Telomere uncapping as a possible mechanism for chemotherapy‐induced vascular toxicity

Abstract

Cardiovascular toxicity is one of the detrimental side effects of chemotherapy that limits treatment. Specifically, chemotherapeutics such as doxorubicin (DOXO) can induce endothelial dysfunction, including reduced endothelial nitric oxide production, cellular senescence, and changes in genes that regulate telomeres, all of which may contribute to toxicity. Telomeres are DNA–protein structures that cap chromosome ends. When telomeres are damaged, they become uncapped and recognized as damaged DNA leading to cellular senescence. Importantly, telomere dysfunction is a biomarker of aging and is associated with numerous diseases including cardiovascular disease. To examine the effects of chemotherapy on vascular endothelial cells (ECs), we used two common chemotherapeutics, DOXO and etoposide (ETOP), both of which act as topoisomerase II inhibitors. Moreover, DOXO intercalates with DNA base pairs resulting in DNA strand breaks. However, whether chemotherapeutics induce telomere dysfunction associated with senescence and, therefore, promote endothelial dysfunction remains elusive. Here, we test the hypothesis that DOXO and ETOP induce endothelial cell telomere uncapping using doses of DOXO (0.1uM) and ETOP (10uM) shown by us and others to induce senescence. We examined telomere uncapping using colocalization of 53BP1, a double-stranded DNA (dsDNA) break marker, and telomeres, by performing immunofluorescence fluorescent in situ hybridization (IF-FISH) on human umbilical vein ECs. At confluence, ECs were treated with either DOXO or ETOP for 24 hours, and 24 and 48 hours later, ECs were collected for RNA isolation or were fixed for IF-FISH. To evaluate senescence, we examined mRNA expression of the senescence marker p21. p21 expression tended to be elevated after 24 hours of treatment (both p > 0.09) and was significantly elevated 48 hours after both DOXO and ETOP treatment (Fig. 1, p<0.001). These results corroborate prior findings that chemotherapeutics such as DOXO and ETOP induce senescence. Twenty-four hours post-treatment, the percentage of cells with ≥ 1 dsDNA break was elevated to 72.5 ± 6.3% in DOXO (n=175 cells) and 70.4 ± 13.6% in ETOP (n=97 cells) treated ECs compared to 13.2 ± 4.8% in the vehicle control group (n=310 cells, Fig. 2a, both p<0.0001). Moreover, the percentage of cells with ≥ 1 Telomere dysfunction-induced Foci (TIF) also increased to 46.6 ± 5.3% in DOXO treated ECs, and 52.2 ± 9.5% in ETOP treated ECs, compared to 7.6 ± 2.8% in the vehicle control group (Fig. 2b, p<0.0001). To determine if dsDNA breaks and TIFs were associated with DOXO and ETOP induced EC senescence, we performed a bivariate correlation analysis. For both treatments, the percentage of ECs with ≥ 1 dsDNA break and ≥ 1 TIF were correlated to p21 mRNA expression at 48 hours post treatment (DOXO: r=0.77 and r=0.78, p<0.01; ETOP: r=0.88 and r=0.91, all p<0.01). These data suggest that DOXO and ETOP may lead to senescence induced endothelial dysfunction via telomere uncapping, providing mechanistic insight into the vascular consequences of chemotherapy.