Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization

Abstract

To investigate the role of oxidative stress–induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light–induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5′ end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair–defective human tumors.

Relationships among smoking, oxidative stress, inflammation, macromolecular damage, and cancer

“…8-oxodG can also be reduced to form other oxidized/ring-opened purines, such as 2,6-diamino-4-hydroxy-5formamidopyrimidine (FapyG), although to a much lesser extent [131,132]. Oxidation of dNTPs and their subsequent misincorporation into the DNA can be followed by erroneous replication of the oxidized nucleotides during translesion DNA synthesis, thereby giving rise to mutation [124,133].…”

“…Mice deficient in MTH1 displayed enhanced tumor formation in the lung, liver and stomach, suggesting a contributory role of oxidized dNTP pool in carcinogenesis [193]. Oxidative damage to the nucleotide pool is increasingly considered a key factor in the genesis and progression of cancer [133,168].…”

Cellular aging beyond cellular senescence: Markers of senescence prior to cell cycle arrest in vitro and in vivo

“…Telomere shortening and chromosomal instabilities are well‐established drivers of senescence (Bernadotte et al, 2016; Busuttil et al, 2004). Although it is not known how the deletions or mutations contribute to senescence, an increase in an average number of mutations has been observed during replicative aging (Busuttil et al, 2003; Caliri et al, 2020). Notably, although DSBs affect genome integrity only locally (i.e., at the site where the break occurs), even such insults could lead to global consequences; for example, damage‐induced shortening of a small fraction of telomeres can lead to cell cycle arrest (Zou et al, 2004).…”

Hydroxychloroquine induces oxidative DNA damage and mutation in mammalian cells

“…Generation of primary and spontaneously immortalized Big Blue® mouse embryonic fibroblasts (MEFs) was done according to our published protocols [37,40]. Briefly, cultures of primary and spontaneously immortalized MEFs (C57BL/6 genetic background) were prepared for HCQ cytotoxicity examination, prior to DNA damage and mutagenicity experiments, as described previously [37].…”

“…More specifically, we have performed the λ Select-cII assay on both primary and spontaneously immortalized Big Blue® mouse embryonic fibroblasts (MEFs) treated in vitro with increasing concentrations of HCQ. Fast-proliferating spontaneously immortalized MEFs, which are more resistant to genotoxins than primary MEFs [37], were chosen to model conditions in which rapidly dividing and highly stressed cancer cells in patients are treated with HCQ. To examine the formation of ROS-induced DNA damage in the HCQ-treated cells, we have also measured the levels of 8-oxo-7,8-dihydro-2 ′ -deoxyguanosine (8-oxodG), the most extensively studied oxidative base lesion [38,39], using an enzyme-linked immunosorbent assay (ELISA).…”