrow indicates internal control of the assay. Positive and negative controls are not shown.</p

Cancer cells expressing _NES-hTERT show changes in morphology and delays in cell cycle progression.

<p>(A) SQ20B (upper panels) LNCaP (middle panels) and their derivatives stably expressing _NES-hTERT were plated on dishes in equal numbers and analyzed 72 hours later. Phase contrast images were taken on an Olympus IX70 microscope. Note enlarged morphology of SQ20B _NES-hTERT and cells harboring multiple nuclei (arrows). Clustering is observed in LNCaP (see box), but not seen in LNCaP _NES-hTERT. Bottom panels show HeLa cells that were transiently transfected with the _NES-hTERT mutant. Images were taken 48 hours after transfections. Arrows indicate enlarged and multinucleated cells (middle) observed only upon transfection with the mutant hTERT. (B) SQ20B, LNCaP and their _NES-hTERT derivatives were plated and allowed to grow for up to 144 hours. At various times cells were harvested and counted using a hemocytometer. In the case of LNCaP, cells were replated and counted again 72 hours later. Mean of three analyses is shown, error bars represent s.e.m. (*p≤0.05) (C) Percentage of cells in each phase of the cell cycle was calculated by flow cytometry based on PI staining. (D) Cells were serum starved overnight, then released by serum addition. Cells were labeled with [³H]-thymidine and analyzed at scheduled time intervals for thymidine incorporation. Mean of two independent experiments is shown, error bars are s.d.</p

Anchorage-independent growth is diminished by expression of _NES-hTERT.

<p>5×10³ cells/well of SQ20B, LNCaP and their _NES-hTERT derivatives were grown in soft agar for up to 3 weeks. Colony growth was evaluated every week and colonies counted based on crystal violet staining. Graphs show results from colonies counted at 1 week when individual colonies, especially in the control cells, were still easily distinguishable. Colonies were scored by two independent observers. Data shown are the average of two independent experiments done in triplicates. Bars represent mean ± sd. Representative images of the plates are shown below each graph.</p

_NES-hTERT sensitizes skin cancer cells to genotoxic stress.

<p>(A) Nuclear DNA damage was estimated in SQ20B and its _NES-hTERT derivative immediately after exposure to 1 Gy of gamma radiation using QPCR. Results represent the average of three independent experiments ± s.e.m. (B) Cells were treated with 200 µM of H₂O₂ for 60 minutes and allowed to recover for 24 hours in conditioned medium. At this point, cells were harvested and the number of apoptotic, dead and viable cells was evaluated by flow cytometry using PI and YOPRO-1. Results are representative of three independent experiments. (C) The same amount of viable cells (500,000) were replated after the H₂O₂ exposures and their growth rate was followed for 2 weeks. The number of cells was counted using a hemocytometer at 24 hours and every time cells became confluent thereafter. As the number of treated SQ20B _NES-hTERT did not change in the following 2 weeks, only data for 24 hours post-treatment are shown. Results are mean of three independent experiments ± s.e.m. (* p≤0.05).</p