Inactive Atm abrogates DSB repair in mouse cerebellum more than does Atm loss, without causing a neurological phenotype.

The genome instability syndrome, ataxia-telangiectasia (A-T) is caused by null mutations in the ATM gene, that lead to complete loss or inactivation of the gene's product, the ATM protein kinase. ATM is the primary mobilizer of the cellular response to DNA double-strand breaks (DSBs) - a broad signaling network in which many components are ATM targets. The major clinical feature of A-T is cerebellar atrophy, characterized by relentless loss of Purkinje and granule cells. In Atm-knockout (Atm-KO) mice, complete loss of Atm leads to a very mild neurological phenotype, suggesting that Atm loss is not sufficient to markedly abrogate cerebellar structure and function in this organism. Expression of inactive ("kinase-dead") Atm (AtmKD) in mice leads to embryonic lethality, raising the question of whether conditional expression of AtmKD in the murine nervous system would lead to a more pronounced neurological phenotype than Atm loss. We generated two mouse strains in which AtmKD was conditionally expressed as the sole Atm species: one in the CNS and one specifically in Purkinje cells. Focusing our analysis on Purkinje cells, the dynamics of DSB readouts indicated that DSB repair was delayed longer in the presence of AtmKD compared to Atm loss. However, both strains exhibited normal life span and displayed no gross cerebellar histological abnormalities or significant neurological phenotype. We conclude that the presence of AtmKD is indeed more harmful to DSB repair than Atm loss, but the murine central nervous system can reasonably tolerate the extent of this DSB repair impairment. Greater pressure needs to be exerted on genome stability to obtain a mouse model that recapitulates the severe A-T neurological phenotype.

AN-7, a butyric acid prodrug, sensitizes cutaneous T-cell lymphoma cell lines to doxorubicin via inhibition of DNA double strand breaks repair.

We previously found that the novel histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) had greater selectivity against cutaneous T-cell lymphoma (CTCL) than SAHA. AN-7 synergizes with doxorubicin (Dox), an anthracycline antibiotic that induces DNA breaks. This study aimed to elucidate the mechanism underlying the effect of AN-7 on Dox-induced double-strand DNA breaks (DSBs) in CTCL, MyLa and Hut78 cell lines. The following markers/assays were employed: comet assay; western blot of γH2AX and p-KAP1; immunofluorescence of γH2AX nuclear foci; Western blot of repair protein; quantification of DSBs-repair through homologous recombination. DSB induction by Dox was evidenced by an increase in DSB markers, and DSBs-repair, by their subsequent decrease. The addition of AN-7 slightly increased Dox induction of DSBs in MyLa cells with no effect in Hut78 cells. AN-7 inhibited the repair of Dox-induced DSBs, with a more robust effect in Hut78. Treatment with AN-7 followed by Dox reduced the expression of DSB-repair proteins, with direct interference of AN-7 with the homologous recombination repair. AN-7 sensitizes CTCL cell lines to Dox, and when combined with Dox, sustains unrepaired DSBs by suppressing repair protein expression. Our data provide a mechanistic rationale for combining AN-7 with Dox or other DSB inducers as a therapeutic modality in CTCL.

Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures.

The ATM gene and its protein product, the ATM protein kinase, were identified as a result of attempts to understand the molecular basis of the genetic disorder, ataxia-telangiectasia (A-T). The cardinal symptom of A-T is neurodegeneration expressed primarily as progressive cerebellar atrophy. A major tool in the investigation of ATM functions in the cerebellum is cerebellar organotypic cultures, which allow cerebellar slices to live in culture for several weeks without losing their viability and organization. These cultures are amenable to various treatments and manipulations and provide a close look at Purkinje cells in their almost natural environment. We optimized the protocol for establishing and maintaining these cultures and provide here examples of readouts of the DNA damage response in cerebellar organotypic cultures treated with a DNA-damaging agent.