ARTICLE Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization Received for publication, May 19, 2020, and in revised form, June 2, 2020 Published, Papers in Press, June 2, 2020, DOI 10.1074/jbc.RA120.014465 Andrew W. Caliri1, Stella Tommasi1, Steven E. Bates2,†, and Ahmad Besaratinia1,* From the 1Department of Preventive Medicine, USC Keck School of Medicine, University of Southern California, Los Angeles, California, USA and 2Department of Cancer Biology, Beckman Research Institute of the City of Hope, Duarte, California, USA Edited by Patrick Sung To investigate the role of oxidative stress–induced DNA dam- death (apoptosis) (4). Evasion of cellular senescence gives rise age and mutagenesis in cellular senescence and immortaliza- to an “indefinite” cell proliferation state, otherwise known as tion, here we profiled spontaneous and methylene blue plus immortalization (5). Immortalization allows cells to continually Downloaded from light–induced mutations in the cII gene from l phage in trans- divide and accumulate additional oncogenic alterations, which genic mouse embryonic fibroblasts during the transition from may lead to a fully malignant phenotype (3, 5). Understanding primary culture through senescence and immortalization. the underlying mechanisms of cellular senescence bypass and Consistent with detection of characteristic oxidized guanine immortalization can fundamentally improve our knowledge of lesions (8-oxodG) in the treated cells, we observed signifi- cancer biology (5). http://www.jbc.org/ cantly increased relative cII mutant frequency in the treated Cellular senescence, first described by Hayflick in 1961 (6), is pre-senescent cells which was augmented in their immortal- a stress condition in which cells, despite being alive, cease to ized counterparts. The predominant mutation type in the further proliferate (4). Normal adult somatic mammalian cells treated pre-senescent cells was G:CfiT:A transversion, whose frequency was intensified in the treated immortalized cells. capable of dividing in vivo exhibit a limited life span in vitro as they undergo senescence after successive rounds of replication Conversely, the prevailing mutation type in the treated im- at USC Norris Medical Library on July 17, 2020 mortalized cells was A:TfiC:G transversion, with a unique (5). At variable frequency, however, cultivated cells may escape sequence-context specificity, i.e. flanking purines at the 59 senescence and become immortalized (5, 7). The immortaliza- end of the mutated nucleotide. This mutation type was also tion frequency seems to be primarily species-dependent (4, 7). enriched in the treated pre-senescent cells, although to a Whereas rodent cells efficiently undergo spontaneous immor- lower extent. The signature mutation of G:CfiT:A transver- talization in vitro, primary human and avian cells rarely, if ever, sions in the treated cells accorded with the well-established become spontaneously immortalized in culture (4, 5). Although translesion synthesis bypass caused by 8-oxodG, and the hallmark the overwhelming majority of cultured rodent cells eventually A:TfiC:G transversions conformed to the known replication senesce, few may grow past this barrier, also known as the “cri- errors because of oxidized guanine nucleosides (8-OHdGTPs). sis” phase, and acquire the ability to grow indefinitely and The distinctive features of photosensitization-induced mutagene- become immortalized cell lines (5). sis in the immortalized cells, which were present at attenuated lev- Mouse embryonic fibroblasts (MEF) are a classic model sys- els, in spontaneously immortalized cells provide insights into the tem to study replicative senescence and immortalization (5, 8). role of oxidative stress in senescence bypass and immortalization. Primary MEF, grown in standard cell culture conditions and Our results have important implications for cancer biology passaged serially, typically undergo senescence after several because oxidized purines in the nucleoside pool can significantly rounds of passaging, remain in the “crisis” phase for a while, contribute to genetic instability in DNA mismatch repair–defec- then override the senescence block, re-enter the cell cycle, and tive human tumors. switch to an immortalized phenotype (9). It is widely believed that prolonged exposure of cells to supraphysiological concen- trations of atmospheric oxygen (20%) used in standard cell cul- Malignant transformation is a complex multistep process ture incubators is a main contributor to senescence and during which genetic, epigenetic, and/or environmental factors immortalization (10). The nonphysiological O tension, which trigger a cascade of events, disrupting critical cellular and sub- 2 imposes a significant burden of oxidative stress on the cells, cellular targets (1, 2). Of these, disruption of key cell growth leads to generation of reactive oxygen species (ROS) that regulatory pathways, resulting in uncontrolled cell prolifera- induce promutagenic DNA damage, among other macromo- tion, is an early event and a hallmark of carcinogenesis (1, 3). lecular changes, such as epigenetic modifications (8, 9). The Abrogation of cell-cycle checkpoint controls during malignant ROS-induced genetic mutations and epigenetic alterations in transformation releases the cells from senescence, a state of key genes involved in cell-cycle checkpoints and related regula- permanent growth arrest without undergoing programmed cell tory pathways are thought to play a crucial role in senescence bypass and spontaneous immortalization of MEF (3–5, 10). ® †Deceased. Transgenic Big Blue MEF represent a versatile system for * For correspondence: Ahmad Besaratinia, [email protected]. studying mutagenesis and immortalization simultaneously 9974 J. Biol. Chem. (2020) 295(29) 9974–9985 © 2020 Caliri et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc. Mutagenesis, senescence, and immortalization ® (11, 12). The genome of Big Blue MEF contains multiple copies of the chromosomally integrated, and easily recoverable, lLIZ shuttle vector, which carries two mutation reporter genes, namely the LacI and cII transgenes (11). In the present study, we have constructed a comprehensive catalogue of spontaneous and photosensitization-induced mutations in a mammalian ge- nome by interrogating the cII gene from l phage in transgenic ® Big Blue MEF during the transition from primary culture through senescence and immortalization. More specifically, we have determined the mutant frequency of cII transgene as well ® as characterized the spectra of cII mutation in Big Blue MEF treated with methylene blue plus visible light and counterpart untreated cells both before senescence and after immortaliza- tion. Methylene blue is a photosensitizer that, upon excitation with visible light, produces reactive oxygen species (mainly sin- 1 glet oxygen ( O2)) which primarily cause oxidation of purine and pyrimidine bases in the DNA, with oxidized guanine resi- Downloaded from dues, such as 8-oxo-7,8-dihydro-29-deoxyguanosine (8-oxodG), being the predominant and highly mutagenic lesion (13–15). To verify the effectiveness of methylene blue plus light treatment in inducing promutagenic DNA damage in vitro, we subjected the genomic DNA of the treated and untreated cells to enzymatic http://www.jbc.org/ digestion with formamidopyrimidine DNA glycosylase (Fpg), which cleaves 8-oxodG and other oxidized/ring-opened purines (16, 17), followed by visualization with alkaline gel electrophore- sis. A flowchart of the study is shown in Fig. 1. at USC Norris Medical Library on July 17, 2020 Results Cytotoxicity examination As shown in Fig. 2A, treatment of MEF with methylene blue Figure 1. Flow chart of the study design. T0, immediately after methylene plus light was dose-dependently cytotoxic. Dependent on both blue plus light treatment, cells were used for DNA damage analysis. T1,24h posttreatment (primary MEF), cells were used for cytotoxicity examination. the concentration of methylene blue and the duration of light T2, 8 days posttreatment (early passage pre-senescent MEF), cells were used exposure, cell viability decreased as the administered dose for the first mutagenicity analysis. T3, cells were used for the second mutage- nicity analysis (immortalized MEF). increased. Treatment with 2 mM methylene blue plus 5 min light exposure resulted in 80.0 6 2.7% cell viability that met the threshold limit of cytotoxicity set for our mutagenicity experi- that such treatment is effective in inducing promutagenic DNA ments. Higher doses of methylene blue plus light were prohibi- damage for our mutagenicity experiments. tively cytotoxic as they caused high to excessive cell death in the treated MEF (Fig. 2A). As indicated earlier, moderate to Mutation analysis high cell survival post treatment is optimal and most amenable To investigate the role played by oxidative stress–induced for mutagenicity studies (12, 18, 19). We also note that methyl- DNA damage and mutagenesis in cellular senescence and ene blue treatment alone or light exposure only at the tested immortalization, we have determined the cII mutant frequency ® conditions did not cause any detectable cytotoxicity in MEF and mutation spectrum in Big Blue MEF treated with methyl- (data not shown). ene blue plus light and counterpart untreated cells both before senescence (T2) and after immortalization (T3). Based on the DNA damage detection cytotoxicity