Investigation of INDEL variants in apoptosis: the relevance to gastric cancer.

BACKGROUND: Apoptosis is a type of cell death involved in different pathways inherent to the cell and the evasion from this mechanism has been related to cancer, although this process remains not very well comprehended. Gastric cancer (GC) is one of the most incident and aggressive types of cancer worldwide. In this study, we analyzed the distribution of INDEL variants in GC patients (Case) and individuals from the general population (Control) from the Amazon region, in which GC is remarkably frequent. METHODS: A panel of nine INDEL markers in apoptosis-related genes (BCL2 rs11269260, CASP3 rs4647655, CASP8 rs3834129 and rs59308963, CASP9 rs4645982 and rs61079693, FADD rs4197, FAS rs10562972 and TP53 rs17880560) was developed and genotyped by multiplex PCR in both groups. RESULTS: In our analyses, only marker rs4197 (FADD gene) was associated to GC development as follows: INS/DEL genotype of rs4197 increasing in about 2-fold the chances of developing this type of cancer (P = 0.046; OR = 1.940; 95%CI = 1.011-3.725). CONCLUSION: Our results suggest that rs4197 (FADD gene) might play a role in gastric carcinogenesis in the investigated population. More studies are needed to clarify this relation. Here, we highlight the importance of investigating INDEL variants in genes involved in apoptosis.

Caspase-8 and FADD: Where Cell Death and Inflammation Collide.

Caspase-8 is a master regulator of cell death pathways, although its regulation during inflammation remains elusive. Using elegant mouse genetic approaches, Schwarzer et al. and Tummers et al. revealed the importance of FADD in regulating caspase-8-mediated inflammatory responses and gut homeostasis.

DNA methylation profiles of immune response-related genes in apical periodontitis.

AIM: To investigate the DNA methylation profiles of immune response-related genes in apical periodontitis (AP) lesions. METHODOLOGY: The methylation profiles on the cytosine-phosphate-guanine (CpG) regions of 22 gene promoters involved in inflammation and autoimmunity were assessed in 60 human AP lesions and 24 healthy periodontal ligaments (controls) using a pathway-specific real-time polymerase chain reaction array (EpiTect® Methyl Signature PCR Array Human Inflammatory Response). Differentially methylated genes were subsequently assessed for their mRNA expression. Data analyses (One-way anova, Tukey's multiple comparisons tests and Mann-Whitney tests) were performed using GraphPad Prism 6 software. P values ≤ 0.05 were considered statistically significant. RESULTS: Significant DNA hypermethylation was observed for CXCL3 and FADD gene promoters in AP lesions when compared to control tissues (P < 0.001) and among other genes (P < 0.05). In contrast, IL12B and IL4R were associated with significant hypomethylation in comparison to other genes (P < 0.05). IL12B, IL4R, CXCL3 and FADD had differential mRNA expression in AP lesions and controls (P < 0.001). CONCLUSIONS: Differential methylation profiles of immune response-related genes, such as FADD, CXCL3, IL12B and IL4R, may have an influence on individual AP susceptibility and patient treatment outcomes, through their potential contributions to altered expression of disease-relevant genes. Methylation and/or genetic variations in additional genes may also contribute to the dynamics of AP development and should be considered in future studies.

Copy number and expression analysis of FOSL1, GSTP1, NTSR1, FADD and CCND1 genes in primary breast tumors with axillary lymph node metastasis.

In breast cancer, lymph node (LN) metastasis is one of the strongest prognostic factors at diagnosis. Therefore the identification of molecular markers with metastatic potential that promote the development of LN metastasis is of critical clinical relevance. In this study, we evaluated the copy number status of the FOSL1, GSTP1, NTSR1, FADD and CCND1 genes by TaqMan assays in 137 breast cancer patients, 84 with LN metastasis (LN+) and 53 with no LN metastasis (LN-). The copy number data for four of these genes (FOSL1, GSTP1, FADD and CCND1) were integrated with their mRNA expression levels in 31 patients. In both groups of patients, gains were the most frequent copy number alteration (CNA) observed, involving mainly the CCND1, NTSR1 and FADD genes; mRNA overexpression was more commonly observed for the CCND1 and FADD genes. For the FADD gene in the LN+ group, gene expression was shown to be dependent on CNAs; for the other genes no association was found. In conclusion, increase copy number and mRNA overexpression of FOSL1, GSTP1, FADD, NTSR1 and CCND1 genes are frequently observed in primary breast tumors, and except for the FADD gene, they occur independently and irrespectively of the patients' LN axillary metastatic status.

Necroptotic Cell Death Signaling and Execution Pathway: Lessons from Knockout Mice.

Under stress conditions, cells in living tissue die by apoptosis or necrosis depending on the activation of the key molecules within a dying cell that either transduce cell survival or death signals that actively destroy the sentenced cell. Multiple extracellular (pH, heat, oxidants, and detergents) or intracellular (DNA damage and Ca(2+) overload) stress conditions trigger various types of the nuclear, endoplasmic reticulum (ER), cytoplasmatic, and mitochondrion-centered signaling events that allow cells to preserve the DNA integrity, protein folding, energetic, ionic and redox homeostasis, thus escaping from injury. Along the transition from reversible to irreversible injury, death signaling is highly heterogeneous and damaged cells may engage autophagy, apoptotic, or necrotic cell death programs. Studies on multiple double- and triple- knockout mice identified caspase-8, flip, and fadd genes as key regulators of embryonic lethality and inflammation. Caspase-8 has a critical role in pro- and antinecrotic signaling pathways leading to the activation of receptor interacting protein kinase 1 (RIPK1), RIPK3, and the mixed kinase domain-like (MLKL) for a convergent execution pathway of necroptosis or regulated necrosis. Here we outline the recent discoveries into how the necrotic cell death execution pathway is engaged in many physiological and pathological outcome based on genetic analysis of knockout mice.

Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS gene.

We present a case of autoimmune lymphoproliferative syndrome (ALPS) caused by a previously undescribed minimal deletion in the death domain of the FAS gene. ALPS is an uncommon disease associated with an impaired Fas-mediated apoptosis. The patient presented with a history of splenomegaly since 4 months of age, associated with cervical lymphadenopathy, which improved with oral corticosteroid treatment. Relevant laboratory findings were the presence of anemia, thrombocytopenia, and positive direct and indirect Coombs tests. He was not an offspring of consanguineous parents. Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. In both lymph nodes, there was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation and a very high cell proliferating index. Some clear cells were also present, raising the suspicion of malignant lymphoma. In one of the lymph nodes, there was also a focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease. Immunostaining showed numerous CD3+ cells, many of which were double-negative (CD4- CD8-) and expressed CD57, especially around the follicles. Molecular studies of the lymph node biopsy showed a point deletion (4-base pair deletion) in exon 9 of the FAS gene (930del TGCT), which results in 3 missense amino acids.

Induction of apoptosis on human hepatocarcinoma cell lines by an alkyl resorcinol isolated from Lithraea molleoides.

AIM: To study the mechanism of cytotoxicity of a new active 5-alkyl resorcinol [1, 3-dihydroxy-5- (tridec-4', 7'-dienyl) benzene] isolated from Lithraea molleoides leaves on liver tumor cells. METHODS: Human hepatocarcinoma cell lines (HepG2 and Hep3B) in culture were treated with inhibitory concentrations, 50% of the compound, for 24 h. The induction of apoptosis was detected in treated cells by analysis of DNA fragmentation, DNA content, and acridine orange and propidium iodide staining. RESULTS: After 24 h of 5-alkyl resorcinol treatment, both cell lines showed: (1) the typical morphological alterations of apoptosis; (2) DNA fragmentation, detected by laddering and appearance of a subG0 population by flow cytometry; and (3) condensed and fragmented nuclei by acridine orange-propidium iodide staining. CONCLUSION: Based on the results, this compound exerts its cytotoxic effect in both hepatocellular cell lines through apoptotic cell death. For Hep3B, cells with mutated p53 and Fas, apoptosis would proceed by p53- or Fas-independent pathways.

Effect of a vascular endothelial growth factor (VEGF) inhibitory treatment on the folliculogenesis and ovarian apoptosis in gonadotropin-treated prepubertal rats.

In the present study, we investigated whether vascular endothelial growth factor A (VEGFA) plays a critical intraovarian survival role in gonadotropin-dependent folliculogenesis. The effect of an intrabursal administration of a VEGFA antagonist on follicular development, apoptosis, and levels of pro- and antiapoptotic proteins of BCL2 family members (BAX, BCL2, and BCL2L1), as well as of TNFRSF6 (also known as FAS) and FAS ligand (FASLG), was examined. To inhibit VEGFA, a soluble FLT1/Fc Chimera (Trap) was administered to prepubertal eCG-treated rats. Injection of 0.5 mug of Trap per ovary did not change the number of preantral follicles (PFs) or early antral follicles (EAFs); however, it significantly decreased the number of periovulatory follicles 48 h after surgery and significantly increased the number of atretic follicles. No significant differences were found in any stage of the follicles either 12 or 24 h after injection. Cells undergoing DNA fragmentation were quantified by performing TUNEL on ovarian sections. Trap treatment caused a twofold increase in the number of apoptotic cells in EAFs. DNA isolated from antral follicles incubated for 24 h exhibited the typical apoptotic DNA pattern. Follicles obtained from Trap-treated ovaries showed a significant increase in the spontaneous onset of apoptotic DNA fragmentation. The injection of Trap significantly increased the levels of BAX and decreased the levels of BCL2 protein. The ratio of BCL2L1L:BCL2L1s was significantly diminished in follicles obtained from ovaries treated with Trap. No changes in the levels of TNFRSF6 or FASLG were observed after treatment. We concluded that the local inhibition of VEGFA activity appears to produce an increase in ovarian apoptosis through an imbalance among the BCL2 family members, thus leading a larger number of follicles to atresia.