Institute of Fundamental Technological Research
Polish Academy of Sciences

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Ewa Sikora

Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Supervision of doctoral theses
1.  2012-09-28 Korwek Zbigniew  
(IBD PAN)
Mechanizm śmierci i starzenia ludzkich limfocytów T – rola ścieżki sygnalizacyjnej odpowiedzi na uszkodzenia DNA (DDR) 

Recent publications
1.  Was H., Barszcz K., Czarnecka J., Kowalczyk A., Bernas T., Uzarowska E., Koza P., Klejman A., Piwocka K., Kaminska B., Sikora E., Bafilomycin A1 triggers proliferative potential of senescent cancer cells in vitro and in NOD/SCID mice, Oncotarget, ISSN: 1949-2553, DOI: 10.18632/oncotarget.14066, Vol.8, No.6, pp.9303-9322, 2017

Abstract:
Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls. Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.

Keywords:
colon cancer, chemotherapy, senescence, autophagy, angiogenesis

Affiliations:
Was H. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Barszcz K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Czarnecka J. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kowalczyk A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Bernas T. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Uzarowska E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Koza P. - other affiliation
Klejman A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Piwocka K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kaminska B. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Sikora E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
2.  Alster O., Bielak-Żmijewska A., Mosieniak G., Moreno-Villaneuva M., Dudka-Ruszkowska W., Wojtala A., Kusio-Kobiałka M., Korwek Z., Burkle A., Piwocka K., Siwicki J.K., Sikora E., The Role of Nibrin in Doxorubicin-Induced Apoptosis and Cell Senescence in Nijmegen Breakage Syndrome Patients Lymphocytes, PLOS ONE, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0104964, Vol.9, No.8, pp.e104964-1-13, 2014

Abstract:
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.

Affiliations:
Alster O. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Bielak-Żmijewska A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Mosieniak G. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Moreno-Villaneuva M. - University of Konstanz (DE)
Dudka-Ruszkowska W. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Wojtala A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Kusio-Kobiałka M. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Korwek Z. - other affiliation
Burkle A. - University of Konstanz (DE)
Piwocka K. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Siwicki J.K. - Institute of Oncology (PL)
Sikora E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
3.  Bielak-Żmijewska A., Wnuk M., Przybylska D., Grabowska W., Lewinska A., Alster O., Korwek Z., Cmoch A., Myszka A., Pikula S., Mosieniak G., Sikora E., A comparison of replicative senescence and doxorubicin-induced premature senescence of vascular smooth muscle cells isolated from human aorta, BIOGERONTOLOGY, ISSN: 1389-5729, DOI: 10.1007/s10522-013-9477-9, Vol.15, pp.47-64, 2014

Abstract:
Senescence of vascular smooth muscle cells (VSMCs) contributes to aging as well as age-related diseases of the cardiovascular system. Senescent VSMCs have been shown to be present in atherosclerotic plaques. Both replicative (RS) and stress-induced premature senescence (SIPS) accompany cardiovascular diseases. We aimed to establish the signature of RS and SIPS of VSMCs, induced by a common anticancer drug, doxorubicin, and to discover the so far undisclosed features of senescent cells that are potentially harmful to the organism. Most of the senescence hallmarks were common for both RS and SIPS; however, some differences were observed. 32 % of doxorubicin-treated cells were arrested in the G2/M phase of the cell cycle, while 73 % of replicatively senescing cells were arrested in the G1 phase. Moreover, on the basis of alkaline phosphatase activity measurements, we show that a 7-day treatment with doxorubicin (dox), does not cause precocious cell calcification, which is a characteristic feature of RS. We did not observe calcification even though after 7 days of dox-treatment many other markers characteristic for senescent cells were present. It can suggest that dox-induced SIPS does not accelerate the mineralization of vessels. We consider that detailed characterization of the two types of cellular senescence can be useful in in vitro studies of potential anti-aging factors.

Keywords:
Senescence, VSMCs, Doxorubicin, Aging, Cardiovascular diseases, Calcification

Affiliations:
Bielak-Żmijewska A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Wnuk M. - other affiliation
Przybylska D. - other affiliation
Grabowska W. - other affiliation
Lewinska A. - other affiliation
Alster O. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Korwek Z. - other affiliation
Cmoch A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Myszka A. - other affiliation
Pikula S. - other affiliation
Mosieniak G. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Sikora E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
4.  Korwek Z., Bielak-Żmijewska A., Mosieniak G., Alster O., Moreno-Villaneuva M., Burkle A., Sikora E., DNA damage-independent apoptosis induced by curcumin in normal resting human T cells and leukaemic Jurkat cells, MUTAGENESIS, ISSN: 0267-8357, DOI: 10.1093/mutage/get017, Vol.4, pp.1-6, 2013

Abstract:
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.

Affiliations:
Korwek Z. - other affiliation
Bielak-Żmijewska A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Mosieniak G. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Alster O. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Moreno-Villaneuva M. - University of Konstanz (DE)
Burkle A. - University of Konstanz (DE)
Sikora E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
5.  Korwek Z., Sewastianik T., Bielak-Żmijewska A., Mosieniak G., Alster O., Moreno-Villaneuva M., Burkle A., Sikora E., Inhibition of ATM blocks the etoposide-induced DNA damage response and apoptosis of resting human T cells, DNA REPAIR, ISSN: 1568-7864, DOI: 10.1016/j.dnarep.2012.08.006, Vol.11, pp.864-873, 2012

Abstract:
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.

Keywords:
FADU, γH2AX, DSBs, Caspases, KU 55933

Affiliations:
Korwek Z. - other affiliation
Sewastianik T. - other affiliation
Bielak-Żmijewska A. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Mosieniak G. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Alster O. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)
Moreno-Villaneuva M. - University of Konstanz (DE)
Burkle A. - University of Konstanz (DE)
Sikora E. - Nencki Institute of Experimental Biology, Polish Academy of Sciences (PL)

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