DNA damage-independent apoptosis induced by curcumin in normal resting human T cells and leukaemic Jurkat cells

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

Inhibition of ATM blocks the etoposide-induced DNA damage response and apoptosis of resting human T cells

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

The role of nibrin in Doxorubicin-induced apoptosis and cell senescence in nijmegen breakage syndrome patients lymphocytes

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

FADU analysis in L5, S3R and S4 cells treated with doxorubicin.

<p>The percentage of double-stranded DNA, was measured in all of the cell lines using the FADU method. All of the cell lines were treated with 1 and 10 µM doxorubicin and the measurements were performed 30, 60 and 90 min after treatment with this agent. Data is calculated as the percentage of control. The values are means ± SD obtained from three independent experiments. Statistical significance was estimated using the Student’s T test.</p

The role of nibrin in doxorubicin-induced senescence of human Vascular Smooth Muscle Cells (VSMCs).

<p>Cells were transfected with negative siRNA (−) or <i>NBN</i> siRNA (+) and afterwards cultured for three days in the presence of doxorubicin (100 nM). <b>A.</b> Downregulation of the NBS1 protein level in VSMCs using specific siRNA (60 nM). Whole cell extracts were prepared at indicated time points after treatment with doxorubicin. Expression of the indicated proteins was estimated by Western blotting, β-actin was used as a loading control. The amount of the protein in cells transfected with <i>NBN</i> siRNA was calculated by densitometry as a fraction of that present in cells transfected with negative siRNA (1). <b>B.</b> 53BP1 staining in doxorubicin-treated control cells and cells with silenced nibrin. Representative images from one of three independent experiments. Magnification 100x. <b>C.</b> 53BP1 staining in doxorubicin treated control cells and cells with silenced nibrin. Cells with DNA damage were divided into four groups based on the number of 53BP1 foci: cells without 53BP1 foci, with one focus, with 2–5 foci, with more than 5 foci. Means from three independent experiments. <b>D.</b> SA-β-Gal activity in doxorubicin treated VSMC cells. Representative images from one of three independent experiments, magnification 100x. <b>E.</b> The percentage of SA-β-Gal positive cells (a mean ± SD) from three independent experiments. <b>F.</b> BrdU incorporation assay. Control cells and cells transfected with negative siRNA or <i>NBN</i> si RNA were cultured with BrdU for 24 h. Data presented as means ± SD from three independent experiments.</p

Levels of nibrin, p70-nibrin, MRE11, ATM and BRCA1 in the DDR complex estimated by immunoprecipitation assay.

<p><b>A.</b> Level of nibrin: wild-type (p95) and the truncated form (p70) in control (C) and doxorubicin treated (D, 1 µM/1 h) S3R, S4 and VSMCs. Expression of nibrin was analyzed by immunoprecipitation using an anti-NBS1 antibody followed by Western blotting with anti-NBS1 (upper panel). Alternatively, IP using anti-MRE11 antibody was performed followed by WB with anti-NBS1 (lower panel). MRE11 was used as a loading control. The last lane (C IP) shows the negative IP control. Note that p95 is only present in VSMCs, in which there is no p70-nibrin. <b>B.</b> ATM binding to nibrin in control and doxorubicin-treated S3R and S4 cells analyzed by immunoprecipitation using anti-NBS1 antibody (upper panel) or anti-ATM antibody (lower panel). Levels of ATM and p70 were detected by WB. Loading controls were performed in both variants of IP. <b>C.</b> Expression of BRCA1 in control and dox-treated S3R and S4 cells was analyzed by immunoprecipitation using anti-ATM antibody followed by WB using an anti-BRCA1 antibody. Loading and negative IP controls were performed as above.</p

Dose-dependent influence of doxorubicin on cell cycle arrest, apoptosis and activation of the DNA damage response (DDR).

<p><b>A.</b> DNA content, analyzed by flow cytometry, 24 h after treatment with different concentrations of doxorubicin (0–250 nM). Representative histograms from one of three independent experiments. <b>B.</b> Concentration-dependent apoptosis measured 24 h after treatment with doxorubicin (0–250 nM). The percentage of apoptotic cells was estimated by the Annexin V/7-AAD flow cytometry assay in three independent experiments. The bars show means ± SD values. Data was analyzed using the CellQuest software. Statistical significance was estimated using the Student’s T test. <b>C.</b> Expression of the DDR proteins analyzed by Western blotting in control (C) and treated with doxorubicin (D) S3R, S4 and L5 cells. Whole cell extracts were prepared 24 h after cell treatment with the following cytostatic concentrations of dox: 50 nM (L5), 10 nM (S3R), 100 nM (S4) and β-actin was used as a loading control.</p

The induction of cellular senescence and apoptosis upon doxorubicin treatment.

<p><b>A.</b> SA-β-Gal activity. Cells were treated for 1 day with doxorubicin (L5 - 50 nM, S3R - 10 nM and S4 - 100 nM) and then cultured in doxorubicin-free medium (1+n; n- are days of culture without doxorubicin). The bars show means ± SD. The percentage of SA-β-Gal positive cells from at least three independent experiments and representative images from one of three independent experiments. <b>B.</b> Magnification 200x. <b>C.</b> Expression of protein markers of senescence (p-p53, p53, p21 and p16) analyzed by Western blotting in untreated (0) cells and on the following days after culturing in doxorubicin-free medium (1+n), β-actin was used as a loading control. <b>D.</b> The percentage of apoptotic cells after treatment with doxorubicin estimated by the AnnexinV/7-AAD flow cytometry assay. The bars show means ± SD values. Data were obtained from three independent experiments. Statistical significance was estimated using the Student’s T test.</p