Telomerase inhibition causes premature senescence of fetal guinea pig muscle cells

Premature senescence in low birth weight rodents is associated with later life metabolic disease, including the development of insulin resistance. Telomerase, a reverse transcriptase enzyme with telomeric and non-telomeric functions, is present at high levels during development to maintain and repair long telomere lengths and to protect cells from oxidative stress-induced growth arrest. Adverse In utero environments are often associated with increased reactive oxygen species (ROS), and ROS have been documented to impair/alter telomerase function. We postulate that telomerase protects cells against oxidative stress-induced damage, and its inhibition could lead to premature senescence. A primary cell line of fetal guinea pig muscle cells was differentiated under high (20%) and low (1-2%) oxygen concentrations and telomerase activity was pharmacologically inhibited using a synthetic tea catechin. Following 48 hours, ROS detection was conducted with MitoSOX, Mitotracker and 6-carboxy-2’,7’-dichlorodihydrofluorescein diacetate staining. Cells cultured at 20% O2 and treated with a telomerase activity inhibitor displayed reduced cell growth rates and increased levels of senescence markers, including p21 and p53. Telomeric DNA damage, measured by phosphorylated-γH2A.X staining at telomeres, was significantly increased in cells cultured at all oxygen concentrations with telomerase inhibition. Telomerase inhibition altered metabolic signaling (e.g. mTOR, p66Shc) and increased mitochondrial ROS levels. Telomerase may protect cells during development from adverse in utero environments that cause premature senescence.

1 between oxygen concentrations was apparent at the different Telomerase Inhibitor IX 2 treatments (p<0.05) except at the highest dose of 2.0μM (Fig 1). 12 TI-IX compared to controls. Cells cultured in 2% O 2 conditions exhibited significantly 13 higher telomerase activity levels in control cell groups and cells treated with TI-IX at every 14 concentration, except for 2.0µM, compared to 20% O 2 grown/treated cells. Results (n=3) 15 were analyzed with a two-way ANOVA and different letters above the histogram bars 16 represent significant differences (p<0.05) in mean telomerase activities. 17 18 Senescent characteristics displayed by 20% oxygen 19 cultured cells treated with telomerase inhibitor 20 Fetal guinea pig muscle cells treated with increasing concentrations of telomerase inhibitor 21 IX (TI-IX) displayed altered cell morphology including larger, flatter cells and reduced 22 confluency that are typical morphological characteristics of senescent cells (Fig 2). This 23 effect was observed to a greater extent at 20% O 2 compared to 2% O 2 culture conditions.

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To strengthen the observed senescent phenotype, cells were seeded into 6-well 1 dishes at different concentrations of 25,000, 35,000 and 50,000 cells and were counted 2 every 24 hours for 3 days to determine population-doubling time. There was no significant 3 (p>0.05) difference between cells grown at 2% O 2 and 0.5% O 2 with TI-IX treatment, but 4 there was a significant increase (p<0.001) in population doubling time for fetal guinea pig 5 muscle cells grown at 20% O 2 and treated with 1 μM TI-IX (Fig 2B). Cell viability was 6 determined after 48 hours in both control and telomerase inhibited cells using trypan blue 7 and hemocytometer counts. There was no significant difference (p>0.05) in fetal muscle 8 cell viability at different oxygen tensions or Telomerase Inhibitor IX concentrations (Fig 2C). 9 Annexin V and propidium iodide (PI) staining were used to determine if telomerase 10 inhibition triggered apoptosis in fetal guinea pig muscle cells. There was no significant 11 difference between cells cultured at the same oxygen concentration, however cells cultured  2 Cells were incubated for 48 hr at 20% and 2% O 2 with or without TI-IX with a positive 3 control of a 24 hr treatment with 500 µM H 2 O 2 . Cells were trypsinized and stained for 4 extracellular apoptotic marker Annexin V, and an intracellular fluorescent marker 5 propidium iodide (PI) to label apoptotic/necrotic cells. The cells were analyzed in the Accuri 6 C6 flow cytometer, and mean fluorescence of each stain was determined. Mean 7 fluorescence was graphed and shown for Annexin V (D), and for PI (E). Results were 8 analyzed with two-way ANOVA, n=4. Labelled bars with the same letter were not 9 significantly different (p>0.05) from each other. Labelled bars with a and b indicate a 10 significant difference of p<0.05, and c labelled bars indicate a significant difference of 11 p<0.01 compared to a and b. 12 13 Telomerase inhibition causes alteration in tumor 14 suppressor proteins that signify cell cycle arrest 15 DNA damage-induced activation of ATM/ATR and Chk1/Chk2 phosphorylates the tumor 16 suppressor protein p53, which affects transcription of many downstream proteins including 17 the cell cycle inhibitor p21. Together p53 and p21 mediates cellular senescence when cells 18 are exposed to certain stressors and damage [67]. With the addition of Telomerase 19 Inhibitor IX, p53 protein levels were significantly elevated in fetal muscle cells cultured in 20 20% O 2 (p<0.05), 2% O 2 (p<0.01) and 0.5% O 2 (p<0.05) atmospheres (Fig 3A). Increases in 21 p53 protein were accompanied with elevated phospho-p53 at serine-15, but these increases 22 were not significant (Fig 3B). Following the expression levels of p53, p21 was significantly 23 increased two-fold at each oxygen concentration with Telomerase Inhibitor IX treatment of 24 muscle cells cultured in 20% O 2 (p<0.001), 2% O 2 and 0.5% O 2 (p<0.05) conditions ( Fig 3C).
1 Retinoblastoma protein (Rb) is a critical regulator of the G1/S checkpoint and must be 2 phosphorylated (pRb) in order to be inactivated and release of the E2F transcription factors 3 to allow the cell to continue through the cell cycle. There was no difference between any of 4 the groups when immunoblotting for non-phosphorylated Rb protein levels ( Fig 3E).
5 However, cells cultured in 20% O 2 and treated with Telomerase Inhibitor IX showed 6 significantly decreased (p<0.05) levels of phosphorylated (S795) Rb protein (Fig 3F), which 7 decreased more significantly (p<0.001) when the ratio of pRb to total Rb levels were 8 calculated (Fig 3G). Cells cultured in 2% O 2 displayed no significant difference (p>0.05) 9 between non-treated controls and inhibitor treated cells. 12 with telomerase inhibition. Fetal guinea pig muscle cells were treated for 48 hr at 20%, 13 2%, and 0.5% oxygen (O 2 ) tensions with or without Telomerase Inhibitor (TI-IX). Total 14 protein extractions were immunoblotted followed by densitometric analyses of blots 15 exposed to antibodies specific to (A) p53, (B) phospho-p53 (Ser15), and (C) p21. β-actin 16 protein levels were detected as a loading control. A representative western blot is shown in 17 (D). Results were analyzed with a two-way ANOVA, n=5, with (***), (**) and (*) indicating 18 p<0.001, p<0.01, and p<0.05, respectively. (E-H) Cells were collected and nuclear protein 19 extractions were used in immunoblotting followed by densitometric analyses of blots 20 exposed to antibodies specific to (E) Rb, and (F) phospho-Rb (Ser795). A ratio of pRb/Rb 21 was calculated to determine amount of total Rb that was phosphorylated (G). Total histone 22 H3 protein levels were detected as a loading control. Results were analyzed with a two-way 23 ANOVA, n=3, with (***) and (*) indicating p<0.001, and p<0.05, respectively. A 24 representative Western blot is shown in (H). 25 1 Significant increase in telomere-damage induced foci in 2 telomerase inhibited fetal guinea pig muscle cells 3 Telomerase has been shown to play a role in the DNA damage response, and its inhibition 4 may be a potential reason for the increase in cell senescence markers. To determine if there 5 is an increased amount of DNA damage, the DNA double strand break marker 6 phosphorylated ɣ-H2AX was assayed by immunoblotting and immuno-FISH with a telomere 7 DNA probe. Total protein levels of phospho-ɣH2AX showed a slight increase when treated 8 with Telomerase Inhibitor IX, and were increased as the oxygen concentration decreased 9 ( Fig 4C). However, densitometric analysis of the immunoblots revealed that these 10 differences were not significant (p>0.05). Telomere induced foci (TIFs) are comprised of 11 co-localization of DNA damage markers, such as phospho-ɣH2AX with a fluorescent 12 telomeric probe (Fig 4A). A significant increase in TIF-positive cells was observed in TI-IX 13 treated cells at both 20% O 2 (p<0.01) and 2% O 2 (p<0.05) tensions compared to non-treated 14 controls (Fig 4B). Furthermore, there was greater TIF-positive cells for cells grown under 15 20% O 2 conditions compared to those cultured in 2% O 2 (p<0.05).

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The DNA damage response is initially sensed at the DNA level by the MRN complex 17 and the ATM/ATR proteins. ATM and ATR are kinases that phosphorylate many 18 downstream targets involved in cell cycle arrest during DNA damage and DNA repair 19 proteins. To determine if ATR was active, a phospho-ATR (S428) antibody was utilized for 20 immunoblotting. There were no significant differences between control and cells treated 21 with Telomerase Inhibitor IX, but there was a significant increase in phospho-ATR (S428) 22 levels within fetal muscle cells cultured at lower oxygen concentrations compared to those 23 at 20% O 2 (p<0.001) (Fig 4D). 10 Significant differences between groups were calculated with a two-way ANOVA, n=4, with 11 (*) and (**) representing p<0.05 and p<0.01, respectively. (C-E) Altered DNA damage 12 response proteins ɣH2AX and ATR. Fetal guinea pig muscle cells were treated for 48 hours 13 at 20%, 2%, and 0.5% O 2 with or without TI-IX. Cells were collected and total protein 14 extractions were used in immunoblotting followed by densitometric analyses of blots 15 exposed to antibodies specific to (C) phospho-ɣH2AX (Ser139), and (D) phospho-ATR 16 (Ser428). Total histone H3 protein levels were detected as a loading control. A 17 representative western blot is shown in (E). Results were analyzed with a two-way ANOVA,  24 briefly with telomerase inhibitor, mitochondrial ROS was evaluated due to its known 1 involvement in regulating the senescence state [29]. MitoTracker® Red CMXRos is a red-2 fluorescent dye that fluoresces upon oxidation in live cells and is sequestered into 3 mitochondria based on its membrane potential. Guinea pig muscle cells displayed red 4 fluorescence in the mitochondria, shown as small punctate points surrounding the nucleus 5 ( Fig 5A). Flow cytometric analysis of MitoTracker fluorescence was significantly (p<0.001) 6 increased in cells cultured in 20% O 2 compared to those grown in 2% O 2 (Fig 5B).

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The antioxidant proteins SOD1 and catalase were also evaluated by immunoblotting.
3 SOD1 is a soluble cytoplasmic and mitochondrial intermembrane space enzyme responsible 4 for converting superoxide radicals into molecular oxygen and hydrogen peroxide (H 2 O 2 ), 5 while catalase is the major enzyme for converting H 2 O 2 into water and oxygen in a cell.   24 Antioxidant proteins catalase and SOD1 are elevated in fetal muscle cells cultured in higher 25 oxygen concentrations. Fetal muscle cells were treated for 48 hours at 20%, 2%, and 0.5% 1 O 2 with or without TI-IX. Total protein extractions were used in immunoblotting followed 2 by densitometric analyses of blots exposed to antibodies specific to catalase (E) and SOD1 3 (F). Total β-actin protein levels were detected as a loading control. A representative 4 western blot is shown in (G). Results were analyzed with a two-way ANOVA, n=3, with (*) 5 indicating p<0.05. 6 7 Altered metabolic signaling in telomerase inhibited fetal 8 guinea pig muscle cells 9 The mTOR pathway is necessary for integrating different stimuli and regulating cell growth 10 and metabolism, and may play a role in triggering senescence [68]. Immunoblotting for 11 mTOR and phopsho-mTOR revealed no difference in mTOR protein levels between 12 treatment groups, nevertheless there was a significant decrease in phospho-mTOR at 20% 13 O 2 (p<0.05) compared to cells grown in both 2% O 2 and 0.5% O 2 conditions (Fig 6A, B).

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S6K is the downstream kinase in the mTOR pathway that regulates ribosomal 17 protein synthesis. Total S6K protein levels were increased in cells grown at 2% O 2 18 compared to cells grown in 20% O 2 and 0.5% O 2 (p<0.01), but was not different with 19 telomerase inhibition (Fig 6D). Phosphorylated S6K (T421/S424) was not significantly 20 (p>0.05) up regulated between control cells and cells treated with the telomerase inhibitor 21 at both 2% and 0.5% O 2 , but was significantly (p<0.01) elevated in 20% O 2 cultures (Fig 6E).

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P66Shc is a unique adaptor protein as it has been reported to have roles in both 4 sensing oxidative stress, mediating its effects and even causing more ROS to be generated at

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The specific mechanism of action of TI-IX is currently unclear, but it may inhibit 24 telomerase by transcriptional and translational inhibition of telomerase reverse

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Since telomerase inhibition may also trigger apoptosis [80, 81], markers of 4 apoptosis were analyzed by flow cytometry to strengthen the cellular growth arrest 5 phenotype. The incidence of apoptosis did not increase upon telomerase inhibition since 6 both Annexin V and PI fluorescence levels did not differ significantly between control cells 7 and those treated with TI-IX. However, there was a significant increase in apoptotic cells 8 incubated at 20% O 2 compared to those at 2% O 2 further supporting that atmospheric 9 oxygen is inherently a more stressful in vitro environment that can lead to cellular 10 apoptosis/senescence when cells are exposed to another challenge such as telomerase 11 inhibition. Importantly, cell viability between oxygen concentrations and treatments was 12 not significantly affected. Since telomerase inhibited fetal muscle cells cultured in 20%      18 Surprisingly, we detected decreased cytoplasmic ROS levels in telomerase inhibited cells.

Contrary to reports that telomerase improves mitochondrial function [39], mitochondrial
20 TERT also appears to exacerbate free-radical-mediated mtDNA damage [110]. These 21 results support differing roles for telomerase in regulating ROS production, but further 22 research is needed to fully elucidate its role in the mitochondria.

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The transcription factor PGC-1α regulates genes involved in energy metabolism and 24 mitochondrial biogenesis [73], and was significantly increased in 20% O 2 compared to 2% 25 O 2 cells. Elevated PGC-1α correlated with increased mitochondrial number in cells grown at 1 20% O 2 and could be responsible for increased ROS levels [111]. Coupled with results 2 indicating higher levels of DNA damage and senescent markers, 20% O 2 promotes a 3 hyperoxic environment [112]. Low 2% O 2 is more physiological to what fetal muscle cells 4 are exposed to in utero, allowing "normal" function without being hypoxic [113]. Hypoxia      11 These oxygen tensions estimate the oxygen concentrations found in adult arterial PO 2 (~80-12 100mmHg), fetal arterial PO 2 (~15mmHg), and a lower oxygen tension to model a hypoxic 13 arterial PO 2 (~3mmHg), respectively [113]. Cells were grown in the same conditions over