Anh Le

Treatment with siRNA and antisense oligonucleotides targeted to HIF-1α induced apoptosis in human tongue squamous cell carcinomas

Overexpression of hypoxia inducible factor‐1α (HIF‐1α) in cancers has been correlated to a more aggressive tumor phenotype. We investigated the effect of HIF‐1α knockout on the in vitro survival and death of human tongue squamous cell carcinomas (SCC‐4 and SCC‐9). Under normoxic condition, a basal level of HIF‐1α protein was constitutively expressed in SCC‐9 cells, albeit an undetectable level of HIF‐1α messages. Exposure to hypoxia induced only a transient increase in mRNA transcript but a prolonged elevation of HIF‐1α protein and its immediate downstream target gene product, VEGF. Under normoxic or hypoxic conditions, treatment of SCC‐9 cells with AS‐HIF‐1α ODN suppressed both constitutive and hypoxia‐induced HIF‐1α expression at both mRNA and protein levels. Knockout of HIF‐1α gene expression via either AS‐HIF‐1α ODN or siRNA (siRNAHIF‐1α) treatment resulted in inhibition of cell proliferation and induced apoptosis in SCC‐4 and SCC‐9 cells. We also demonstrated that exposure of SCC‐9 cells to hypoxia led to a time‐dependent increase in the expression of bcl‐2 and IAP‐2, but not p53. The attenuated levels of bcl‐2 and IAP‐2, and the enhanced activity of caspase‐3 after treatment with AS‐HIF‐1α ODN may contribute partly to the effects of HIF‐1α blockade on SCC‐9 cell death. Collectively, our data suggest that a constitutive or hypoxia‐induced expression of HIF‐1α in SCC‐9 and SCC‐4 cells is sufficient to confer target genes expression essential for tumor proliferation and survival. As a result, interfering with HIF‐1α pathways by antisense or siRNA strategy may provide a therapeutic target for human tongue squamous cell carcinomas.

Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures

Proteolytic degradation of the provisional fibrin matrix and subsequent substitution by fibroblast-produced collagen are essential features of injury repair. Immunohistochemical studies revealed that although dermal fibroblasts of normal scars and keloids expressed both urokinase type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1), keloid fibroblasts had a much higher PAI-1 expression. In long-term three-dimensional fibrin gel cultures (the in vitro fibroplasia model), normal fibroblasts expressed moderate and modulated activity levels of uPA and PAI-1. In contrast, keloid fibroblasts expressed a persistently high level of PAI-1 and a low level of uPA. The high PAI-1 activity of keloid fibroblasts correlated with their elevated collagen accumulation in fibrin gel cultures. Substituting collagen for fibrin in the gel matrix resulted in increased uPA activity and reduced collagen accumulation of keloid fibroblasts. Furthermore, decreasing PAI-1 activity of keloid fibroblasts in fibrin gel cultures with anti-PAI-1-neutralizing antibodies also resulted in a reduction in collagen accumulation by keloid fibroblasts. Cumulatively, these results suggest that PAI-1 overexpression is a consistent feature of keloid fibroblasts both in vitro and in vivo, and PAI-1 may play a causative role in elevated collagen accumulation of keloid fibroblasts.

Elevated Vascular Endothelial Growth Factor in Keloids: Relevance to Tissue Fibrosis

Excessive scar or keloid shares common features of a benign dermal growth. Yet, in contrast to malignant tumor, a keloid does not expand beyond the dermis. What triggers the continuing growth of a benign lesion? Deficient or overabundant levels of vascular endothelial growth factor have been reported to contribute to impaired or excessive wound healing. Although numerous studies have examined the pathophysiology of impaired wounds, little information has been provided on mechanisms of exuberant healing. The molecular basis of keloid formation is governed by the interplay of cellular signaling pathways, specific target gene activation, and the nature of the microenvironment. Recent works have demonstrated an accumulation of hypoxia-inducible factor-1α protein in freshly biopsied keloid tissues, thus providing first evidence that a local state of hypoxia exists in keloids. Our findings and the findings of others support at least two plausible mechanisms implicated in the development of fibrotic wounds, a state of ongoing fibroplasia or inflammation and an excessive accumulation of extracellular matrix. This article will review recent works examining the potential role of vascular endothelial growth factor in keloid pathogenesis with particular focus on its involvement in the two proposed pathological processes, a prolonged inflammation and an altered balance in extracellular matrix metabolism.

Activation of NFκB signal pathways in keloid fibroblasts

Keloids are characterized as an “over-exuberant” healing response resulting in a disproportionate extracellular matrix (ECM) accumulation and tissue fibrosis. In view of the integral role of inflammation and cytokines in the healing response, it is logical to assume that they may play a part in orchestrating the pathology of this “abnormal” healing process. Tumor necrosis factor-α (TNF-α) is a potent proinflammatory cytokine involved in activation of signaling events and transcriptional programs, such as NFκB. This study attempts to determine the difference in NFκB and its related genes expression and DNA binding activity between keloid and normal skin fibroblasts. Three keloid and normal skin tissues (NSk) and their derived fibroblasts were used to determine NFκB signaling pathway expression using specific cDNA microarrays, Western blot analysis and immunohistochemistry. Electrophoretic mobility gel shift assay (EMSA) was used to assess NFκB-binding activity, all assays were performed in the presence and absence of TNF-α. TNF-α up-regulated 15% of NFκB signal pathway related genes in keloid fibroblast compared to normal skin. At the protein level, keloid fibroblasts and tissues showed higher basal levels of TNF- receptor–associated factors—TRAF1, TRAF2—TNF-α, inhibitor of apoptosis (c-IAP-1), and NFκB, compared with NSk. Keloid fibroblasts showed a constitutive increase in NFκB-binding activity in comparison to NSk both with and without TNF-α treatment. NFκB and its targeted genes, especially the antiapoptotic genes, could play a role in keloid pathogenesis; targeting NFκB could help in developing therapeutic interventions for the treatment of keloid scarring.

The Geographic Distribution of Obesity in the US and the Potential Regional Differences in Misreporting of Obesity

Objective: State‐level estimates of obesity based on self‐reported height and weight suggest a geographic pattern of greater obesity in the Southeastern US; however, the reliability of the ranking among these estimates assumes errors in self‐reporting of height and weight are unrelated to geographic region.

Crosstalk of hypoxia-mediated signaling pathways in upregulating plasminogen activator inhibitor-1 expression in keloid fibroblasts.

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor‐1 (PAI‐1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia‐mediated PAI‐1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia‐induced upregulation of PAI‐1 expression is mainly controlled by hypoxia inducible factors‐1α (HIF‐1α) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol‐3‐kinase/Akt (PI3‐K/Akt) and extracellular signal‐regulated kinases 1/2 (ERK1/2). Treatment of cells with PI‐3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia‐induced PAI‐1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia‐induced stabilization of HIF‐1α protein, attenuation of the steady‐state level of HIF‐1α mRNA, and its DNA‐binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia‐induced PAI‐1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia‐mediated stabilization of HIF‐1α protein and the steady‐state level of HIF‐1α mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3‐K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia‐mediated induction of PAI‐1 expression via activation of HIF‐1α in keloid fibroblasts. J. Cell. Physiol. 199: 89–97, 2004© 2003 Wiley‐Liss, Inc.

[6] Derivation and assay of biological effects of monoclonal antibodies to epidermal growth factor receptors

Publisher Summary In recent years, a considerable number of monoclonal antibodies have been raised against epidermal growth factor (EGF) receptors. A table illustrated in this chapter shows EGF receptor monoclonal antibodies. A431 cells and A431 plasma membrane vesicles, as well as partially purified EGF receptors, have been successfully used to raise EGF receptor monoclonal antibodies. The chapter explains the production of EGF receptor monoclonal antibodies and discusses the screening of hybridomas and the characterization of EGF-receptor antibodies. The purification methods of monoclonal antibodies include protein A-agarose and DEAE-Affi-Gel Blue. As an intensively studied peptide growth factor, EGF induces several well-defined immediate and delayed responses in receptor-bearing target cells. The knowledge of the effects of EGF on responsive cells has made it possible to test EGF receptor monoclonal antibodies for agonistic and antagonistic biological activities.

Effect of TGF-beta 1 on PDGF receptors expression in human scar fibroblasts.

This study examined the effect of exogenous TGF -beta1 on platelet derived growth factor alpha and beta (PDGF-alpha, beta) receptor expression in human dermal fibroblasts derived from both normal cutaneous tissues (normal skin [NSk]) and (normal scar [NSc]) and abnormal scar (keloid). TGF-beta and PDGF are present in the early phases of wound healing and are implicated in tissue fibrosis. In this study, replicate samples of NSk, NSc and keloid fibroblasts were grown to subconfluency in DMEM/10% FBS followed by replacement of media with DMEM/0.1%FBS for 24 hrs. One group of cells (NSk, NSc and keloid) were exposed to 10 ng/mL of exogenous TGF-beta1 for 24 hours, while the other group was used as control with no exposure to exogenous TGF-beta1. RadioImmunoBinding assays, Western and Northern blot analysis were performed to examine both PDGF-alpha and PDGF-beta receptor expression at the transcriptional and post-transcriptional levels. cDNA receptor probes were synthesized using polymerase chain reaction (PCR) with selected primer sets derived from published sequences. Beta-actin probe was used as a control to confirm that the same quantity of RNA was used for each experimental condition. TGF-beta1 was found to upregulate the expression of PDGF-alpha receptor for keloid fibroblasts but not for NSk or NSc fibroblasts. No effect was observed for TGF-beta 1 on PDGF-beta receptor expression for any of the cell lines examined.

Cats differ from other species in their cytokine and antioxidant enzyme response when developing obesity.

Objectives: Obese cats show many similarities to obese people, including insulin resistance and an increased diabetes risk. However, atherosclerosis and cardiovascular disease are not seen in cats. In people, they are associated with the development of an inflammatory response, which, we hypothesized, does not occur in cats.

Autoimmune lymphoproliferative syndrome: A cause of chronic splenomegaly, lymphadenopathy, and cytopenias in children—report on diagnosis and management of five patients

Autoimmune lymphoproliferative syndrome (ALPS) usually manifests in early childhood with splenomegaly, lymphadenopathy, and cytopenias. In most patients, it results from mutations in genes that regulate lymphocyte apoptosis via the Fas pathway. Here, we report five children with ALPS. All five children had splenomegaly, cytopenias, and hypertriglyceridemia at presentation; four had lymphadenopathy. Mutations in the Fas receptor gene were demonstrated in three children. Clinical picture is variable: in only one child manifestations are severe enough to require immunosuppressive therapy. Diagnosis of ALPS can be challenging and increased awareness of the disease can result in more directed diagnostic approaches as well as earlier initiation of treatment. Pediatr Blood Cancer 2004;43:164–169.