Charles Giardina

miR-449a targets HDAC-1 and induces growth arrest in prostate cancer

Histone deacetylases (HDACs) are frequently overexpressed in broad range of cancer types, where they alter cellular epigenetic programming to promote cell proliferation and survival. However, the mechanism by which HDACs become overexpressed in human cancers remains somewhat of a mystery. In this study, we investigated the expression and functional significance of miR-449a in prostate cancer cells. Using real-time PCR, we found that miR-449a is downregulated in prostate cancer tissues relative to patient-matched control tissue. Introduction of miR-449a into PC-3 prostate cancer cells resulted in cell-cycle arrest, apoptosis and a senescent-like phenotype. In silico analysis of 3′-UTR regions identified a number of genes involved in cell-cycle regulation as putative targets of miR-449a. Using a luciferase 3′-UTR reporter system, we established that HDAC-1 (histone deacetylase 1), a gene that is frequently overexpressed in many types of cancer, is a direct target of miR-449a. Further, our data indicate that miR-449a regulates cell growth and viability in part by repressing the expression of HDAC-1 in prostate cancer cells. Our findings provide new insight into the function of miRNA in regulating HDAC expression in normal versus cancerous tissue.

The luminal short-chain fatty acid butyrate modulates NF-κB activity in a human colonic epithelial cell line

BACKGROUND & AIMS The transcription factor nuclear factor-kappaB (NF-kappaB) plays a central role in regulating immune and inflammatory responses. Because butyrate deficiency has been associated with inflammatory bowel disease, we examined the effect of butyrate on NF-kappaB activity in the human HT-29 colonic cell line. METHODS The influence of butyrate (4 mmol/L) on NF-kappaB activity was determined using the gel mobility shift assay. The effect of butyrate on the expression of NF-kappaB subunits and inhibitory proteins was determined by immunoblotting. NF-kappaB-regulated gene expression was assayed by primer extension of intercellular adhesion molecule 1 and Mn superoxide dismutase messenger RNA, and by analysis of a transfected luciferase reporter. RESULTS Exposure of HT-29 cells to butyrate eliminated their constitutive NF-kappaB, p50 dimer activity. This inhibition corresponded with a reduction in p50 nuclear localization, without a reduction in expression. Butyrate also selectively modulated activation of NF-kappaB, suppressing its activation by tumor necrosis factor alpha and phorbol ester more than 10-fold, without affecting the activity induced by interleukin (IL)-1beta. Butyrate did, however, enhance formation of the stronger p65-p50 transcriptional activator in IL-1beta-stimulated cells. The changes in NF-kappaB activation did not correlate with changes in IkappaBalpha levels. Gene expression reflected DNA binding. The influence of butyrate on NF-kappaB may result in part from its ability to inhibit deacetylases because the specific deacetylase inhibitor trichostatin A has a similar effect. CONCLUSIONS These findings suggest that the influences of butyrate on colonic inflammatory responses may result in part from its influence on NF-kappaB activation. This activity of butyrate apparently involves its ability to inhibit deacetylases.

Mouse models for the study of colon carcinogenesis

The study of experimental colon carcinogenesis in rodents has a long history, dating back almost 80 years. There are many advantages to studying the pathogenesis of carcinogen-induced colon cancer in mouse models, including rapid and reproducible tumor induction and the recapitulation of the adenoma-carcinoma sequence that occurs in humans. The availability of recombinant inbred mouse panels and the existence of transgenic, knock-out and knock-in genetic models further increase the value of these studies. In this review, we discuss the general mechanisms of tumor initiation elicited by commonly used chemical carcinogens and how genetic background influences the extent of disease. We will also describe the general features of lesions formed in response to carcinogen treatment, including the underlying molecular aberrations and how these changes may relate to the pathogenesis of human colorectal cancer.

Dynamic protein-DNA architecture of a yeast heat shock promoter.

Here we present an in vivo footprinting analysis of the Saccharomyces cerevisiae HSP82 promoter. Consistent with current models, we find that yeast heat shock factor (HSF) binds to strong heat shock elements (HSEs) in non-heat-shocked cells. Upon heat shock, however, additional binding of HSF becomes apparent at weak HSEs of the promoter as well. Recovery from heat shock results in a dramatic reduction in HSF binding at both strong and weak HSEs, consistent with a model in which HSF binding is subject to a negative feedback regulation by heat shock proteins. In vivo KMnO4 footprinting reveals that the interaction of the TATA-binding protein (TBP) with this promoter is also modulated: heat shock slightly increases TBP binding to the promoter and this binding is reduced upon recovery from heat shock. KMnO4 footprinting does not reveal a high density of polymerase at the promoter prior to heat shock, but a large open complex between the transcriptional start site and the TATA box is formed rapidly upon activation, similar to that observed in other yeast genes.

NF-κB regulates transcription of the mouse telomerase catalytic subunit

Expression of the telomerase catalytic subunit (TERT) is the rate-limiting determinant of telomerase activity in most cells. Analysis of the mouse TERT promoter revealed a potential NF-κB binding site 350 base pairs upstream from the translational start site. An oligonucleotide from this region of the TERT promoter bound to proteins in a nuclear extract prepared from a mouse hepatoma cell line. These proteins were identified as NF-κB by a number of criteria: 1) the protein complex formed on the TERT oligonucleotide had an electrophoretic mobility similar to that formed on an NF-κB consensus oligonucleotide; 2) protein binding to this site was enhanced by NF-κB activators tumor necrosis factor-α, phorbol 12-myristate 13-acetate, and interleukin-1β; and 3) the complex was specific and could be supershifted with antibodies against the p50 or p65 NF-κB subunits. The NF-κB binding site from the mouse TERT promoter activated transcription when fused to a basal SV40 promoter and enhanced the activity of the native TERT promoter in mouse hepatoma cells stimulated with phorbol 12-myristate 13-acetate. Transcriptional activation by the TERT NF-κB site could also be enhanced by co-transfection with an NF-κB1 expression vector. NF-κB may therefore contribute to the activation of TERT expression observed in mouse tissue.

NSAIDs and butyrate sensitize a human colorectal cancer cell line to TNF-α and Fas ligation: the role of reactive oxygen species

The nonsteroidal antiinflammatory drugs (NSAIDs) indomethacin and salicylic acid and the short chain fatty acid butyrate are effective colon cancer chemopreventive agents that increase reactive oxygen species (ROS) generation in colon cancer cells. Here we demonstrate that these agents sensitize the normally resistant human HT-29 colon cancer cell line to apoptosis induced by TNF-alpha or a Fas ligating antibody. The role of ROS in this sensitization is supported by the finding that direct exposure of the cells to H2O2 is sufficient for sensitization. Neither TNF-alpha nor Fas ligation alter basal or chemopreventive agent-activated ROS generation, suggesting that the death ligands and chemopreventive agents act in a complementary fashion. The dual chemopreventive agent/death ligand treatments do not increase Fas, TNF receptor 1, Bak or c-myc expression (although salicylic acid moderately induces of Fas expression). Cell death does correlate with alterations in NF-kappa B activity: the NSAIDs, butyrate and H2O2 enhance c-Rel complex formation by TNF-alpha and provide an overall enhancement of NF-kappa B activation by Fas. The antioxidant N-acetylcysteine (NAC) blocks cell death and NF-kappa B activation induced by Fas ligation, suggesting a potential role for NF-kappa B in Fas-induced apoptosis in these cells. The effects of NAC on TNF-alpha-induced cell death are more complex, with NAC being marginally protective and itself enhancing the formation of c-Rel containing complexes at higher concentrations (25 mM). The influence of NSAIDs and butyrate on ROS generation and death ligand sensitivity may be relevant to their ability to suppress colon carcinogenesis.

Hsp70B′ regulation and function

Abstract Heat shock protein (Hsp) 70B′ is a human Hsp70 chaperone that is strictly inducible, having little or no basal expression levels in most cells. Using siRNAs to knockdown Hsp70B′ and Hsp72 in HT-29, SW-480, and CRL-1807 human colon cell lines, we have found that the two are regulated coordinately in response to stress. We also have found that proteasome inhibition is a potent activator of hsp70B′. Flow cytometry was used to assay hsp70B′ promoter activity in HT-29eGFP cells in this study. Knockdown of both Hsp70B′ and Hsp72 sensitized cells to heat stress and increasing concentrations of proteasome inhibitor. These data support the conclusion that Hsp72 is the primary Hsp70 family responder to increasing levels of proteotoxic stress, and Hsp70B′ is a secondary responder. Interestingly ZnSO4 induces Hsp70B′ and not Hsp72 in CRL-1807 cells, suggesting a stressor-specific primary role for Hsp70B′. Both Hsp70B′ and Hsp72 are important for maintaining viability under conditions that increase the accumulation of damaged proteins in HT-29 cells. These findings are likely to be important in pathological conditions in which Hsp70B′ contributes to cell survival.

Green Tea Extract Suppresses NFκB Activation and Inflammatory Responses in Diet-Induced Obese Rats with Nonalcoholic Steatohepatitis

Nonalcoholic steatohepatitis (NASH) is characterized by oxidative stress and inflammatory responses that exacerbate liver injury. The objective of this study was to determine whether the antioxidant and antiinflammatory activities of green tea extract (GTE) would protect against NASH in a model of diet-induced obesity. Adult Wistar rats were fed a low-fat (LF) diet or high-fat (HF) diet containing no GTE or GTE at 1% or 2% (HF+2GTE) for 8 wk. The HF group had greater (P ≤ 0.05) serum alanine (ALT) and aspartate aminotransferases and hepatic lipids than the LF group. Both GTE groups had lower ALT and hepatic lipid than the HF group. In liver and epididymal adipose, the HF group had lower glutathione as well as greater mRNA and protein expression of TNFα and monocyte chemoattractant protein-1 (MCP-1) and NFκB binding activity than the LF group. Compared to the HF group, the HF+2GTE group had greater glutathione and lower protein and mRNA levels of inflammatory cytokines in both tissues. NFκB binding activities at liver and adipose were also lower, likely by inhibiting the phosphorylation of inhibitor of NFκB. NFκB binding activities in liver and adipose (P ≤ 0.05; r = 0.62 and 0.46, respectively) were correlated with ALT, and hepatic NFκB binding activity was inversely related to liver glutathione (r = -0.35). These results suggest that GTE-mediated improvements in glutathione status are associated with the inhibition of hepatic and adipose inflammatory responses mediated by NFκB, thereby protecting against NASH.

Anti-inflammatory effects of freeze-dried black raspberry powder in ulcerative colitis

Ulcerative colitis (UC) is a chronic inflammatory disease of the colonic mucosa that can dramatically increase the risk of colon cancers. In the present study, we evaluated the effects of a dietary intervention of freeze-dried black raspberries (BRB), a natural food product with antioxidant and anti-inflammatory bioactivities, on disease severity in an experimental mouse model of UC using 3% dextran sodium sulfate (DSS). C57BL/6J mice were fed either a control diet or a diet containing BRB (5 or 10%) for 7-14 days and then the extent of colonic injury was assessed. Dietary BRB markedly reduced DSS-induced acute injury to the colonic epithelium. This protection included better maintenance of body mass and reductions in colonic shortening and ulceration. BRB treatment, however, did not affect the levels of either plasma nitric oxide or colon malondialdehyde, biomarkers of oxidative stress that are otherwise increased by DSS-induced colonic injury. BRB treatment for up to 7 days suppressed tissue levels of several key pro-inflammatory cytokines, including tumor necrosis factor α and interleukin 1β. Further examination of the inflammatory response by western blot analysis revealed that 7 day BRB treatment reduced the levels of phospho-IκBα within the colonic tissue. Colonic cyclooxygenase 2 levels were also dramatically suppressed by BRB treatment, with a concomitant decrease in the plasma prostaglandin E₂ (276 versus 34 ng/ml). These findings demonstrate a potent anti-inflammatory effect of BRB during DSS-induced colonic injury, supporting its possible therapeutic or preventive role in the pathogenesis of UC and related neoplastic events.

NF-κB is involved in the survival of cerebellar granule neurons: association of Iκβ phosphorylation with cell survival: NF-κB in neuronal survival

The NF‐κB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c‐Rel. NF‐κB activity is tightly controlled by IκB proteins which bind to NF‐κB preventing its translocation to the nucleus. Activation of NF‐κB is most often mediated by IκB degradation, which permits NF‐κB to enter the nucleus. We investigated the role of NF‐κB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF‐κB activity: SN‐50, N‐tosyl‐l‐phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF‐κB is required for neuronal survival. Gel‐shift assays reveal three complexes that bind to the NF‐κB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium‐induced apoptosis, whereas overexpression of IκBα promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IκBα and IκBβ is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c‐Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IκBβ is, however, reduced by low potassium treatment. Besides being necessary for high potassium‐mediated neuronal survival, NF‐κB is also involved in the survival‐promoting effects of IGF‐1 and cAMP as judged by the ability of SN‐50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium‐induced alterations in the DNA‐binding activity of NF‐κB. Taken together, our results show that NF‐κB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF‐κB‐mediated survival of cerebellar granule neurons.