Ataxia telangiectasia mutated (ATM) kinase is critical in sensing and repairing DNA double-stranded breaks (DSBs) such as those induced by temozolomide (TMZ). ATM deficiency increases TMZ sensitivity, which suggests that ATM inhibitors may be effective TMZ sensitizing agents. In this study, the TMZ sensitizing effects of 2 ATM specific inhibitors were studied in established and xenograft-derived glioblastoma (GBM) lines that are inherently sensitive to TMZ and derivative TMZ-resistant lines. In parental U251 and U87 glioma lines, the addition of KU-55933 to TMZ significantly increased cell killing compared to TMZ alone [U251 survival: 0.004 ± 0.0015 vs. 0.08 ± 0.01 (p<0.001), respectively, and U87 survival: 0.02 ± 0.005 vs. 0.04 ± 0.002 (p<0.001), respectively] and also elevated the fraction of cells arrested in G2/ M [U251 G2/ M fraction: 61.8 ± 1.1% vs. 35 ± 0.8% (p<0.001), respectively, and U87 G2/ M fraction 25 ± 0.2% vs.18.6 ± 0.4% (p<0.001), respectively]. In contrast, KU-55933 did not sensitize the resistant lines to TMZ, and neither TMZ alone or combined with KU-55933 induced a G2/ M arrest. While KU-55933 did not enhance TMZ induced Chk1/ Chk2 activation, it increased TMZ-induced residual γ-H2AX foci in the parental cells but not in the TMZ resistant cells. Similar sensitization was observed with either KU-55933 or CP-466722 combined with TMZ in GBM12 xenograft line but not in GBM12TMZ, which is resistant to TMZ due to MGMT overexpression. These findings are consistent with a model where ATM inhibition suppresses the repair of TMZ-induced DSBs in inherently TMZ-sensitive tumor lines, which suggests an ATM inhibitor potentially could be deployed with an improvement in the therapeutic window when combined with TMZ.

Blocking calcium entry may prevent normal and pathological cell proliferation. There is evidence suggesting that molecules such as carboxyamidotriazole, widely used in anti-cancer therapy based on its ability to block calcium entry in nonexcitable cells, also have antiproliferative properties. We found that carboxyamidotriazole and the capacitative calcium entry blocker 2-aminoethoxydiphenyl borate inhibited proliferation in HEK-293 cells with IC50 values of 1.6 and 50 μM, respectively. Capacitative calcium entry is activated as a result of intracellular calcium store depletion. However, non-capacitative calcium entry pathways exist that are independent of store depletion and are activated by arachidonic acid and diacylglycerol, generated subsequent to G protein coupled receptor stimulation. We found that carboxyamidotriazole completely inhibited the capacitative calcium entry and had no effect on the amplitude of arachidonic-acid-activated non-capacitative calcium entry. However, investigation of the effects of carboxyamidotriazole on mitochondrial calcium dynamics induced by carbachol, capacitative calcium entry and exogenously set calcium loads in intact and digitonin-permeabilized cells revealed that carboxyamidotriazole inhibited both calcium entry and mitochondrial calcium uptake in a time-dependent manner. Mitochondrial inner-membrane potential was altered by carboxyamidotriazole treatment, suggesting that carboxyamidotriazole antagonizes mitochondrial calcium import and thus local calcium clearance, which is crucial for the maintenance of capacitative calcium entry.

DNA adducts derived from carcinogenic polycyclic aromatic hydrocarbons like benzo[a]pyrene (B[a]P) and benzo[c]phenanthrene (B[c]Ph) impede replication and transcription, resulting in aberrant cell division and gene expression. Global nucleotide excision repair (NER) and transcription-coupled DNA repair (TCR) are among the DNA repair pathways that evolved to maintain genome integrity by removing DNA damage. The interplay between global NER and TCR in repairing the polycyclic aromatic hydrocarbon-derived DNA adducts (+)-trans-anti-B[a]P-N6-dA, which is subject to NER and blocks transcription in vitro, and (+)-trans-anti-B[c]Ph-N6-dA, which is a poor substrate for NER but also blocks transcription in vitro, was tested. The results show that both adducts inhibit transcription in human cells that lack both NER and TCR. The (+)-trans-anti-B[a]P-N6-dA lesion exhibited no detectable effect on transcription in cells proficient in NER but lacking TCR, indicating that NER can remove the lesion in the absence of TCR, which is consistent with in vitro data. In primary human cells lacking NER, (+)-trans-anti-B[a]P-N6-dA exhibited a deleterious effect on transcription that was less severe than in cells lacking both pathways, suggesting that TCR can repair the adduct but not as effectively as global NER. In contrast, (+)-trans-anti-B[c]Ph-N6-dA dramatically reduces transcript production in cells proficient in global NER but lacking TCR, indicating that TCR is necessary for the removal of this adduct, which is consistent with in vitro data showing that it is a poor substrate for NER. Hence, both global NER and TCR enhance the recovery of gene expression following DNA damage, and TCR plays an important role in removing DNA damage that is refractory to NER.

The most common, oxidatively generated lesion in cellular DNA is 8-oxo-7,8-dihydroguanine, which can be oxidized further to yield highly mutagenic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) in DNA. In human cell-free extracts, both lesions can be excised by base excision repair and global genomic nucleotide excision repair. However, it is not known if these lesions can be removed by transcription-coupled DNA repair (TCR), a pathway that clears lesions from DNA that impede RNA synthesis. To determine if Sp or Gh impede transcription, which could make them viable substrates for TCR, either an Sp or a Gh lesion was positioned on the transcribed strand of DNA under the control of a promoter that supports transcription by human RNA polymerase II. These constructs were incubated in HeLa nuclear extracts that contained active RNA polymerase II, and the resulting transcripts were resolved by denaturing polyacrylamide gel electrophoresis. The structurally rigid Sp strongly blocks transcription elongation, permitting nominal lesion bypass of 1.6 ± 0.5%. In contrast, the conformationally flexible Gh poses less of a block to human RNAPII, allowing 9 ± 2% bypass. Furthermore, fractional lesion bypass for Sp and Gh is minimally affected by glycosylase activity found in the HeLa nuclear extract. These data specifically suggest that both Sp and Gh may well be susceptible to TCR since each poses a significant block to human RNA polymerase II progression. A more general principle is also proposed: Conformational flexibility may be an important structural feature of DNA lesions that enhances their transcriptional bypass.

Our findings suggest that the E233G variant affects RAD51D functions and protein interactions and increases cellular chemoresistance. This study is the first to analyze the functional effects of a clinically relevant RAD51D amino acid substitution. Further study of this variant will provide mechanistic insight into the role of RAD51D in cellular response to anticancer agents and as a molecular target for cancer therapy.