Instytut Podstawowych Problemów Techniki

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Nibrin plays an important role in the DNA damage response (DDR) and DNA repair. DDR is a crucial signaling pathway in apoptosis and senescence. To verify whether truncated nibrin (p70), causing Nijmegen Breakage Syndrome (NBS), is involved in DDR and cell fate upon DNA damage, we used two (S4 and S3R) spontaneously immortalized T cell lines from NBS patients, with the founding mutation and a control cell line (L5). S4 and S3R cells have the same level of p70 nibrin, however p70 from S4 cells was able to form more complexes with ATM and BRCA1. Doxorubicin-induced DDR followed by cell senescence could only be observed in L5 and S4 cells, but not in the S3R ones. Furthermore the S3R cells only underwent cell death, but not senescence after doxorubicin treatment. In contrary to doxorubicin treatment, cells from all three cell lines were able to activate the DDR pathway after being exposed to γ-radiation. Downregulation of nibrin in normal human vascular smooth muscle cells (VSMCs) did not prevent the activation of DDR and induction of senescence. Our results indicate that a substantially reduced level of nibrin or its truncated p70 form is sufficient to induce DNA-damage dependent senescence in VSMCs and S4 cells, respectively. In doxorubicin-treated S3R cells DDR activation was severely impaired, thus preventing the induction of senescence.

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Curcumin, a phytochemical derived from the rhizome of Curcuma longa, is a very potent inducer of cancer cell death. It is believed that cancer cells are more sensitive to curcumin treatment than normal cells. Curcumin has been shown to act as a prooxidant and induce DNA lesions in normal cells. We were interested in whether curcumin induces DNA damage and the DNA damage response (DDR) signalling pathway leading to apoptosis in normal resting human T cells. To this end, we analysed DNA damage after curcumin treatment of resting human T cells (CD3+) and of proliferating leukaemic Jurkat cells by the fluorimetric detection of alkaline DNA unwinding (FADU) assay and immunocytochemical detection of γ-H2AX foci. We showed that curcumin-treated Jurkat cells and resting T cells showed neither DNA lesions nor did they activate key proteins in the DDR signalling pathway, such as phospho-ATM and phospho-p53. However, both types of cell were equally sensitive to curcumin-induced apoptosis and displayed activation of caspase-8 but not of DNA damage-dependent caspase-2. Altogether, our results revealed that curcumin can induce apoptosis of normal resting human T cells that is not connected with DNA damage.

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It is believed that normal cells with an unaffected DNA damage response (DDR) and DNA damage repair machinery, could be less prone to DNA damaging treatment than cancer cells. However, the anticancer drug, etoposide, which is a topoisomerase II inhibitor, can generate DNA double strand breaks affecting not only replication but also transcription and therefore can induce DNA damage in non-replicating cells. Indeed, we showed that etoposide could influence transcription and was able to activate DDR in resting human T cells by inducing phosphorylation of ATM and its substrates, H2AX and p53. This led to activation of PUMA, caspases and to apoptotic cell death. Lymphoblastoid leukemic Jurkat cells, as cycling cells, were more sensitive to etoposide considering the level of DNA damage, DDR and apoptosis. Next, we used ATM inhibitor, KU 55933, which has been shown previously to be a radio/chemo-sensitizing agent. Pretreatment of resting T cells with KU 55933 blocked phosphorylation of ATM, H2AX and p53, which, in turn, prevented PUMA expression, caspase activation and apoptosis. On the other hand, KU 55933 incremented apoptosis of Jurkat cells. However, etoposide-induced DNA damage in resting T cells was not influenced by KU 55933 as revealed by the FADU assay. Altogether our results show that KU 55933 blocks DDR and apoptosis induced by etoposide in normal resting T cells, but increased cytotoxic effect on proliferating leukemic Jurkat cells. We discuss the possible beneficial and adverse effects of drugs affecting the DDR in cancer cells that are currently in preclinical anticancer trials.

Słowa kluczowe:

FADU, γH2AX, DSBs, Caspases, KU 55933

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