Effect of testosterone on E1S-sulfatase activity in non-malignant and cancerous breast cells in vitro

INTRODUCTION: Testosterone (T) is a therapeutic option for women with hypoactive sexual desire disorder. T may have an impact on the mammary gland by altering local estrogen synthesis. The aim of the present study was to measure the effect of T on estrone-sulfate (E1S)-sulfatase (STS) expression, and activity using hormone-dependent BC cells with high and low aggressive potential (BT-474, MCF-7), and HBL-100 as a breast cell line of non-malignant origin. METHODS: Cells were incubated in RPMI 1640 medium containing 5% steroid-depleted fetal calf serum for 3d, and subsequently incubated in absence or presence of T alone, and combined with anastrozole (A) at 10(-8)M, and 10(-6)M at 37 degrees C for either 24h or directly in cell extracts ("direct"). STS protein expression was measured by dot-blot (immunoblotting), and STS, HSD17B1 and HSD17B2 mRNA levels by quantitative RT-PCR. STS activity was evaluated by incubating homogenized breast cells with [(3)H]-E1S and separating the products E1, and E2 by thin layer chromatography. RESULTS: Basal STS mRNA expression did not reveal group differences. However, STS mRNA was decreased by T+A in MCF-7 cells. 17HSDB1 expression was decreased by T+A in BT-474 cells, and 17HSDB2 expression was decreased by A and T+A treatment in MCF-7 cells. Basal and T treated STS protein expression was significantly higher in malignant compared to non-malignant breast cells. However, T did not induce significant intra-cell line differences. Similarly, basal and T treated STS activity was significantly higher in highly malignant compared to non-malignant breast cells. Regardless of cell lines, T slightly decreased STS activity after "direct" incubation, but led to an increase of local estrogen formation after 24h which was attenuated, and partly reversed by A, respectively. CONCLUSIONS: The more aggressive the breast cell line, the higher the local estrogen formation. The transition from normal to malignant seems to be accompanied by an altered autoregulation. The given local endocrine milieu seems to be essential for response to T.

Impact of beta-galactosidase mutations on the expression of the canine lysosomal multienzyme complex

beta-galactosidase (GLB1) forms a functional lysosomal multienzyme complex with lysosomal protective protein (PPCA) and neuraminidase 1 (NEU1) which is important for its intracellular processing and activity. Mutations in the beta-galactosidase gene cause the lysosomal storage disease G(M1)-gangliosidosis. In order to identify additional molecular changes associated with the presence of beta-galactosidase mutations, the expression of canine lysosomal multienzyme complex components in GLB1(+/+), GLB1(+/-) and GLB1(-/-) fibroblasts was investigated by quantitative RT-PCR, Western blot and enzymatic assays. Quantitative RT-PCR revealed differential regulation of total beta-galactosidase, beta-galactosidase variants and protective protein for beta-galactosidase gene (PPGB) in GLB1(+/-) and GLB1(-/-) compared to GLB1(+/+) fibroblasts. Furthermore, it was shown that PPGB levels gradually increased with the number of mutant beta-galactosidase alleles while no change in the NEU1 expression was observed. This is the first study that simultaneously examine the effect of GLB1(+/+), GLB1(+/-) and GLB1(-/-) genotypes on the expression of lysosomal multienzyme complex components. The findings reveal a possible adaptive process in GLB1 homozygous mutant and heterozygous individuals that could facilitate the design of efficient therapeutic strategies.

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses

REASONS FOR PERFORMING STUDY: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. OBJECTIVES: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. METHODS: We performed quantitative RT-PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO-affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. RESULTS: Neutrophils were increased in RAO-affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO-affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO-affected animals overexpressing CLCA1 in relation to eqMUC5AC. CONCLUSIONS: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.

Tooth eruption: altered gene expression in the dental follicle of patients with cleidocranial dysplasia

OBJECTIVES The dental follicle plays an important role in tooth eruption by providing key regulators of osteogenesis and bone resorption. Patients with cleidocranial dysplasia (CCD) exhibit delayed tooth eruption in combination with increased bone density in the maxilla and mandible, suggesting disturbances in bone remodeling. The aim of this study was to determine the expression of genes relevant for tooth eruption and bone remodeling in the dental follicles of patients with CCD and normal subjects. MATERIAL AND METHODS Thirteen dental follicles were isolated from five unrelated patients with CCD, and fourteen dental follicles were obtained from 10 healthy individuals. All teeth were in the intraosseous phase of eruption. The expression of RANK, RANKL, OPG, and CSF-1 was determined by quantitative RT-PCR. RESULTS In patients with CCD, the mRNA levels of RANK, OPG, and CSF-1 were significantly elevated compared with the control group. Accordingly, the ratios of RANKL/OPG and RANKL/RANK mRNAs were significantly decreased in patients with CCD. CONCLUSION The observed alterations in the expression and ratios of the aforementioned factors in the dental follicle of CCD individuals suggest a disturbed paracrine signaling for bone remodeling that could be responsible for the impaired tooth eruption seen in these patients.

RNA-Seq-based analysis of the physiologic cold shock-induced changes in Moraxella catarrhalis gene expression

BACKGROUND Moraxella catarrhalis, a major nasopharyngeal pathogen of the human respiratory tract, is exposed to rapid downshifts of environmental temperature when humans breathe cold air. The prevalence of pharyngeal colonization and respiratory tract infections caused by M. catarrhalis is greatest in winter. We investigated how M. catarrhalis uses the physiologic exposure to cold air to regulate pivotal survival systems that may contribute to M. catarrhalis virulence. RESULTS In this study we used the RNA-seq techniques to quantitatively catalogue the transcriptome of M. catarrhalis exposed to a 26 °C cold shock or to continuous growth at 37 °C. Validation of RNA-seq data using quantitative RT-PCR analysis demonstrated the RNA-seq results to be highly reliable. We observed that a 26 °C cold shock induces the expression of genes that in other bacteria have been related to virulence a strong induction was observed for genes involved in high affinity phosphate transport and iron acquisition, indicating that M. catarrhalis makes a better use of both phosphate and iron resources after exposure to cold shock. We detected the induction of genes involved in nitrogen metabolism, as well as several outer membrane proteins, including ompA, m35-like porin and multidrug efflux pump (acrAB) indicating that M. catarrhalis remodels its membrane components in response to downshift of temperature. Furthermore, we demonstrate that a 26 °C cold shock enhances the induction of genes encoding the type IV pili that are essential for natural transformation, and increases the genetic competence of M. catarrhalis, which may facilitate the rapid spread and acquisition of novel virulence-associated genes. CONCLUSION Cold shock at a physiologically relevant temperature of 26 °C induces in M. catarrhalis a complex of adaptive mechanisms that could convey novel pathogenic functions and may contribute to enhanced colonization and virulence.

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

BACKGROUND In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). HYPOTHESIS/OBJECTIVES Our aim was to characterize canine TSLP and to assess its expression in CAD. METHODS Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT-PCR. Real-time quantitative RT-PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. RESULTS Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full-length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll-like receptor ligands lipopolysaccharide and poly I:C. CONCLUSIONS AND CLINICAL IMPORTANCE Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.

A specific expression profile of heat-shock proteins and glucose-regulated proteins is associated with response to neoadjuvant chemotherapy in oesophageal adenocarcinomas

BACKGROUND Oesophageal adenocarcinomas often show resistances to chemotherapy (CTX), therefore, it would be of high interest to better understand the mechanisms of resistance. We examined the expression of heat-shock proteins (HSPs) and glucose-regulated proteins (GRPs) in pretherapeutic biopsies of oesophageal adenocarcinomas to assess their potential role in CTX response. METHODS Ninety biopsies of locally advanced adenocarcinomas before platin/5-fluorouracil (FU)-based CTX were investigated by reverse phase protein arrays (RPPAs), immunohistochemistry (IHC) and quantitative RT-PCR. RESULTS CTX response strongly correlated with survival (P=0.001). Two groups of tumours with specific protein expression patterns were identified by RPPA: Group A was characterised by low expression of HSP90, HSP27 and p-HSP27((Ser15, Ser78, Ser82)) and high expression of GRP78, GRP94, HSP70 and HSP60; Group B exhibited the inverse pattern. Tumours of Group A were more likely to respond to CTX, resulting in histopathological tumour regression (P=0.041) and post-therapeutic down-categorisation from cT3 to ypT0-T2 (P=0.040). High HSP60 protein (IHC) and mRNA expression were also associated with tumour down-categorisation (P=0.016 and P=0.004). CONCLUSION Our findings may enhance the understanding of CTX response mechanisms, might be helpful to predict CTX response and might have translational relevance as they highlight the role of potentially targetable cellular stress proteins in the context of CTX response.

Investigation of border disease and bovine virus diarrhoea in sheep from 76 mixed cattle and sheep farms in eastern Switzerland

The purpose of this study was to examine the occurrence of sheep persistently infected with Border disease virus (BDV) on 76 mixed cattle and sheep farms and whether seroconversion to BDV infection occurred in cattle of these farms. Seroprevalence of BDV and bovine viral disease virus (BVDV) infection in sheep was also investigated. Quantitative RT-PCR for pestivirus detection and an ELISA to detect pestivirus antibodies were used in 2'384 and 2'291 ovine blood samples, respectively. Another 27 seropositive sheep from ten flocks underwent serum neutralization testing to differentiate between BDV and BVDV antibodies. A BDV titre that was at least four times higher than the BVDV titre was interpreted as the result of BDV infection. Titres against BVDV were interpreted in an analogous fashion. All examined sheep were pestivirus-negative, 310 sheep were seropositive, 119 had an indeterminate titre and 1'862 were seronegative. The flock seroprevalence ranged from 0.0 to 73.9 %. Three of the 27 flocks that underwent serum neutralization testing were interpreted as BDV-infected because of 6 sheep with higher BDV titres, and 6 flocks were interpreted as BVDV-infected because of 14 sheep with higher BVDV titres.

LDOC1, A NOVEL BIOMARKER OF PROGNOSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA

In chronic lymphocytic leukemia (CLL), one of the best predictors of outcome is the somatic mutation status of the immunoglobulin heavy chain variable region (IGHV) genes. Patients whose CLL cells have unmutated IGHV genes have a median survival of 8 years; those with mutated IGHV genes have a median survival of 25 years. To identify new prognostic biomarkers and molecular targets for therapy in untreated CLL patients, we reanalyzed the raw data from four published gene expression profiling microarray studies. Of 88 candidate biomarkers associated with IGHV somatic mutation status, we identified LDOC1 (Leucine Zipper, Down-regulated in Cancer 1), as one of the most significantly differentially expressed genes that distinguished mutated from unmutated CLL cases. LDOC1 is a putative transcription factor of unknown function in B-cell development and CLL pathophysiology. Using a highly sensitive quantitative RT-PCR (QRT-PCR) assay, we confirmed that LDOC1 mRNA was dramatically down-regulated in mutated compared to unmutated CLL cases. Expression of LDOC1 mRNA was also vii strongly associated with other markers of poor prognosis, including ZAP70 protein and cytogenetic abnormalities of poor prognosis (deletions of chromosomes 6q21, 11q23, and 17p13.1, and trisomy 12). CLL cases positive for LDOC1 mRNA had significantly shorter overall survival than negative cases. Moreover, in a multivariate model, LDOC1 mRNA expression predicted overall survival better than IGHV mutation status or ZAP70 protein, among the best markers of prognosis in CLL. We also discovered LDOC1S, a new LDOC1 splice variant. Using isoform-specific QRT-PCR assays that we developed, we found that both isoforms were expressed in normal B cells (naïve > memory), unmutated CLL cells, and in B-cell non-Hodgkin lymphomas with unmutated IGHV genes. To investigate pathways in which LDOC1 is involved, we knocked down LDOC1 in HeLa cells and performed global gene expression profiling. GFI1 (Growth Factor-Independent 1) emerged as a significantly up-regulated gene in both HeLa cells and CLL cells that expressed high levels of LDOC1. GFI1 oncoprotein is implicated in hematopoietic stem cell maintenance, lymphocyte development, and lymphomagenesis. Our findings indicate that LDOC1 mRNA is an excellent biomarker of overall survival in CLL, and may contribute to B-cell differentiation and malignant transformation.

Characterization of the inflammatory infiltrate and cytokine expression in the skin of horses with recurrent urticaria.

BACKGROUND Recurrent urticaria (RU) is a common skin disease of horses, but little is known about its pathogenesis. HYPOTHESIS/OBJECTIVE The aim of this study was to characterize the inflammatory cell infiltrate and cytokine expression pattern in the skin of horses with RU. ANIMALS   Biopsies of lesional and nonlesional skin of horses with RU (n = 8) and of skin from healthy control horses (n = 8) were evaluated. METHODS The inflammatory cell infiltrate was analysed by routine histology. Immunohistochemistry was used to identify T cells (CD3), B  ells (CD79), macrophages (MAC387) and mast cells (tryptase). Expression of T-helper 2 cytokines (interleukins IL-4, IL-5 and IL-13), a T-helper 1 cytokine (interferon-γ), IL-4 receptor α and thymic stromal lymphopoietin was assessed by quantitative RT-PCR. Results -  In subepidermal lesional skin of RU-affected horses, increased numbers of eosinophils (P ≤ 0.01), CD79-positive (P ≤ 0.01), MAC387-positive (P ≤ 0.01) and tryptase-positive cells (P ≤ 0.05) were found compared with healthy horses. Subepidermal lesional skin of RU-affected horses contained more eosinophils (P ≤ 0.05) and tryptase-positive cells (P ≤ 0.05) compared with nonlesional skin. There was no significant difference in infiltrating cells between nonlesional skin and skin of healthy horses. Expression of IL-4 (P ≤ 0.01), IL-13 (P ≤ 0.05), thymic stromal lymphopoietin (P ≤ 0.05) and IL-4 receptor α (P ≤ 0.05) was increased in lesional skin of RU-affected horses compared with control horses. Expression of IL-4 was higher (P ≤ 0.05) in lesional compared with nonlesional RU skin. CONCLUSIONS AND CLINICAL IMPORTANCE Analysis of cytokine expression and inflammatory infiltrate suggests that T-helper 2 cytokines, eosinophils, mast cells and presumptive macrophages play a role in the pathogenesis of equine RU.

Biomechanical loading modulates proinflammatory and bone resorptive mediators in bacterial-stimulated PDL cells.

The present study aimed to evaluate in vitro whether biomechanical loading modulates proinflammatory and bone remodeling mediators production by periodontal ligament (PDL) cells in the presence of bacterial challenge. Cells were seeded on BioFlex culture plates and exposed to Fusobacterium nucleatum ATCC 25586 and/or cyclic tensile strain (CTS) of low (CTSL) and high (CTSH) magnitudes for 1 and 3 days. Synthesis of cyclooxygenase-2 (COX2) and prostaglandin E2 (PGE2) was evaluated by ELISA. Gene expression and protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) were evaluated by quantitative RT-PCR and ELISA, respectively. F. nucleatum increased the production of COX2 and PGE2, which was further increased by CTS. F. nucleatum-induced increase of PGE2 synthesis was significantly (P < 0.05) increased when CTSH was applied at 1 and 3 days. In addition, CTSH inhibited the F. nucleatum-induced upregulation of OPG at 1 and 3 days, thereby increasing the RANKL/OPG ratio. OPG and RANKL mRNA results correlated with the protein results. In summary, our findings provide original evidence that CTS can enhance bacterial-induced syntheses of molecules associated with inflammation and bone resorption by PDL cells. Therefore, biomechanical, such as orthodontic or occlusal, loading may enhance the bacterial-induced inflammation and destruction in periodontitis.

Cholesterol metabolism, transport, and hepatic regulation in dairy cows during transition and early lactation

The transition from the nonlactating to the lactating state represents a critical period for dairy cow lipid metabolism because body reserves have to be mobilized to meet the increasing energy requirements for the initiation of milk production. The purpose of this study was to provide a comprehensive overview on cholesterol homeostasis in transition dairy cows by assessing in parallel plasma, milk, and hepatic tissue for key factors of cholesterol metabolism, transport, and regulation. Blood samples and liver biopsies were taken from 50 multiparous Holstein dairy cows in wk 3 antepartum (a.p.), wk 1 postpartum (p.p.), wk 4 p.p., and wk 14 p.p. Milk sampling was performed in wk 1, 4, and 14 p.p. Blood and milk lipid concentrations [triglycerides (TG), cholesterol, and lipoproteins], enzyme activities (phospholipid transfer protein and lecithin:cholesterol acyltransferase) were analyzed using enzymatic assays. Hepatic gene expression patterns of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGC) synthase 1 (HMGCS1) and HMGC reductase (HMGCR), sterol regulatory element-binding factor (SREBF)-1 and -2, microsomal triglyceride transfer protein (MTTP), ATP-binding cassette transporter (ABC) A1 and ABCG1, liver X receptor (LXR) α and peroxisome proliferator activated receptor (PPAR) α and γ were measured using quantitative RT-PCR. Plasma TG, cholesterol, and lipoprotein concentrations decreased from wk 3 a.p. to a minimum in wk 1 p.p., and then gradually increased until wk 14 p.p. Compared with wk 4 p.p., phospholipid transfer protein activity was increased in wk 1 p.p., whereas lecithin:cholesterol acyltransferase activity was lowest at this period. Total cholesterol concentration and mass, and cholesterol concentration in the milk fat fraction decreased from wk 1 p.p. to wk 4 p.p. Both total and milk fat cholesterol concentration were decreased in wk 4 p.p. compared with wk 1 and 14 p.p. The mRNA abundance of genes involved in cholesterol synthesis (SREBF-2, HMGCS1, and HMGCR) markedly increased from wk 3 a.p. to wk 1 p.p., whereas SREBF-1 was downregulated. The expression of ABCA1 increased from wk 3 a.p. to wk 1 p.p., whereas ABCG1 was increased in wk 14 p.p. compared with other time points. In conclusion, hepatic expression of genes involved in the biosynthesis of cholesterol as well as the ABCA1 transporter were upregulated at the onset of lactation, whereas plasma concentrations of total cholesterol, phospholipids, lipoprotein-cholesterol, and TG were at a minimum. Thus, at the gene expression level, the liver seems to react to the increased demand for cholesterol after parturition. Whether the low plasma cholesterol and TG levels are due to impaired hepatic export mechanisms or reflect an enhanced transfer of these compounds into the milk to provide essential nutrients for the newborn remains to be elucidated.

Common target genes of palatal and gingival fibroblasts for EMD: the microarray approach.

BACKGROUND AND OBJECTIVE Connective tissue grafts are frequently applied, together with Emdogain(®) , for root coverage. However, it is unknown whether fibroblasts from the gingiva and from the palate respond similarly to Emdogain. The aim of this study was therefore to evaluate the effect of Emdogain(®) on fibroblasts from palatal and gingival connective tissue using a genome-wide microarray approach. MATERIAL AND METHODS Human palatal and gingival fibroblasts were exposed to Emdogain(®) and RNA was subjected to microarray analysis followed by gene ontology screening with Database for Annotation, Visualization and Integrated Discovery functional annotation clustering, Kyoto Encyclopedia of Genes and Genomes pathway analysis and the Search Tool for the Retrieval of Interacting Genes/Proteins functional protein association network. Microarray results were confirmed by quantitative RT-PCR analysis. RESULTS The transcription levels of 106 genes were up-/down-regulated by at least five-fold in both gingival and palatal fibroblasts upon exposure to Emdogain(®) . Gene ontology screening assigned the respective genes into 118 biological processes, six cellular components, eight molecular functions and five pathways. Among the striking patterns observed were the changing expression of ligands targeting the transforming growth factor-beta and gp130 receptor family as well as the transition of mesenchymal epithelial cells. Moreover, Emdogain(®) caused changes in expression of receptors for chemokines, lipids and hormones, and for transcription factors such as SMAD3, peroxisome proliferator-activated receptor gamma and those of the ETS family. CONCLUSION The present data suggest that Emdogain(®) causes substantial alterations in gene expression, with similar patterns observed in palatal and gingival fibroblasts.

Towards the identification of a tumor suppressor gene on chromosome 3p12: Physical mapping and candidate gene screening

Renal cell carcinoma (RCC) is the most common malignant tumor of the kidney. Characterization of RCC tumors indicates that the most frequent genetic event associated with the initiation of tumor formation involves a loss of heterozygosity or cytogenetic aberration on the short arm of human chromosome 3. A tumor suppressor locus Nonpapillary Renal Carcinoma-1 (NRC-1, OMIM ID 604442) has been previously mapped to a 5–7 cM region on chromosome 3p12 and shown to induce rapid tumor cell death in vivo, as demonstrated by functional complementation experiments. ^ To identify the gene that accounts for the tumor suppressor activities of NRC-1, fine-scale physical mapping was conducted with a novel real-time quantitative PCR based method developed in this study. As a result, NRC-1 was mapped within a 4.6-Mb region defined by two unique sequences within UniGene clusters Hs.41407 and Hs.371835 (78,545Kb–83,172Kb in the NCBI build 31 physical map). The involvement of a putative tumor suppressor gene Robo1/Dutt1 was excluded as a candidate for NRC-1. Furthermore, a transcript map containing eleven candidate genes was established for the 4.6-Mb region. Analyses of gene expression patterns with real-time quantitative RT-PCR assays showed that one of the eleven candidate genes in the interval (TSGc28) is down-regulated in 15 out of 20 tumor samples compared with matched normal samples. Three exons of this gene have been identified by RACE experiments, although additional exon(s) seem to exist. Further gene characterization and functional studies are required to confirm the gene as a true tumor suppressor gene. ^ To study the cellular functions of NRC-1, gene expression profiles of three tumor suppressive microcell hybrids, each containing a functional copy of NRC-1, were compared with those of the corresponding parental tumor cell lines using 16K oligonucleotide microarrays. Differentially expressed genes were identified. Analyses based on the Gene Ontology showed that introduction of NRC-1 into tumor cell lines activates genes in multiple cellular pathways, including cell cycle, signal transduction, cytokines and stress response. NRC-1 is likely to induce cell growth arrest indirectly through WEE1. ^

Retinoid -induced regression of human head and neck squamous cell carcinomas is associated with the induction of a pro -inflammatory response

Retinoids are Vitamin A derivatives that are effective chemopreventative and chemotherapeutic agents for head and neck squamous cell carcinomas (HNSCC). Despite the wide application of retinoids in cancer treatment, the mechanism by which retinoids inhibit head and neck squamous cell carcinomas is not completely understood. While in vitro models show that drugs affect cell proliferation and differentiation, in vivo models, such as tumor xenografts in nude mice drugs affect more complex parameters such as extracellular matrix formation, angiogenesis and inflammation. Therefore, we studied the effects of retinoids on the growth of the 22B HNSCC tumors using a xenograft model. In this system, retinoids had no effect on tumor cell differentiation but caused invasion of the tumor by inflammatory cells. Retinoid induced inflammation lead to tumor cell death and tumor regression. Therefore, we hypothesized that retinoids stimulated the 22B HNSCC xenografts to produce a pro-inflammatory signal such as chemokines that in turn activated host inflammatory responses. ^ We used real time quantitative RT-PCR to measure cytokine and chemokine expression in retinoid treated tumors. Treatment of tumors with an RAR-specific retinoid, LGD1550, had no effect on the expression of TNFα, IL-1α, GROα, IP-10, Rantes, MCP-1 and MIP-1α but induced IL-8 mRNA 5-fold. We further characterized the retinoid effect on IL-8 expression on the 22B HNSCC and 1483 HNSCC cells in vitro. Retinoids increased IL-8 expression and enhanced TNFα-dependent IL-8 induction. In addition, retinoids increased the basal and TNFα-dependent expression of MCP-1 but decreased the basal and TNFα dependent expression of IP-10. The effect of retinoids on IL-8 and MCP-1 expression was very rapid with increased levels of mRNA detected within 1–2 hours. This effect did not require new protein synthesis and did not result from mRNA stabilization. Both RAR and RXR ligands increased IL-8 expression whereas only RAR ligands activated MCP-1 expression. ^ We identified a functional retinoid response element in the IL-8 promoter that was located adjacent to the C/EBP-NFkB response element. TNFα treatment of the 22B cells caused rapid, transient and selective acetylation of regions of the IL-8 promoter associated with the NFkB response element. Co-treatment of the cells with retinoids plus TNF increased the acetylation of chromatin in this region without altering the kinetics of acetylation. These results demonstrate that ligand activated retinoid receptors can cooperate with NFkB in histone acetylation and chromatin remodeling. We believe that in certain HNSCC tumors this cooperation and the resulting enhancement of IL-8 expression can induce an inflammatory response that leads to tumor regression. We believe that the induction of inflammation in susceptible tumors, possibly coupled with cytotoxic interventions may be an important component in the use of retinoids to treat human squamous cancers. ^