No abstract

Aging is characterized by progressive loss of physiological and cellular functions, but the molecular basis of this decline remains unclear. We explored how aging impacts transcriptional dynamics using single-cell RNA-sequencing of unstimulated and stimulated naive and effector memory CD4 + T cells from young and old mice from two divergent species. In young animals, immunological activation drives a conserved transcriptomic switch resulting in tightly regulated gene expression, characterized by a strong up-regulation of a core activation program, coupled with a decrease in cell-to-cell variability. Aging perturbed the activation of this core program, and increased expression heterogeneity across populations of cells in both species. These discoveries suggest that increased cell-to-cell transcriptional variability will be a hallmark feature of aging across most, if not all, mammalian tissues.

S N 1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O 6 -methylguanine ( 6Me G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N -nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G 2 phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S*/T*Q substrate, ␥-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.

The mismatch repair (MMR) gene hMLH1 is mutated in ∼50% of hereditary non‐polyposis colon cancers and transcriptionally silenced in ∼25% of sporadic tumours of the right colon. Cells lacking hMLH1 display microsatellite instability and resistance to killing by methylating agents. In an attempt to study the phenotypic effects of hMLH1 downregulation in greater detail, we designed an isogenic system, in which hMLH1 expression is regulated by doxycycline. We now report that human embryonic kidney 293T cells expressing high amounts of hMLH1 were MMR‐proficient and arrested at the G2/M cell cycle checkpoint following treatment with the DNA methylating agent N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG), while cells not expressing hMLH1 displayed a MMR defect and failed to arrest upon MNNG treatment. Interestingly, MMR proficiency was restored even at low hMLH1 concentrations, while checkpoint activation required a full complement of hMLH1. In the MMR‐proficient cells, activation of the MNNG‐induced G2/M checkpoint was accompanied by phosphorylation of p53, but the cell death pathway was p53 independent, as the latter polypeptide is functionally inactivated in these cells by SV40 large T antigen.