Local hypoxia is produced at sites of intratumour injection

Intratumour injection, commonly used for gene or drug delivery but also associated with needle biopsy or insertion of invasive measuring devices, may damage tumour microvessels. To examine this possibility, SCCVII tumours grown subcutaneously in C3H mice were injected with a 26 gauge needle containing 0.1 ml of the fluorescent dye Hoechst 33342 to label cells lining the track of the needle. Hoechst-labelled cells sorted from these tumours were more sensitive to killing by hypoxic cell cytotoxins (tirapazamine, RSU-1069) and less sensitive to damage by ionizing radiation. Hoechst-labelled cells also bound the hypoxia marker pimonidazole when given by i.p. injection. Intratumour injection transiently increased hypoxia from 18 to 70% in the tumour cells adjacent to the track of the needle. The half-time for return to pre-treatment oxygenation was about 30 min; oxygenation of tumour cells along the track had recovered by 20 h after intratumour injection. This effect could have significant implications for intratumour injection of drugs, cytokines or vectors that are affected by the oxygenation status of the tumour cells as well as potential effects on biodistribution via local microvasculature

Residual γH2AX foci as an indication of lethal DNA lesions

<p>Abstract</p> <p>Background</p> <p>Evidence suggests that tumor cells exposed to some DNA damaging agents are more likely to die if they retain microscopically visible γH2AX foci that are known to mark sites of double-strand breaks. This appears to be true even after exposure to the alkylating agent MNNG that does not cause direct double-strand breaks but does produce γH2AX foci when damaged DNA undergoes replication.</p> <p>Methods</p> <p>To examine this predictive ability further, SiHa human cervical carcinoma cells were exposed to 8 DNA damaging drugs (camptothecin, cisplatin, doxorubicin, etoposide, hydrogen peroxide, MNNG, temozolomide, and tirapazamine) and the fraction of cells that retained γH2AX foci 24 hours after a 30 or 60 min treatment was compared with the fraction of cells that lost clonogenicity. To determine if cells with residual repair foci are the cells that die, SiHa cervical cancer cells were stably transfected with a RAD51-GFP construct and live cell analysis was used to follow the fate of irradiated cells with RAD51-GFP foci.</p> <p>Results</p> <p>For all drugs regardless of their mechanism of interaction with DNA, close to a 1:1 correlation was observed between clonogenic surviving fraction and the fraction of cells that retained γH2AX foci 24 hours after treatment. Initial studies established that the fraction of cells that retained RAD51 foci after irradiation was similar to the fraction of cells that retained γH2AX foci and subsequently lost clonogenicity. Tracking individual irradiated live cells confirmed that SiHa cells with RAD51-GFP foci 24 hours after irradiation were more likely to die.</p> <p>Conclusion</p> <p>Retention of DNA damage-induced γH2AX foci appears to be indicative of lethal DNA damage so that it may be possible to predict tumor cell killing by a wide variety of DNA damaging agents simply by scoring the fraction of cells that retain γH2AX foci.</p

H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues

A sequence variant of histone H2A called H2AX is one of the key components of chromatin involved in DNA damage response induced by different genotoxic stresses. Phosphorylated H2AX (γH2AX) is rapidly concentrated in chromatin domains around DNA double-strand breaks (DSBs) after the action of ionizing radiation or chemical agents and at stalled replication forks during replication stress. γH2AX foci could be easily detected in cell nuclei using immunofluorescence microscopy that allows to use γH2AX as a quantitative marker of DSBs in various applications. H2AX is phosphorylated in situ by ATM, ATR, and DNA-PK kinases that have distinct roles in different pathways of DSB repair. The γH2AX serves as a docking site for the accumulation of DNA repair proteins, and after rejoining of DSBs, it is released from chromatin. The molecular mechanism of γH2AX dephosphorylation is not clear. It is complicated and requires the activity of different proteins including phosphatases and chromatin-remodeling complexes. In this review, we summarize recently published data concerning the mechanisms and kinetics of γH2AX loss in normal cells and tissues as well as in those deficient in ATM, DNA-PK, and DSB repair proteins activity. The results of the latest scientific research of the low-dose irradiation phenomenon are presented including the bystander effect and the adaptive response estimated by γH2AX detection in cells and tissues

CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.

G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016).This work was supported by the Canadian Breast Cancer Foundation BC/Yukon, BC Cancer Foundation, Stand Up to Cancer Canada (SU2C-AACR-DT-18-15), TFRI Grant 1021, CCSRI Grant 701584, CIHR Grant MOP-126119, Canada Foundation for Innovation and Cancer Research UK. Grant Brown lab is supported by CCSRI Impact Grant 702310 (to G.W.B.) and Ontario Government Scholarship (to B.H.). S.A. is supported by a Canada Research Chair in Molecular Oncology. The Balasubramanian lab is supported by a programme grant (C14303/A17197) and core funding (C14303/A17197) from Cancer Research UK

Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks

Mouse embryonic stem (mES) cells will give rise to all of the cells of the adult mouse, but they failed to rejoin half of the DNA double-strand breaks (dsb) produced by high doses of ionizing radiation. A deficiency in DNA-PKcs appears to be responsible since mES cells expressed <10% of the level of mouse embryo fibroblasts (MEFs) although Ku70/80 protein levels were higher than MEFs. However, the low level of DNA-PKcs found in wild-type cells appeared sufficient to allow rejoining of dsb after doses <20 Gy even in G1 phase cells. Inhibition of DNA-PKcs with wortmannin and NU7026 still sensitized mES cells to radiation confirming the importance of the residual DNA-PKcs at low doses. In contrast to wild-type cells, mES cells lacking H2AX, a histone protein involved in the DNA damage response, were radiosensitive but they rejoined double-strand breaks more rapidly. Consistent with more rapid dsb rejoining, H2AX¯/¯ mES cells also expressed 6 times more DNA-PKcs than wild-type mES cells. Similar results were obtained for ATM−/− mES cells. Differentiation of mES cells led to an increase inDNA-PKcs, an increase in dsb rejoining rate, and a decrease in Ku70/80. Unlike mouse ES, human ES cells were proficient in rejoining of dsb and expressed high levels of DNA-PKcs. These results confirm the importance of homologous recombination in the accurate repair of double-strand breaks in mES cells, they help explain the chromosome abnormalities associated with deficiencies in H2AX and ATM, and they add to the growing list of differences in the way rodent and human cells deal with DNA damage