The PAI-1 swing: microenvironment and cancer cell migration

Cancer is a complex and dynamic process caused by a cellular dysfunction leading to a whole organ or even organism vital perturbation. To better understand this process, we need to study each one of the levels involved, which allows the scale change, and to integrate this knowledge. A matricellular protein, PAI-1, is able to induce in vitro cell behaviour modifications, morphological changes, and to promote cell migration. PAI-1 influences the mesenchymo-amaeboid transition. This matricellular protein should be considered as a potential 'launcher' of the metastatic process acting at the molecular, cellular, tissular levels and, as a consequence, at the organism's level.     

Enhanced Anti-tumor Activity by the Combination of the Natural Compounds (?)-Epigallocatechin-3-gallate and Luteolin: POTENTIAL ROLE OF p53*

Natural dietary agents have drawn a great deal of attention toward cancer prevention because of their wide safety margin. However, single agent intervention has failed to bring the expected outcome in clinical trials; therefore, combinations of chemopreventive agents are gaining increasingly popularity. In the present study, we investigated a combinatorial approach using two natural dietary polyphenols, luteolin and EGCG, and found that their combination at low doses (at which single agents induce minimal apoptosis) synergistically increased apoptosis (3–5-fold more than the additive level of apoptosis) in both head and neck and lung cancer cell lines. This combination also significantly inhibited growth of xenografted tumors in nude mice. The in vivo findings also were supported by significant inhibition of Ki-67 expression and increase in TUNEL-positive cells in xenografted tissues. Mechanistic studies revealed that the combination induced mitochondria-dependent apoptosis in some cell lines and mitochondria-independent apoptosis in others. Moreover, we found more efficient stabilization and ATM-dependent Ser¹⁵ phosphorylation of p53 due to DNA damage by the combination, and ablation of p53 using shRNA strongly inhibited apoptosis as evidenced by decreased poly(ADP-ribose) polymerase and caspase-3 cleavage. In addition, we observed mitochondrial translocation of p53 after treatment with luteolin or the combination of EGCG and luteolin. Taken together, our results for the first time suggest that the combination of luteolin and EGCG has synergistic/additive growth inhibitory effects and provides an important rationale for future chemoprevention trials of head and neck and lung cancers.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

Calcium-dependent Conformational Changes in Inositol Trisphosphate Receptors

We have used limited trypsin digestion and reactivity with PEG-maleimides (MPEG) to study Ca²⁺-induced conformational changes of IP₃Rs in their native membrane environment. We found that Ca²⁺ decreased the formation of the 95-kDa C-terminal tryptic fragment when detected by an Ab directed at a C-terminal epitope (CT-1) but not with an Ab recognizing a protected intraluminal epitope. This suggests that Ca²⁺ induces a conformational change in the IP₃R that allows trypsin to cleave the C-terminal epitope. Half-maximal effects of Ca²⁺ were observed at ∼0.5 μm and was sensitive to inhibition by IP₃. Ca²⁺ also stimulated the reaction of MPEG-5 with an endogenous thiol in the 95-kDa fragment. This effect was eliminated when six closely spaced cysteine residues proximal to the transmembrane domains were mutated (C2000S, C2008S, C2010S, C2043S, C2047S, and C2053S) or when the N-terminal suppressor domain (amino acids 1–225) was deleted. A cysteine substitution mutant introduced at the C-terminal residue (A2749C) was freely accessible to MPEG-5 or MPEG-20 in the absence of Ca²⁺. However, cysteine substitution mutants in the interior of the tail were poorly reactive with MPEG-5, although reactivity was enhanced by Ca²⁺. We conclude the following: a) that large conformational changes induced by Ca²⁺ can be detected in IP₃Rs in situ; b) these changes may be driven by Ca²⁺ binding to the N-terminal suppressor domain and expose a group of closely spaced endogenous thiols in the channel domain; and c) that the C-terminal cytosol-exposed tail of the IP₃R may be relatively inaccessible to regulatory proteins unless Ca²⁺ is present.     

Dimerization Is Essential for 14-3-3�� Stability and Function in Vivo

Members of the conserved 14-3-3 protein family spontaneously self-assemble as homo- and heterodimers via conserved sequences in the first four (αA-αD) of the nine helices that comprise them. Dimeric 14-3-3s bind conserved motifs in diverse protein targets involved in multiple essential cellular processes including signaling, intracellular trafficking, cell cycle regulation, and modulation of enzymatic activities. However, recent mostly in vitro evidence has emerged, suggesting functional and regulatory roles for monomeric 14-3-3s. We capitalized on the simplicity of the 14-3-3 family in Drosophila to investigate in vivo 14-3-3ζ monomer properties and functionality. We report that dimerization is essential for the stability and function of 14-3-3ζ in neurons. Moreover, we reveal the contribution of conserved amino acids in helices A and D to homo- and heterodimerization and their functional consequences on the viability of animals devoid of endogenous 14-3-3ζ. Finally, we present evidence suggesting endogenous homeostatic adjustment of the levels of the second family member in Drosophila, D14-3-3ϵ, to transgenic monomeric and dimerization-competent 14-3-3ζ.     

Correlation between bacterial indicators and bacteriophages in sewage and sludge

The use of bacteriophages as potential indicators of faecal pollution has recently been studied. The correlation of the number of bacterial indicators and the presence of three groups of bacteriophages, namely somatic coliphages (SOMCPH), F-RNA-specific phages (FRNAPH) and phages of Bacteroides fragilis (BFRPH), in raw and treated wastewater and sludge is presented in this study. Raw and treated wastewater and sewage sludge samples from two wastewater treatment plants in Athens were collected on a monthly basis, over a 2-year period, and analysed for total coliforms, Escherichia coli, intestinal enterococci and the three groups of bacteriophages. A clear correlation between the number of bacterial indicators and the presence of bacteriophages was observed. SOMCPH may be used as additional indicators, because of their high densities and resistance to various treatment steps.