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Abstract
Cellular senescence, a form of cell cycle arrest, is one of the cellular responses to different types of exogenous and endogenous damage. The senescence phenotype can be induced in vitro by oncogene overexpression and/or DNA damage. Recently, we have reported a novel mechanism of cellular senescence induction by mild genotoxic stress. Specifically, we have shown that the formation of a small number of DNA lesions in normal and cancer cells during S phase leads to cellular senescence-like arrest within the same cell cycle. Here, based on this mechanism, we suggest an approach to remotely induce premature senescence in human cell cultures using short-term light irradiation. We used the genetically encoded photosensitizers, tandem KillerRed and miniSOG, targeted to chromatin by fusion to core histone H2B to induce moderate levels of DNA damage by light in S phase cells. We showed that the cells that express the H2B-fused photosensitizers acquire a senescence phenotype upon illumination with the appropriate light source. Furthermore, we demonstrated that both chromatin-targeted tandem KillerRed (produces O2−) and miniSOG (produces 1O2) induce single-stranded DNA breaks upon light illumination. Interestingly, miniSOG was also able to induce double-stranded DNA breaks.
Highlights
Cellular senescence is growth arrest characterized by complex phenotypic changes and loss of re-proliferative potential [1, 2]
We showed that formation of a small number of DNA lesions in normal and cancer cells during S phase leads to cellular senescence-like arrest within the same cell cycle
The procedure used to induce cellular senescence includes the following steps: i) establishment of the cell line that transiently or stably expresses either tKR or miniSOG fused to core histone H2B to direct them to chromatin, ii) synchronization of the cells in S phase, and iii) light illumination of the cells (Figure 1B)
Summary
Cellular senescence is growth arrest characterized by complex phenotypic changes and loss of re-proliferative potential [1, 2]. One can transfect cells with a plasmid that encodes a constitutively active oncogene, inducing the socalled oncogene-induced senescence (OIS) [3, 4]. It is thought that in course of OIS cells, which are cell cyclearrested due to oncogene-induced DNA damage, undergo geroconversion stimulated by activation of mTOR pathway [1, 5]. The expression of activated oncogenes induces an excessively complex composition of senescence-inducing stimuli, making OIS a difficult-to-interpret model [4, 12]. A topoisomerase II inhibitor, doxorubicin, which is known to induce premature senescence, stimulates ROS overproduction and histone eviction, as well as topoisomerase-dependent DNA damage [14,15,16]. It is reasonable to develop a simple and versatile method to induce cellular senescence in normal and cancer cell lines
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