Alex Lyakhovich

Histone H2AX and Fanconi anemia FANCD2 function in the same pathway to maintain chromosome stability

Fanconi anemia (FA) is a chromosome fragility syndrome characterized by bone marrow failure and cancer susceptibility. The central FA protein FANCD2 is known to relocate to chromatin upon DNA damage in a poorly understood process. Here, we have induced subnuclear accumulation of DNA damage to prove that histone H2AX is a novel component of the FA/BRCA pathway in response to stalled replication forks. Analyses of cells from H2AX knockout mice or expressing a nonphosphorylable H2AX (H2AXS136A/S139A) indicate that phosphorylated H2AX (γH2AX) is required for recruiting FANCD2 to chromatin at stalled replication forks. FANCD2 binding to γH2AX is BRCA1‐dependent and cells deficient or depleted of H2AX show an FA‐like phenotype, including an excess of chromatid‐type chromosomal aberrations and hypersensitivity to MMC. This MMC hypersensitivity of H2AX‐deficient cells is not further increased by depleting FANCD2, indicating that H2AX and FANCD2 function in the same pathway in response to DNA damage‐induced replication blockage. Consequently, histone H2AX is functionally connected to the FA/BRCA pathway to resolve stalled replication forks and prevent chromosome instability.

New roads to FA/BRCA pathway: H2AX.

We have recently described an involvement of H2AX into the Fanconi anemia (FA) BRCA pathway through recruitment of FA protein FANCD2 to the sites of stalled replication forks. We showed that BRCA1 mediates the recruitment of FANCD2 by γH2AX to damaged chromatin and cells deficient or depleted of H2AX exhibit an FA-like phenotype, including an excess of chromatid-type chromosomal aberrations and hypersensitivity to MMC. Here, we discuss a model for the FA pathway and how it could partially explain the common phenotypes of H2AX, BRCA2 and FA deficiencies.

Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells

Fanconi anemia (FA) is a rare syndrome characterized by developmental abnormalities, progressive bone marrow failure, and cancer predisposition. Cells from FA patients exhibit hypersensitivity to DNA cross-linking agents and oxidative stress that may trigger apoptosis. Damage-induced activation of caspases and poly ADP ribose polymerase (PARP) enzymes have been described for some of the FA complementation groups. Here, we show the constitutive activation of caspase-3 and PARP cleavage in the FA cells without exposure to exogenous DNA-damaging factors. These effects can be reversed in the presence of reactive oxygen species scavenger N-acetylcystein. We also show the accumulation of oxidized proteins in FA cells, which is accompanied by the tumor necrosis factor (TNF)-α oversecretion and the upregulation of early stress response kinases pERK1/2 and p-P38. Suppression of TNF-α secretion by the extracellular signal-regulated kinase inhibitor PD98059 results in reduction of caspase-3 cleavage, suggesting a possible mechanism of caspases-3 activation in FA cells. Thus, the current study is the first evidence demonstrating the damage-independent activation of caspase-3 and PARP in FA cells, which seems to occur through mitogen-activated protein kinase activation and TNF-α oversecretion.Mol Cancer Res; 8(1); 46–56

Mitochondrial dysfunction and potential anticancer therapy

Mitochondrial dysfunction (MDF) has been identified as an important factor in various diseases ranging from neurological disorders, to diseases of the cardiovascular system and metabolic syndromes. MDF was also found in cancer as well as in cancer predisposition syndromes with defective DNA damage response (DDR) machinery. Moreover, a recent highlight arises from the detection of MDF in eukaryotic cells upon treatment with antibiotics. In this review, we focus on recent studies of MDF in pathological conditions with a particular emphasis on the effects of various classes of antibiotics on mitochondria. Special attention is given to the role of autophagy/mitophagy in MDF and repurposing antibiotics as anticancer drugs.

The interplay between autophagy and tumorigenesis: exploiting autophagy as a means of anticancer therapy.

In wild‐type cells, autophagy represents a tumour‐suppressor mechanism, and dysfunction of the autophagy machinery increases genomic instability, DNA damage, oxidative stress and stem/progenitor expansion, which are events associated with cancer onset. Autophagy occurs at a basal level in all cells depending on cell type and cellular microenvironment. However, the role of autophagy in cancer is diverse and can promote different outcomes even in a single tumour. For example, in hypoxic tumour regions, autophagy emerges as a protective mechanism and allows cancer cell survival. By contrast, in cancer cells surrounding the tumour mass, the induction of autophagy by radio‐ or chemotherapy promotes cell death and significantly reduces the tumour mass. Importantly, inhibition of autophagy compromises tumorigenesis by mechanisms that are not entirely understood. The aim of this review is to explain the apparently contradictory role of autophagy as a mechanism that both promotes and inhibits tumorigenesis using different models. The induction/inhibition of autophagy as a mechanism for cancer treatment is also discussed.

Targeting cancer cells through antibiotics-induced mitochondrial dysfunction requires autophagy inhibition

A significant part of current research studies utilizes various cellular models which imply specific antibiotics-containing media as well as antibiotics used for clonal selection or promoter de/activation. With the great success of developing such tools, mitochondria, once originated from bacteria, can be effectively targeted by antibiotics. For that reason, some studies propose antibiotics-targeting of mitochondria as part of anticancer therapy. Here, we have focused on the effects of various classes of antibiotics on mitochondria in cancer and non-cancer cells and demonlow mitochondrial membrane potential, reduced ATP production, altered morphology and lowered respiration rate which altogether suggested mitochondrial dysfunction (MDF). This was in parallel with increased level of reactive oxygen species (ROS) and decreased activity of mitochondrial respiration complexes. However, both survival and repopulation capacity of cancer cells was not significantly affected by the antibiotics, perhaps due to a glycolytic shift or activated autophagy. In turn, simultaneous inhibition of autophagy and treatment with antibiotics largely reduced tumorigenic properties of cancer cells suggesting potential strategy for anticancer therapy.

Quick two-dimensional differential in gel electrophoresis-based method to determine length and secondary structures of telomeric DNA

A novel differential in gel electrophoresis (DIGE)-based method has been developed and applied to measure telomere length and appearance of two-dimensional structural DNA changes. It can be applied to any area requiring quick and evident measurement of structural DNA changes.