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. 2016 Aug 23:6:186.
doi: 10.3389/fonc.2016.00186. eCollection 2016.

Cancer Cells with Alternative Lengthening of Telomeres Do Not Display a General Hypersensitivity to ATR Inhibition

Katharina I Deeg  1 , Inn Chung  1 , Caroline Bauer  1 , Karsten Rippe  1
Affiliations

Cancer Cells with Alternative Lengthening of Telomeres Do Not Display a General Hypersensitivity to ATR Inhibition

Katharina I Deeg et al. Front Oncol. .

Abstract

Telomere maintenance is a hallmark of cancer as it provides cancer cells with cellular immortality. A significant fraction of tumors uses the alternative lengthening of telomeres (ALT) pathway to elongate their telomeres and to gain an unlimited proliferation potential. Since the ALT pathway is unique to cancer cells, it represents a potentially valuable, currently unexploited target for anti-cancer therapies. Recently, it was proposed that ALT renders cells hypersensitive to ataxia telangiectasia- and RAD3-related (ATR) protein inhibitors (Flynn et al. , Science 347, 273). Here, we measured the response of various ALT- or telomerase-positive cell lines to the ATR inhibitor VE-821 VSports手机版. In addition, we compared the effect of the inhibitor on cell viability in isogenic cell lines, in which ALT was active or suppressed. In these experiments, a general ATR inhibitor sensitivity of cells with ALT could not be confirmed. We rather propose that the observed variations in sensitivity reflect differences between cell lines that are unrelated to ALT. .

Keywords: ATR inhibitor; VE-821; alternative lengthening of telomeres; ataxia telangiectasia- and RAD3-related V体育安卓版. .

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Figure 1
Figure 1
Cell viability of ALT- and telomerase-positive (TEL) cell lines upon treatment with the ATR inhibitor VE-821. (A) Different cell numbers of HCT116 (TEL) cells were seeded in a 96-well plate and treated with increasing concentrations of the ATR inhibitor VE-821 for 6 days to determine the influence of the starting cell number on the cell viability assay. Cell viability was analyzed using CellTiter Glo and is depicted as percentage of control (n = 3). (B) Same as panel A but for the U2OS ALT cell line. (C) Analysis of cell viability for different cell lines with starting cell numbers that led to 70–90% confluency after 6 days in the absence of inhibitor (n = 3). Cell numbers used for seeding were 500 cells/well for U2OS, HeLa, HCT116, and MG63 and 1500 cells/well for the CAL72 and SAOS2 cell lines. Error bars represent SD of triplicate experiments. (D) IC50 concentrations determined from cell viability curves shown in panel C. (E) FACS analysis of cell death fraction in ALT and TEL cell lines treated with 3 μM VE-821 for 6 days in comparison to the DMSO control. Dead cells were determined by annexin V staining. Error bars represent SEM (n = 3). (F) Percentage of induced cell death determined from FACS analysis as in panel E. The induced cell death fraction was calculated as described in the section “Materials and Methods.”
Figure 2
Figure 2
ALT features and sensitivity to ATR inhibitor treatment upon ectopic expression of ATRX in ATRX-deficient U2OS cells. (A) Western blot showing the expression of HA-tagged ATRX in the generated U2OSATRX-1 cell clone upon doxycycline induction for 48 h and a HeLa reference. In addition to the full-length ATRX band at about 260 kDa, shorter ATRX variants between 200 and 220 kDa were also detected in the HA blot that might correspond to degraded or alternatively spliced products of ATRX, as described previously (15, 16). (B) Average number of APBs per cell in uninduced and for 7 days induced U2OSATRX-1 cells (n = 350) analyzed by 3D confocal image analysis of PML immunofluorescence and telomere FISH stainings as described previously (10). (C) C-circle assay as a marker of ALT activity in uninduced and induced U2OSATRX-1 cells. Samples without polymerase (no pol) and uninduced U2OSATRX-1 were included as controls. The bar plot shows a quantification of C-circle levels in uninduced and induced U2OSATRX-1 cells from three experiments. (D) ATR inhibitor sensitivity in dependence of ALT activity in the U2OSATRX-1 cell line. ATRX-induced U2OSATRX-1 (+) and uninduced U2OSATRX-1 (−) cells were analyzed using the CellTiter Glo assay in the presence of increasing concentrations of the ATR inhibitor VE-821 for 6 days. No changes in ATR inhibitor sensitivity were observed when ALT was silenced by ATRX expression. (E) Western blot showing the expression of ATRX in the U2OSATRX-2 cell line from (8) after induction for 7 days. (F) C-circle assay to test ALT activity in U2OSATRX-2 after 7 and 13 days of doxycycline induction. Samples without polymerase (no pol) and from uninduced cells were included as controls. (G) ATR inhibitor sensitivity in U2OSATRX-2 with (-ATRX) and without ALT activity (+ATRX). The viability assay was performed as described in the legend to panel D. No changes in ATR inhibitor sensitivity were observed when ALT was silenced by ATRX expression.
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