The accumulation of DNA damage over time in somatic cells leads to senescence and, ultimately, cell death. In a publication from 2012 focusing on histone H2A family members, Lopez et al. induced DNA damage in HCA2 fibroblasts by treating them with the chemotherapeutic drug bleomycin.1 Their investigation sought to find evidence of changes in chromatin in response to the DNA damage.
HCA2 fibroblasts were treated for 3 hours to induce DNA damage. Drug-induced senescence occurred after culturing the cells for 3 to 5 days. A preliminary western blot performed on these cells revealed a significant increase in the phosphorylated histone variant γH2A.X in whole cell extracts during acute DNA damage but only a marginal increase in γH2A.X following drug-evoked senescence, when compared with the proliferating cells. There were no changes to H2A levels; however, antibody-based analysis staining is known to be less specific when compared with other methods of quantification, such as mass spectrometry (MS).
To further quantify HCA2 gene products, the researchers developed a multiplexed selected reaction monitoring (SRM) assay. The assay focused on three aspects of the histone H2A family: an H2A variant unbiased approach to represent the histone H2A family proteins, a variant-specific approach focusing on the histone variant H2A.X, and a phosphorylation post-translational modification approach for yH2A.X.
The HCA2 SRM analysis was performed on a TSQ Vantage triple stage quadrupole mass spectrometer equipped with an Accela MS pump, a CTC autosampler, and an IonMax source and a Hypersil Gold 3.0-µm C18-particle column (all Thermo Scientific). Thermo Scientific’s Pinpoint software was also used to predict candidate peptides, select fragment ions, and build the MS instrument method.
As a result of their experiments, the researchers observed a statistically significant loss of the histone H2A family proteins in the HCA2 human fibroblasts under conditions of chronic DNA damage, and less of the variant γH2A.X upon senescence than immediately following acute DNA damage. The researchers propose that the decrease in protein levels was not due to DNA damage accumulation, but that the senescent HCA2 phenotype instead might be a result of drastic chromatin relaxation. This conclusion was also suggested in related work by another lab.
Lopez et al. also propose that the apparent chromatin relaxation and histone deficiency prevents proper nucleosomal reassembly at the last stage of DNA damage repair, with the result that DNA damage signaling continues in the senescent cells. In addition, changes in the histone biosynthesis and chromatin assembly may directly contribute to cellular aging.
1. Lopez, M.F., et al. (2012) “Depletion of nuclear histone H2A variants is associated with chronic DNA damage signaling upon drug-evoked senescence of human somatic cells,” Aging, 4(11) (pp. 823–42).