SET protein accumulates in HNSCC and contributes to cell survival: Antioxidant defense, Akt phosphorylation and AVOs acidification

Objectives: Determination of the SET protein levels in head and neck squamous cell carcinoma (HNSCC) tissue samples and the SET role in cell survival and response to oxidative stress in HNSCC cell lineages. Materials and Methods: SET protein was analyzed in 372 HNSCC tissue samples by immunohistochemistry using tissue microarray and HNSCC cell lineages. Oxidative stress was induced with the pro-oxidant tert-butylhydroperoxide (50 and 250 mu M) in the HNSCC HN13 cell lineage either with (siSET) or without (siNC) SET knockdown. Cell viability was evaluated by trypan blue exclusion and annexin V/propidium iodide assays. It was assessed caspase-3 and -9, PARP-1, DNA fragmentation, NM23-H1, SET, Akt and phosphorylated Akt (p-Akt) status. Acidic vesicular organelles (AVOs) were assessed by the acridine orange assay. Glutathione levels and transcripts of antioxidant genes were assayed by fluorometry and real time PCR, respectively. Results: SET levels were up-regulated in 97% tumor tissue samples and in HNSCC cell lineages. SiSET in HN13 cells (i) promoted cell death but did not induced caspases, PARP-1 cleavage or DNA fragmentation, and (ii) decreased resistance to death induced by oxidative stress, indicating SET involvement through caspase-independent mechanism. The red fluorescence induced by siSET in HN13 cells in the acridine orange assay suggests SET-dependent prevention of AVOs acidification. NM23-H1 protein was restricted to the cytoplasm of siSET/siNC HN13 cells under oxidative stress, in association with decrease of cleaved SET levels. In the presence of oxidative stress, siNC HN13 cells showed lower GSH antioxidant defense (GSH/GSSG ratio) but higher expression of the antioxidant genes PRDX6, SOD2 and TXN compared to siSET HN13 cells. Still under oxidative stress, p-Akt levels were increased in siNC HN13 cells but not in siSET HN13, indicating its involvement in HN13 cell survival. Similar results for the main SET effects were observed in HN12 and CAL 27 cell lineages, except that HN13 cells were more resistant to death. Conclusion: SET is potential (i) marker for HNSCC associated with cancer cell resistance and (ii) new target in cancer therapy. (C) 2012 Elsevier Ltd. All rights reserved.

Roles of Ecdysone signaling in cell survival and epithelium morphogenesis during Drosophila melanogaster development

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I’ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival

A critical role for Tie1, an orphan endothelial receptor, in blood vessel morphogenesis has emerged from mutant mouse studies. Moreover, it was recently demonstrated that certain angiopoietin (Ang) family members can activate Tie1. We report here that Ang1 induces Tie1 phosphorylation in endothelial cells. Tie1 phosphorylation was, however, Tie2 dependent because 1) Ang1 failed to induce Tie1 phosphorylation when Tie2 was down-regulated in endothelial cells; 2) Tie1 phosphorylation was induced in the absence of Ang1 by either a constitutively active form of Tie2 or a Tie2 agonistic antibody; 3) in HEK 293 cells Ang1 phosphorylated a form of Tie1 without kinase activity when coexpressed with Tie2, and Ang1 failed to phosphorylate Tie1 when coexpressed with kinase-defective Tie2. Ang1-mediated AKT and 42/44MAPK phosphorylation is predominantly Tie2 mediated, and Tie1 down-regulates this pathway. Finally, based on a battery of in vitro and in vivo data, we show that a main role for Tie1 is to modulate blood vessel morphogenesis by virtue of its ability to down-regulate Tie2-driven signaling and endothelial survival. Our new observations help to explain why Tie1 null embryos have increased capillary densities in several organ systems. The experiments also constitute a paradigm for how endothelial integrity is fine-tuned by the interplay between closely related receptors by a single growth factor.

Red cell survival in relation to changes in the hematocrit: more important than you think

The management of anemia in patients with chronic renal failure has greatly improved with the availability of recombinant human erythropoietin in the late 1980s, leading to a considerable reduction in mortality and morbidity and to an improvement in quality of life. The findings from recent controlled clinical outcome trials have resulted in a rather narrow, generally accepted therapeutic hematocrit target range. However, currently available dosing algorithms do not permit achievement and maintenance of target values within the therapeutic range in many patients. One possible explanation for this failure may be the ignorance of a finite erythrocyte lifespan not integrated into most algorithms. The purpose of this article is to underline the essential role played by the erythrocyte lifespan in the erythropoietic response to recombinant human erythropoietin and to encourage the integration of this concept in the future development of computer-assisted decision support systems.

ROLE OF GSH METABOLISM IN MEDIATING STROMAL-LEUKEMIA INTERACTION AND PROMOTING CELL SURVIVAL AND DRUG RESISTANCE IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the western countries. The interaction between CLL cells and the bone marrow stromal environment is thought to play a major role in promoting the leukemia cell survival and drug resistance. My dissertation works proved a novel biochemical mechanism by which the bone marrow stromal cells exert a profound influence on the redox status of primary CLL cells and enhance their ability to sustain oxidative stress and drug treatment. Fresh leukemia cells isolated from the peripheral blood of CLL patients exhibited two major redox alterations when they were cultured alone: a significant decrease in cellular glutathione (GSH) and an increase in basal ROS levels. However, when cultured in the presence of bone marrow stromal cells, CLL cells restored their redox balance with an increased synthesis of GSH, a decrease in spontaneous apoptosis, and an improved cell survival. Further study showed that CLL cells were under intrinsic ROS stress and highly dependent on GSH for survival, and that the bone marrow stromal cells promoted GSH synthesis in CLL cells through a novel biochemical mechanism. Cysteine is a limiting substrate for GSH synthesis and is chemically unstable. Cells normally obtain cysteine by uptaking the more stable and abundant precursor cystine from the tissue environment and convert it to cysteine intracellularly. I showed that CLL cells had limited ability to take up extracellular cystine for GSH synthesis due to their low expression of the transporter Xc-, but had normal ability to uptake cysteine. In the co-culture system, the bone marrow stromal cells effectively took up cystine and reduced it to cysteine for secretion into the tissue microenvironment to be taken up by CLL cells for GSH synthesis. The elevated GSH in CLL cells in the presence of bone marrow stromal cells significantly protected the leukemia cells from stress-induced apoptosis, and rendered them resistant to standard therapeutic agents such as fludarabine and oxaliplatin. Importantly, disabling of this protective mechanism by depletion of cellular GSH using a pharmacological approach potently sensitized CLL cells to drug treatment, and effectively enhanced the cytotoxic action of fludarabine and oxaliplatin against CLL in the presence of stromal cells. This study reveals a key biochemical mechanism of leukemia-stromal cells interaction, and identifies a new therapeutic strategy to overcome drug resistance in vivo.

p75 receptor-mediated neurotrophism: A new mechanism of melanoma cell survival and invasion

Trophism as a "clonal dominance" support mechanism for tumor cells is an unexplored area of tumor progression. This report presents evidence that the human melanoma low-affinity neurotrophin receptor (p75) can signal independently of its high-affinity tyrosine kinase counterparts, the TRK family of kinases. Signaling may be accomplished by a p75-associated purine-analog-sensitive kinase and results in enhanced invasion into a reconstituted basement membrane with a corresponding stimulation of matrix metalloproteinase-2 expression. Additionally, a "stress culture" survival assay was developed to mimic the growth limiting conditions encountered by melanoma cells in a rapidly growing primary tumor or metastatic deposit prior to neoangiogenesis. Under these conditions, p75, promotes the survival of high p75 expressing brain-colonizing melanoma cells. Extensive 70W melanoma cell-cell contact, which downregulates p75, immediately precedes the induction of cell death associated with diminished production of two key cell survival factors, bcl-2 and the p85 subunit of phosphoinositol-3-kinase, and an elevation in apoptosis promoting intracellular reactive oxygen species (ROSs). Since one function of bcl-2 may be to control the generation of ROSs via the antioxidant pathway, these cells may receive a apoptosis-prompting "double hit". 70W melanoma cell death occurred by an apoptotic mechanism displaying classical morphological changes including plasma membrane blebbing, loss of microvilli and redistribution of ribosomes. 70W apoptosis could be pharmacologically triggered following anti-p75 monoclonal antibody-mediated clustering of p75 receptors. 70W cells fluorescently sorted for high-p75 expression (p75$\sp{\rm H}$ cells) exhibited an augmented survival potential and a predilection to sort with the S + G2/M growth phase, relative to their low p75 expressing, p75$\sp{\rm L}$ counterparts. Apoptosis is significantly delayed by p75$\sp{\rm H}$ cells, whereas p75$\sp{\rm L}$ cells are exquisitely prone to initiate apoptosis. Importantly, the p75$\sp{\rm L}$ cells that survive apoptosis, highly re-expressed p75 and were remarkably responsive to exogenous NGF.^ These are the first data to implicate p75-mediated neurotrophism as an invasion and survival support mechanism employed by brain-metastatic cells. In particular, these results may have implications in little understood phenomena of tumor progression, such as the emergence of "clonal dominance" and tumor dormancy. ^

Down-Regulation of Annexin A1 in the Urothelium Decreases Cell Survival After Bacterial Toxin Exposure

PURPOSE: We examined the role of annexins in bladder urothelium. We characterized expression and distribution in normal bladders, biopsies from patients with bladder pain syndrome, cultured human urothelium and urothelial TEU-2 cells. MATERIALS AND METHODS: Annexin expression in bladder layers was analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunofluorescence. We assessed cell survival after exposure to the pore forming bacterial toxin streptolysin O by microscopy and alamarBlue® assay. Bladder dome biopsies were obtained from 8 asymptomatic controls and 28 patients with symptoms of bladder pain syndrome. RESULTS: Annexin A1, A2, A5 and A6 were differentially distributed in bladder layers. Annexin A6 was abundant in detrusor smooth muscle and low in urothelium, while annexin A1 was the highest in urothelium. Annexin A2 was localized to the lateral membrane of umbrella cells but excluded from tight junctions. TEU-2 cell differentiation caused up-regulation of annexin A1 and A2 and down-regulation of annexin A6 mRNA. Mature urothelium dedifferentiation during culture caused the opposite effect, decreasing annexin A1 and increasing annexin A6. Annexin A2 influenced TEU-2 cell epithelial permeability. siRNA mediated knockdown of annexin A1 in TEU-2 cells caused significantly decreased cell survival after streptolysin O exposure. Annexin A1 was significantly reduced in biopsies from patients with bladder pain syndrome. CONCLUSIONS: Several annexins are expressed in human bladder and TEU-2 cells, in which levels are regulated during urothelial differentiation. Annexin A1 down-regulation in patients with bladder pain syndrome might decrease cell survival and contribute to compromised urothelial function.

BIOLOGICAL MECHANISMS AND CLINICAL IMPLICATIONS OF BCR-ABL-INDUCED MITOCHONDRIAL OXIDATIVE STRESS AND CELL SURVIVAL IN CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML), a myeloproliferative disorder, represents approximately 15-20% of all adult leukemia. The development of CML is clearly linked to the constitutively active protein-tyrosine kinase BCR-ABL, which is encoded by BCR-ABL fusion gene as the result of chromosome 9/22 translocation (Philadelphia chromosome). Previous studies have demonstrated that oxidative stress-associated genetic, metabolic and biological alterations contribute to CML cell survival and drug refractory. Mitochondria and NAD(P)H oxidase (NOX) are the major sources of BCR-ABL-induced cellular reactive oxygen species (ROS) production. However, it is still unknown how CML cells maintain the altered redox status, while escaping from the persistent oxidative stress-induced cell death. Therefore, elucidation of the mechanisms by which CML cells cope with oxidative stress will provide new insights into CML leukemogenesis. The major goal of this study is to identify the survival factors protecting CML cells against oxidative stress and develop novel therapeutic strategies to overcome drug resistance. Several experimental models were used to test CML cell redox status and cellular sensitivity to oxidative stress, including BCR-ABL inducible cell lines, BCR-ABL stably transformed cell lines and BCR-ABL-expressing CML blast crisis cells with differential BCL-XL/BCL-2 expressions. Additionally, an artificial CML cell model with heterogenic BCL-XL/BCL-2 expression was established to assess the correlation between differential survival factor expression patterns and cell sensitivity to Imatinib and oxidative stress. In this study, BCL-XL and GSH have been identified as the major survival factors responsive to BCR-ABL-promoted cellular oxidative stress and play a dominant role in regulating the threshold of oxidative stress-induced apoptosis. Cell survival factors BCL-XL and BCL-2 differentially protect mitochondria under oxidative stress. BCL-XL is an essential survival factor in preventing excessive ROS-induced cell death while BCL-2 seems to play a relatively minor role. Furthermore, the redox modulating reagent β-phenethyl isothiocyanate (PEITC) has been found to efficiently deplete GSH and induce potent cell killing effects in drug-resistant CML cells. Combination of PEITC with BCL-XL/BCL2 inhibitor ABT737 or suppression of BCL-XL by BCR-ABL inhibitor Gleevec dramatically sensitizes CML cells to apoptosis. These results have suggested that elevation of BCL-XL and cellular GSH are important for the development of CML, and that redox-directed therapy is worthy of further clinical investigations in CML.

Cell survival in early embryogenesis requires proper dosage of GCN5 and p300

Histone acetylation is a central event in transcriptional activation. The importance of this modification in mammalian development is highlighted by knockout studies that revealed loss of the histone acetyltransferases GCN5, p300, or CBP results in embryonic lethality. Furthermore, early embryogenesis is sensitive to the dosage of p300 and CBP since double p300 +/−CBP+/− heterozygotes die in utero, although either single heterozygote survives. PCAF and GCN5 physically interact with p300 and CBP in vitro. To determine whether these two groups of HATs interact functionally in vivo, we created mice lacking one or more allele of p300, GCN5 or PCAF. As expected, we found that mice heterozygous for any one of these null alleles are viable. The majority of GCN5 p300 double heterozygotes also survive to adulthood with no apparent abnormalities. However, a portion of these mice die prior to birth. These embryos are developmentally stunted and exhibit increased apoptosis compared to wild type or single GCN5 or p300 heterozygous littermates at E8.5. Tissue specification is unaffected in these embryos but organ formation is compromised. In contrast, no abnormalities were observed in mice harboring mutations in both PCAF and p300 , emphasizing the specificity of HAT functions in mammalian development. ^ Since GCN5 null embryos die early in embryogenesis because of a marked increase in apoptosis, studies of its function and mechanism in late development and in tissue specific differentiation are precluded. Here, we also report the establishment of a GCN5 null embryonic stem cell line and a conditional floxGCN5 mouse line, which will serve as powerful genetic tools to examine in depth the function of GCN5 in mammalian development and in adult tissues. ^

NF-κB-dependent inhibition of apoptosis is essential for host cell survival during Rickettsia rickettsii infection

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-κB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-κB was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IκB that rendered NF-κB inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-κB activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-κB activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell’s apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Constitutive Bcl-2 expression throughout the hematopoietic compartment affects multiple lineages and enhances progenitor cell survival

Bcl-2, which can both reduce apoptosis and retard cell cycle entry, is thought to have important roles in hematopoiesis. To evaluate the impact of its ubiquitous overexpression within this system, we targeted expression of the human bcl-2 gene in mice by using the promoter of the vav gene, which is active throughout this compartment but rarely outside it. The vav-bcl-2 transgene was expressed in essentially all nucleated cells of hematopoietic tissues but not notably in nonhematopoietic tissues. Presumably because of enhanced cell survival, the mice displayed increases in myeloid cells as well as a marked elevation in B and T lymphocytes. The spleen was enlarged and the lymphoid follicles expanded. Although total thymic cellularity was normal, T cell development was altered: cells at the very immature and most mature stages were increased, whereas those at the intermediate stage were decreased. Unexpectedly, blood platelets were reduced by half, suggesting that their production from megakaryocytes is regulated by the Bcl-2 family. Colony formation by myeloid progenitor cells in vitro remained cytokine dependent, and the frequency of most progenitor and preprogenitor cells was normal. Macrophage progenitors were less frequent and yielded smaller colonies, however, perhaps reflecting inhibitory effects of Bcl-2 on cell cycling in specific lineages. After irradiation or factor deprivation, Bcl-2 markedly enhanced clonogenic survival of all tested progenitor and preprogenitor cells. Thus, Bcl-2 has multiple effects on the hematopoietic system. These mice should help to further clarify the role of apoptosis in the development and homeostasis of this compartment.