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Patients with chronic lymphocytic leukemia (CLL) with TP53 mutations with a >10% variant allele frequency (VAF) are often refractory to chemotherapy and benefit from targeted therapy. Malcikova and colleagues correlated TP53 mutations with, Key Points Low-burden TP53 mutations in CLL do not significantly affect the response duration to chemo- and/or immunotherapy, but shorten OS.Clonal expansion of low-burden TP53 mutations in CLL is associated with intense chemoimmunotherapy, but not with targeted therapy., Visual Abstract, Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with

The transcription factor c-Myb is an oncoprotein promoting cell proliferation and survival when aberrantly activated/expressed, thus contributing to malignant transformation. Overexpression of c-Myb has been found in leukemias, breast, colon and adenoid cystic carcinoma. Recent studies revealed its expression also in osteosarcoma cell lines and suggested its functional importance during bone development. However, the relevance of c-Myb in control of osteosarcoma progression remains unknown. A retrospective clinical study was carried out to assess a relationship between c-Myb expression in archival osteosarcoma tissues and prognosis in a cohort of high-grade osteosarcoma patients. In addition, MYB was depleted in metastatic osteosarcoma cell lines SAOS-2 LM5 and 143B and their growth, chemosensitivity, migration and metastatic activity were determined. Immunohistochemical analysis revealed that high c-Myb expression was significantly associated with poor overall survival in the cohort and metastatic progression in young patients. Increased level of c-Myb was detected in metastatic osteosarcoma cell lines and its depletion suppressed their growth, colony-forming capacity, migration and chemoresistance in vitro in a cell line-dependent manner. MYB knock-out resulted in reduced metastatic activity of both SAOS-2 LM5 and 143B cell lines in immunodeficient mice. Transcriptomic analysis revealed the c-Myb-driven functional programs enriched for genes involved in the regulation of cell growth, stress response, cell adhesion and cell differentiation/morphogenesis. Wnt signaling pathway was identified as c-Myb target in osteosarcoma cells. Taken together, we identified c-Myb as a negative prognostic factor in osteosarcoma and showed its involvement in the regulation of osteosarcoma cell growth, chemosensitivity, migration and metastatic activity.

Resistance to programmed cell death is one of the hallmarks of cancer cells that affects the process of malignant transformation as well as response to cancer therapy. The goal of this review is to summarize recent information about programmed cell death (PCD) in healthy and cancer cells, as well as new perspectives for anticancer treatments targeting these signaling pathways. Three main types of PCD are described in detail: apoptosis, necrosis/ necroptosis and cell death associated with autophagy. Among them, apoptosis plays the key role in both malignant transformation and response to therapy. In this review, we describe main signaling pathways and molecules participating in apoptosis regulation in healthy cells. In most cancer cells, mutations or aberrant expression of proteins directly or indirectly involved in induction and execution of cell death can be detected - p53, Bcl 2 family proteins, inhibitors of apoptosis, death receptors/ ligands and other proteins. Mutations or changes in expression of these proteins and their relation to certain types of tumors are described. Finally, we provide a review of recently developed treatments that target and reactivate the machinery of programmed cell death and are currently tested in clinical trials.

The Bcl-2 protein is one of the key components of biochemical pathways controlling programmed cell death. The function of this protein can be regulated by posttranslational modifications. Phosphorylation of Bcl-2 has been considered to be significantly associated with cell cycle arrest in the G2/M phase of the cell cycle, and with cell death caused by defects of microtubule dynamics. This study shows that phosphorylation of Bcl-2 can be induced by heavy metals due to activation of the Jun N-terminal kinase pathway that is not linked to the G2/M cell cycle arrest. Furthermore, we demonstrate that hyperphosphorylated Bcl-2 protein is a more potent inhibitor of zinc-induced cell death than its hypophosphorylated mutant form. These data suggest that regulation of Bcl-2 protein function by phosphorylation is an important part of cell responses to stress.

v-myb oncogene of avian myeloblastosis virus (AMV) transforms myelomonocytic cells in vitro and induces acute monoblastic leukemia in vivo. The transforming effect of the v-myb can be suppressed using phorbol ester (TPA) or histone deacetylase inhibitor trichostatin A (TSA), the inducers of cell differentiation that are in clinical trials. In this study, we used proteomics-based approach to identify proteins with variable expression in differentiated BM2 cells. Proteome variations induced by TPA and TSA were compared to examine the mechanism of differentiation-promoting effects of these drugs. We found that expression of several proteins participating in cell cytoskeleton rearrangement, heat shock response, proteosynthesis and cell signaling was altered in TPA- or TSA-treated cells. We present here the first comparative proteome analysis of v-myb-transformed monoblasts BM2 focused on identification of proteins involved in their terminal differentiation.

Cathepsin D (CD), a ubiquitously expressed lysosomal aspartic protease, is upregulated in human breast carcinoma and many other tumor types. CD has been repeatedly reported to act as key mediator of apoptosis induced by various chemotherapeutics. However, there is still controversy over the role of enzymatic/proteolytic versus protein-protein interaction activities of CD in apoptotic signaling. The elucidation of molecular mechanism responsible for the effect of CD in the chemotherapy-induced cell death is crucial for development of an appropriate strategy to target this protease in cancer treatment. Therefore, the objective of this study was to investigate the molecular mechanism behind the CD-mediated regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death. For this purpose, MDA-MB-231 breast carcinoma cells with an increased level of wt CD (CD) or mutant enzymatically inactive CD (ΔCD) were subjected to TRAIL and the frequency of apoptosis was determined. Our results show that CD facilitates the TRAIL-induced apoptosis of MDA-MB-231 breast cancer cells in enzymatic activity-dependent manner. Moreover, the importance of endosomal/lysosomal acidification in this process was documented. Analysis of the potential substrates specifically cleaved by CD during the TRAIL-induced apoptosis confirmed caspase-8 and Bid proteins as the CD targets. Moreover, in search for protein regulators of apoptosis that can be cleaved by CD at physiologically relevant pH, we identified the Bcl-2 protein as a suitable candidate. The modulatory role of CD in cell response to TRAIL was also confirmed in another breast cancer cell line SKBR3. These experiments identified the CD enzymatic activity as a new factor affecting sensitivity of breast cancer cells to TRAIL.

Apoptosis is type I programmed cell death, a process that is essential for development and tissue homeostasis. It is a prevalent form of cell death and it proceeds via two signaling pathways - external (receptor pathway) triggered by death receptors and intrinsic (mitochondrial) apoptotic pathway with major involvement of mitochondria. Mitochondria are important cellular organelles producing energy stored in molecules of adenosine triphosphate that are essential for cell survival. The mitochondrial cell death is characterized by permeabilization of the mitochondrial outer membrane and dissipation of the transmembrane potential. Mitochondria are electronegative organelles and depolarization of the mitochondrial membrane is important for the release of proapoptotic signals. Aberrant control of the mitochondrial cell death might contribute to several diseases including cancer. Mitochondria are also a source of reactive oxygen species, Ca2+ ions and other proteins that affect processes important for the initiation and progression of tumors independently of apoptosis. Current studies focus on research of mitochondrial membrane potential and reactive oxygen species modulating various signaling pathways within the cell, their importance in carcinogenesis, and in treatment of oncological patients. Monitoring of the apoptotic markers, such as the mitochondrial membrane potential (MMP), and the level of reactive oxygen species in samples of oncological patients has a predictive value for the output of treatment protocols.

Cytosine methylation in DNA is an epigenetic mechanism regulating gene expression and plays a vital role in cell differentiation or proliferation. Tumor cells often exhibit aberrant DNA methylation, e.g. hypermethylation of tumor suppressor gene promoters. New methods, capable of determining methylation status of specific DNA sequences, are thus being developed. Among them, MS-HRM (methylation-specific high resolution melting) and electrochemistry offer relatively inexpensive instrumentation, fast assay times and possibility of screening multiple samples/DNA regions simultaneously. MS-HRM is due to its sensitivity and simplicity an interesting alternative to already established techniques, including methylation-specific PCR or bisulfite sequencing. Electrochemistry, when combined with suitable electroactive labels and electrode surfaces, has been applied in several unique strategies for discrimination of cytosines and methylcytosines. Both techniques were successfully tested in analysis of DNA methylation within promoters of important tumor suppressor genes and could thus help in achieving more precise diagnostics and prognostics of cancer. Aberrant methylation of promoters has already been described in hundreds of genes associated with tumorigenesis and could serve as important biomarker if new methods applicable into clinical practice are sufficiently advanced.Key words: DNA methylation - 5-methylcytosine - HRM analysis - melting temperature - DNA duplex - electrochemistry - nucleic acid hybridizationThis work was supported by MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 16. 5. 2016.

Loss of programmed cell death pathways is one of the features of malignancy that complicate the response of cancer cells to a therapy. Activation of alternative cell death pathways offers a promising approach to enhance efficiency of cancer chemotherapy. We analysed programmed cell death pathways of v-myb-transformed BM2 monoblasts induced by arsenic trioxide, cycloheximide and camptothecin with U937 promonocytes as a reference cell line. We show that induced death of BM2 cells is not executed by caspases but rather by alternative cell death pathways. Camptothecin induces the lysosome-dependent cell death, arsenic trioxide induces autophagy, and most of cycloheximide-treated BM2 cells die by necrosis. The fact that alternative cell death pathways can be switched in cells with defects in activation and/or function of caspases suggests that understanding and targeting of these pathways could improve therapy of cancer cells suffering from defective apoptosis.