Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Oct 14;8(10):2449-2462.
doi: 10.18632/aging.101065.

Inducing Cellular Senescence in vitro by Using Genetically Encoded Photosensitizers

Affiliations
Free PMC article

Inducing Cellular Senescence in vitro by Using Genetically Encoded Photosensitizers

Nadezhda V Petrova et al. Aging (Albany NY). .
Free PMC article

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.

Keywords: DNA damage; KillerRed; cellular senescence; miniSOG; optogenetics.

Conflict of interest statement

The authors have no conflict of interests to declare.

Figures

Figure 1
Figure 1. Overview of the method used to optogenetically induce cellular senescence in vitro
(A) Model illustrating how mild genotoxic stress can induce cellular senescence-like proliferation arrest (according to [30]). (B) Overview of the method for inducing cellular senescence using the genetically encoded photosensitizers tandem KillerRed (tKR) and miniSOG that were targeted to chromatin.
Figure 2
Figure 2. DNA damage induced by the activation of miniSOG and tKR targeted to chromatin
(A-B) Asynchronous H2B-miniSOG expressing HeLa cells, along with their non-expressing counterparts, were either blue-light irradiated (“BL”; 465-495 nm, 65 mW/cm2, 5 min) or light irradiated and recovered for 30 min (“BL+rec”). Asynchronous H2B-tKR expressing HeLa cells, along with their non-expressing counterparts, were either green-light irradiated (“GL”; 540-580 nm, 200 mW/cm2, 15 min) or light irradiated and recovered for 30 min (“GL+rec”). Alkaline (A) and neutral (B) comet assays were performed. Non-illuminated cells were used (“control”) as a negative control and cells treated with H2O2 (“H2O2”; 200 μM, 1 hr) were used as a positive control in the alkaline comet assay (A), and cells treated with the topoisomerase II poison etoposide (“VP16”; 10 μg/ml, 1 hr) were used as a positive control in the neutral comet assay (B). Box plots show the tail moments. The boxed region represents the middle 50% of the tail moments, the horizontal lines represent the medians, and the black crosses indicate the means. *P < 0.0001 (two-tailed t-test, n > 70), #P < 0.0001 (two-tailed t-test, n > 150), n.s. – not significant. The results of one of four experiments are shown.
Figure 3
Figure 3. Activated genetically encoded photosensitizers can induce cellular senescence
(A-B) The HeLa Kyoto cell line and its derivatives expressing either H2B-miniSOG or H2B-tKR were synchronized in S phase, illuminated with blue (465-495 nm, 65 mW/cm2, 5 min) or green (540-580 nm, 200 mW/cm2, 15 min) light, allowed to recover for 48 hr, and stained for γH2AX (A) or SA-β-gal (B). Control represents the cells that were synchronized and released for 48 hr (non-illuminated). The DNA was stained with DAPI in (A). Scale bar: 50 μm. (C) Senescent HeLa cells stained for SA-β-gal. Cellular senescence was induced by treatment of S-phase HeLa cells with a DNA topoisomerase I inhibitor camptothecin (1 μM, 1 h). (D) The HeLa Kyoto cell line and its derivatives expressing either H2B-miniSOG or H2B-tKR were synchronized in S phase, illuminated with corresponding light, allowed to recover for 48 hr, and stained with DAPI. Segmentation of cell nuclei was performed using CellProfiler. Boxplots show nuclear area in each case (*P=0.0001, two-tailed t-test).
Figure 4
Figure 4. Locally activated H2B-miniSOG can induce cellular senescence
(A-B) HeLa cells expressing H2B-miniSOG were synchronized in S phase, illuminated with blue (465-495 nm, 65 mW/cm2, 5 min) light, allowed to recover for 24 hr, and stained for γH2AX (A) or SA-β-gal (B). Only part of each specimen was illuminated. Dashed line shows the boundary between illuminated and non-illuminated parts of the specimens. Scale bars: 100 μm (A) and 80 μm (B).
Figure 5
Figure 5. Temporal kinetics of the formation of the persistent DNA damage response foci induced by activation of miniSOG or tKR
(A-B) HeLa cells that express H2B-miniSOG (A) or H2B-tKR (B) were synchronized in S phase, illuminated with blue (465-495 nm, 65 mW/cm2, 5 min) or green (540-580 nm, 200 mW/cm2, 15 min) light, allowed to recover for the indicated time intervals (0, 3, 6 and 24 hr). Histone γH2AX was analyzed by indirect immunofluorescence or WB. Negative control represents the cells that were synchronized but not light illuminated; positive control represents the cells treated with DNA topoisomerase II inhibitor etoposide (VP16; 10 μg/ml, 1 hr). The DNA was stained with DAPI. Scale bar: 20 μm.
Figure 6
Figure 6. Analysis of the expression of p21 and p16 CDK inhibitors in HeLa cells expressing genetically encoded photosensitizers
HeLa cells that express H2B-miniSOG or H2B-tKR were synchronized in S phase, illuminated with blue (465-495 nm, 65 mW/cm2, 5 min) or green (540-580 nm, 200 mW/cm2, 15 min) light, allowed to recover for the indicated time intervals (0, 6, 24 and 48 hr), and subjected to gene expression analysis using qRT-PCR and WB. Control (“C”) represents the non-illuminated cells. The expression of p21CIP1 and p16INK4a was analyzed using EvaGreen-based qRT-PCR. The amplification levels of the cDNA were normalized to the level of the GAPDH cDNA. The results of one representative experiment are shown. WB was performed with an antibody against p21; GAPDH was used as the loading control.

Similar articles

See all similar articles

Cited by 2 articles

References

    1. Blagosklonny MV. Cell cycle arrest is not yet senescence, which is not just cell cycle arrest: terminology for TOR-driven aging. Aging (Albany NY) 2012;4:159–65. doi: 10.18632/aging.100443. - DOI - PMC - PubMed
    1. Blagosklonny MV. Cell cycle arrest is not senescence. Aging (Albany NY) 2011;3:94–101. doi: 10.18632/aging.100281. - DOI - PMC - PubMed
    1. Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell. 1997;88:593–602. doi: 10.1016/S0092-8674(00)81902-9. - DOI - PubMed
    1. Bianchi-Smiraglia A, Nikiforov MA. Controversial aspects of oncogene-induced senescence. Cell Cycle. 2012;11:4147–51. doi: 10.4161/cc.22589. - DOI - PMC - PubMed
    1. Leontieva OV, Blagosklonny MV. Tumor promoter-induced cellular senescence: cell cycle arrest followed by geroconversion. Oncotarget. 2014;5:12715–27. doi: 10.18632/oncotarget.3011. - DOI - PMC - PubMed

Publication types

Substances

LinkOut - more resources

Feedback