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1.  BRCA1/2 mutations perturb telomere biology: characterization of structural and functional abnormalities in vitro and in vivo 
Oncotarget  2015;7(3):2433-2454.
BRCA1 mutation is associated with carcinogenesis, especially of breast tissue. Telomere maintenance is crucial for malignant transformation. Being a part of the DNA repair machinery, BRCA1 may be implicated in telomere biology. We explored the role of BRCA1 in telomere maintenance in lymphocytes of BRCA1/2 mutation carriers and in in vitro system by knocking down its expression in non-malignant breast epithelial cells.
The results in both systems were similar. BRCA1/2 mutation caused perturbation of telomere homeostasis, shortening of the single stranded telomere overhang and increased the intercellular telomere length variability as well as the number of telomere free chromosomal ends and telomeric circles. These changes resulted in an increased DNA damage status. Telomerase activity, inducibility and expression remained unchanged. BRCA1 mutation resulted also in changes in the binding of shelterin proteins to telomeres. DNMT-1 levels were markedly reduced both in the carriers and in in vitro system. The methylation pattern of the sub-telomeric regions in carriers suggested hypomethylation in chromosome 10. The expression of a distinct set of genes was also changed, some of which may relate to pre-disposition to malignancy.
These results show that BRCA gene products have a role in telomere length homeostasis. It is plausible that these perturbations contribute to malignant transformation in BRCA mutants.
PMCID: PMC4823046  PMID: 26515461
telomeres; BRCA1/2; malignant transformation; telomere homeostasis
2.  Proteomic and microRNA data clarifying the effects of telomere shortening on cancer cells 
Data in Brief  2014;2:48-51.
In a previous study, we have shown that shortening of telomeres by telomerase inhibition sensitized cancer cells to cisplatinum, slower their migration, increased DNA damage and impaired DNA repair [1]. In the following study, we present a network model combining microRNA and proteomic profiling attempting to decipher the molecular mechanism underlying the effect of shortened telomeres on the obtained phenotype of cancer cells [2]. The microRNA and proteomic data were used for a network model construction, which provided us with several nodal candidates that may potentially mediate the shortened-telomeres dependent features. These protein expressions were experimentally validated, supporting their potential central role in this system [2]. In this article, we delineate the full proteomic data and a microarray analyses performed on cells with shortened telomeres compared to their cognate parental intact telomere cells. The data is attached as excel files. In principle, clarifying the mechanism behind telomere shortened phenotype may facilitate novel therapeutics development and may also obviate the time consuming process of telomere shortening achieved by telomerase inhibition.
PMCID: PMC4459769  PMID: 26217705
3.  The effect of Bortezomib and Rapamycin on Telomerase Activity in Mantle Cell Lymphoma 
Translational Oncology  2014;7(6):741-751.
Mantle cell lymphoma (MCL) is a hematological malignancy with unfavorable prognosis. Novel therapeutic approaches for treating the disease are aimed at the mechanisms regulating growth signals, cellular proliferation, and survival pathways of the malignant clones. Bortezomib (Brt), a proteasome inhibitor with pleiotropic activities was shown to be active in MCL and is currently implemented in therapeutic combinations for this disease. Telomerase activity is essential for survival of malignant cells and as such is considered a valid therapeutic target. This study evaluated the effects of bortezomib on telomerase activity and its regulation in MCL cells in vitro and ex vivo.
Our study shows that bortezomib exerts a cytotoxic effect in a dose dependent manner in two MCL cell lines, with differential sensitivity. While the IC50 for HBL-2 cells ranged between 2.5 ng/ml to 1.5 ng/ml during 24-72 h respectively, the IC50 for the NCEB cells was twice. Bortezomib differentially inhibited telomerase activity (TA): in HBL-2 cells there was a decline of 20%-55% during 24-72 h respectively. However in NCEB cells the decline was much smaller, and did not exceed 25%. Inhibition of telomerase activity is shown to be operated by two separate mechanisms: reduction of the hTERT mRNA expression (controlled by the binding of transcription factors) and reduction in phosphorylation of the catalytic subunit of hTERT by its kinases, AKT and PKCα.
A decrease in telomerase activity was demonstrated also in mononuclear cells, isolated from three MCL patients following incubation of the cells in the presence of bortezomib for 24-72 h. In one patient the decrease in TA ranged between 17%-37% respectively, in the second patient between 63%-76% and in the third patient between 70-100% for 24-72 h respectively.
The current study indicates that a combination of bortezomib and rapamycin, (an m-Tor pathway inhibitor used in MCL treatment) induced synergistic inhibition of telomerase activity. In HBL-2 cells, the combined treatment of bortezomib and rapamycin decreased TA by 80% compared to the expected value (40%) and for NCEB cells a similar trend was observed. In contrast, there was neither additive nor synergistic effect of this combination on cell proliferation.
In the light of the crucial role of telomerase in cancer cells, it was important to characterize the possible relations between telomerase and bortezomib and to distinguish the biochemical mechanisms of its regulation and its interactions with other signal transduction inhibitors such as rapamycin. The results of this work encourage the in vivo examination of the therapeutic potential of the combination of bortezomib and rapamycin in Mantle Cell Lymphoma patients.
PMCID: PMC4311042  PMID: 25500084
4.  Rituximab associated neutropenia: Description of three cases and an insight into the underlying pathogenesis 
To describe Rituximab associated neutropenia (RAN), and to explore its underlying mechanism.
Case Report
We describe three patients with RAN. The effect of patient’s plasma on colony forming unit, Granulocyte-Monocyte (CFU-GM) was measured by the addition of plasma to the culture of a healthy bone-marrow. Repeated tests were performed after recovery of white count.
In the leukopenic period the patient’s plasma inhibited CFU growth completely. Control plasma did not have such an effect. Addition of patient’s cell supernatant to bone marrow cells did not change the number of CFU. The same effect was demonstrated in normal control. After recovery the patient’s plasma did not inhibit colony formation, similar to control.
RAN is a clinically significant side effect. It may take place during treatment or several months afterwards. Circulating antibodies in the plasma may be responsible for this unique BM toxicity.
PMCID: PMC3539507  PMID: 22037749
rituximab; neutropenia; bone marrow; pathogenesis
5.  Telomere Shortening Sensitizes Cancer Cells to Selected Cytotoxic Agents: In Vitro and In Vivo Studies and Putative Mechanisms 
PLoS ONE  2010;5(2):e9132.
Telomere/telomerase system has been recently recognized as an attractive target for anticancer therapy. Telomerase inhibition results in tumor regression and increased sensitivity to various cytotoxic drugs. However, it has not been fully established yet whether the mediator of these effects is telomerase inhibition per se or telomere shortening resulting from inhibition of telomerase activity. In addition, the characteristics and mechanisms of sensitization to cytotoxic drugs caused by telomerase inhibition has not been elucidated in a systematic manner.
Methodology/Principal Findings
In this study we characterized the relative importance of telomerase inhibition versus telomere shortening in cancer cells. Sensitization of cancer cells to cytotoxic drugs was achieved by telomere shortening in a length dependent manner and not by telomerase inhibition per se. In our system this sensitization was related to the mechanism of action of the cytotoxic drug. In addition, telomere shortening affected also other cancer cell functions such as migration. Telomere shortening induced DNA damage whose repair was impaired after administration of cisplatinum while doxorubicin or vincristine did not affect the DNA repair. These findings were verified also in in vivo mouse model. The putative explanation underlying the phenotype induced by telomere shortening may be related to changes in expression of various microRNAs triggered by telomere shortening.
To our best knowledge this is the first study characterizing the relative impact of telomerase inhibition and telomere shortening on several aspects of cancer cell phenotype, especially related to sensitivity to cytotoxic drugs and its putative mechanisms. The microRNA changes in cancer cells upon telomere shortening are novel information. These findings may facilitate the development of telomere based approaches in treatment of cancer.
PMCID: PMC2817744  PMID: 20161752
6.  Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function 
Journal of Clinical Investigation  2000;106(11):1331-1339.
The chemokine stromal-derived factor-1 (SDF-1) controls many aspects of stem cell function. Details of its regulation and sites of production are currently unknown. We report that in the bone marrow, SDF-1 is produced mainly by immature osteoblasts and endothelial cells. Conditioning with DNA-damaging agents (ionizing irradiation, cyclophosphamide, and 5-fluorouracil) caused an increase in SDF-1 expression and in CXCR4-dependent homing and repopulation by human stem cells transplanted into NOD/SCID mice. Our findings suggest that immature osteoblasts and endothelial cells control stem cell homing, retention, and repopulation by secreting SDF-1, which also participates in host defense responses to DNA damage.
PMCID: PMC381461  PMID: 11104786

Results 1-6 (6)