Inducing cellular senescence in vitro by using genetically encoded photosensitizers

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.

Site-specific targeting of a light activated dCas9-KIllerRed fusion protein generates transient, localized regions of oxidative DNA damage

“…Previous studies have established that KR generates reactive oxidative species that directly damage the DNA (35,49,50). KR-induced damage shows overlap with endogenous DNA damage arising from reactive oxygen species (ROS) that naturally occur during cellular metabolism or from exposure to oxidative agents, including oxidized bases, abasic sites, oxypyrimidines, oxypurines, and single strand breaks (12,33,35,(49)(50)(51)(52)(53)(54)(55)(56). Activation of dCas9-KR produced ROS that locally damaged the DNA, resulting in H2AX and KU70/80 spreading at the dCas9 target sequence (Figure 2D, 3D), suggesting that DSBs result after clustered ROS delivery.…”

“…For this, we used the KillerRed (KR) chromophore, a GFP-related protein isolated and modified from the hydrozoan anm2CP, which generates superoxide when activated by green light (31,32). This superoxide is converted to a variety of reactive oxygen species, including H 2 O 2 and hydroxyl radicals, that can cause direct base damage and DNA strand breaks (31)(32)(33). ROS have short half-lives and limited diffusion, and therefore react very close to their site of formation (34).…”

“…ROS have short half-lives and limited diffusion, and therefore react very close to their site of formation (34). Tethering of KR to transcription activators and repressors (12,35) or histone H2B (33) has previously been demonstrated to induce localized DNA damage, including single and double DNA strand breaks, following light activation in whole cells. Here, we created a fusion protein linking KR to the C-terminus of nuclease inactive Cas9 (dCas9), generating dCas9-KR.…”

Photodynamic Physiology—Photonanomanipulations in Cellular Physiology with Protein Photosensitizers

“…The current consensus is that KillerRed undergoes a Type I photodynamic action to generate superoxide anion, although it was previously thought to generate singlet oxygen by a Type II photodynamic action (Pletnev et al, 2009; Serebrovskaya et al, 2009; Shu et al, 2011; Vegh et al, 2011; Kim et al, 2014). A singlet oxygen-generating capacity cannot be completely ruled out, however (Roy et al, 2010; Petrova et al, 2016). Since KillerRed tends to dimerize, a monomeric mutant, Supernova , has been reported (Figure 2), which has the following 6 mutations compared with KillerRed: G3V, N145S, L160T, F162T, L172K, M204T (Takemoto et al, 2013).…”

“…Therefore, H2B-KR-KR photodynamic action could be used to study cell division, organism development, organogenesis or carcinogenesis in a cell-specific manner in vivo (Serebrovskaya et al, 2011) (Figure 5B). Protein photosensitizers tandem-KillerRed and miniSOG target-expressed at chromatin (H2B-tKR or H2B-miniSOG) in HeLa-Kyoto cells after brief light irradiation (H2B-tandem KillerRed expressing cells with green light 540–580 nm for 15 min at 200 mW/cm 2 ; H2B-miniSOG for 5 min with blue light 465–495 nm at 65 mW/cm 2 ) was able to induce single strand breaks but only miniSOG induced double strand breaks of the genomic DNA, leading to DNA damage response and cell senescence (Petrova et al, 2016). Most interestingly, germline C. elegans expressing Histone-mSOG after exposure to blue light has been found to produce progenies with inheritable phenotypes (Noma and Jin, 2015).…”