Relative Quantification Of Gene Expression Research Articles

Endometriosis does not Alter Aromatase Gene Expression (CYP19A1) in Mural Lutein-granulosa Cells of Women Undergoing Assisted Reproduction Techniques – a Pilot Study

Purpose The aim of this study was to quantify aromatase gene expression in mural lutein-granulosa cells of women with endometriosis undergoing assisted reproduction techniques (ART, IVF or ICSI). Methods: a case-control study was performed on 11 women with endometriosis (all stages, ASRM criteria) and 11 women with male or tubal causes of infertility undergoing ART. There was no difference between the groups regarding age, amount of gonadotrophins used, days of induction, follicles, and eggs collected. Mural lutein-granulosa cells were harvested from pre-ovulatory follicles during oocyte retrieval and correctly isolated. After cells lyses and storage into Trizol LS Reagent®, RNA extraction and cDNA synthesis were performed. Quantification of relative gene expression for CYP19A1 (aromatase) was performed by real time PCR, using SYBR Green reagents. All experiments were performed in duplicates. Results there was no difference between the groups in the quantitative gene expression of CYP19A1 (aromatase) gene on mural lutein-granulosa cells (P>.05, Mann Whitney). Conclusions These results suggest that this enzyme, aromatase, may have a complex and refined control of its gene expression on this population of granulosa cells, and in spite of previous evidence showing its reduced activity on these cells in endometriosis, gene expression per se may not be affected by the disease.

Insulin Induces REDD1 Expression through Hypoxia-inducible Factor 1 Activation in Adipocytes

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

Selective control of primer usage in multiplex one-step reverse transcription PCR

BackgroundMultiplex RT-PCR is a valuable technique used for pathogen identification, disease detection and relative quantification of gene expression. The simplification of this protocol into a one-step procedure saves time and reagents. However, intensive PCR optimization is often required to overcome competing undesired PCR primer extension during the RT step.ResultsHerein, we report multiplex one-step RT-PCR experiments in which the PCR primers contain thermolabile phosphotriester modification groups. The presence of these groups minimizes PCR primer extension during the RT step and allows for control of PCR primer extension until the more stringent, elevated temperatures of PCR are reached. Results reveal that the use of primers whose extension can be controlled in a temperature-mediated way provides improved one-step RT-PCR specificity in both singleplex and multiplex reaction formats.ConclusionsThe need for an accurate and sensitive technique to quantify mRNA expression levels makes the described modified primer technology a promising tool for use in multiplex one-step RT-PCR. A more accurate representation of the abundances in initial template sample is feasible with modified primers, as artifacts of biased PCR are reduced because of greater improvements in reaction specificity.

Endogenous control genes in complex vascular tissue samples

BackgroundGene expression microarrays and real-time PCR are common methods used to measure mRNA levels. Each method has a fundamentally different approach of normalization between samples. Relative quantification of gene expression using real-time PCR is often done using the 2^(-ΔΔCt) method, in which the normalization is performed using one or more endogenous control genes. The choice of endogenous control gene is often arbitrary or bound by tradition. We here present an analysis of the differences in expression results obtained with microarray and real-time PCR, dependent on different choices of endogenous control genes.ResultsIn complex tissue, microarray data and real-time PCR data show the best correlation when endogenous control genes are omitted and the normalization is done relative to total RNA mass, as measured before reverse transcription.ConclusionWe have found that for real-time PCR in heterogeneous tissue samples, it may be a better choice to normalize real-time PCR Ct values to the carefully measured mass of total RNA than to use endogenous control genes. We base this conclusion on the fact that total RNA mass normalization of real-time PCR data shows better correlation to microarray data. Because microarray data use a different normalization approach based on a larger part of the transcriptome, we conclude that omitting endogenous control genes will give measurements more in accordance with actual concentrations.

Age-Related Changes in Pericellular Hyaluronan Organization Leads to Impaired Dermal Fibroblast to Myofibroblast Differentiation

We have previously demonstrated that transforming growth factor-beta1 (TGF-beta1)-mediated fibroblast-myofibroblast differentiation is associated with accumulation of a hyaluronan (HA) pericellular coat. The current study demonstrates failure of fibroblast-myofibroblast differentiation associated with in vitro aging. This is associated with attenuation of numerous TGF-beta1-dependent responses, including HA synthesis and induction of the HA synthase enzyme HAS2 and the hyaladherin tumor necrosis factor-alpha-stimulated gene 6 (TSG-6), which led to an age-related defect in pericellular HA coat assembly. Inhibition of HAS2-dependent HA synthesis by gene silencing, removal of the HA coat by hyaluronidase digestion, or gene silencing of TSG-6 or cell surface receptor CD44 led to abrogation of TGF-beta1-dependent induction of alpha-smooth muscle actin in "young" cells. This result supports the importance of HAS2-dependent HA synthesis and the HA coat during phenotypic activation. Interleukin-1beta stimulation, however, failed to promote phenotypic conversion despite coat formation. A return to basal levels of HA synthesis in aged cells by HAS2 overexpression restored TGF-beta1-dependent induction of TSG-6 and pericellular HA coat assembly. However, this did not lead to the acquisition of a myofibroblast phenotype. Coordinated induction of HAS2 and TSG-6 facilitation of pericellular HA coat assembly is necessary for TGF-beta1-dependent activation of fibroblasts, and both components of this response are impaired with in vitro aging. In conclusion, the HA pericellular coat is integral but not sufficient to correct for the age-dependent defect in phenotypic conversion.

The Non-coding RNA gadd7 Is a Regulator of Lipid-induced Oxidative and Endoplasmic Reticulum Stress

In obesity and diabetes, an imbalance in fatty acid uptake and fatty acid utilization leads to excess accumulation of lipid in non-adipose tissues. This lipid overload is associated with cellular dysfunction and cell death, which contribute to organ failure, a phenomenon termed lipotoxicity. To elucidate the molecular mechanism of lipid-mediated cell death, we generated and characterized a mutant Chinese hamster ovary cell line that is resistant to palmitate-induced cell death. In this mutant, random insertion of a retroviral promoter trap has disrupted the gene for the non-coding RNA, growth arrested DNA-damage inducible gene 7 (gadd7). Here we report that gadd7 is induced by lipotoxic stress in a reactive oxygen species (ROS)-dependent fashion and is necessary for both lipid- and general oxidative stress-mediated cell death. Depletion of gadd7 by mutagenesis or short hairpin RNA knockdown significantly reduces lipid and non-lipid induced ROS. Furthermore, depletion of gadd7 delays and diminishes ROS-induced endoplasmic reticulum stress. Together these data are the first to implicate a non-coding RNA in a feed-forward loop with oxidative stress and its induction of the endoplasmic reticulum stress response.

Emprego da RT-PCR em tempo real para a quantificaÇão da expressão de genes associados à resposta imune em bezerros bovinos experimentalmente infectados por Neospora caninum

Neospora caninum is one of the main causes of abortion and natimortality in cattle. Host immune defense is capable to inhibit tachyzoite activity during acute infection, but there is no action against bradyzoites in tissue cysts. Activation and modulation of this response is controlled by cell mediators. The real-time RT-PCR technique was employed to detect some of those mediators during N. caninum infection. Holstein and Nelore calves intramuscularly infected with tachyzoites and uninfected controls were slaughtered at the sixth day post-infection and popliteal lymph node, liver and brain cortex samples were analyzed. Real-time RT-PCR detected gene expression in all tissues. No significant variation of GAPDH gene expression was detected among groups, its amplification efficiency was similar to the other genes tested and it was used as the endogenous control for the analysis. Comparisons between infected and uninfected groups allowed the relative gene expression quantification. IFN-gamma and TNF-alpha genes showed increased expression in some samples. iNOS and TGF-beta1 genes had some non-significant variations and IL-4 and IL-10 stayed practically unaltered.

Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.

Involvement of Highly Sulfated Chondroitin Sulfate in the Metastasis of the Lewis Lung Carcinoma Cells

The altered expression of cell surface chondroitin sulfate (CS) and dermatan sulfate (DS) in cancer cells has been demonstrated to play a key role in malignant transformation and tumor metastasis. However, the functional highly sulfated structures in CS/DS chains and their involvement in the process have not been well documented. In the present study, a structural analysis of CS/DS from two mouse Lewis lung carcinoma (3LL)-derived cell lines with different metastatic potentials revealed a higher proportion of Delta(4,5)HexUA-GalNAc(4,6-O-disulfate) generated from E-units (GlcUA-GalNAc(4, 6-O-disulfate)) in highly metastatic LM66-H11 cells than in low metastatic P29 cells, although much less CS/DS is expressed by LM66-H11 than P29 cells. This key finding prompted us to study the role of CS-E-like structures in experimental lung metastasis. The metastasis of LM66-H11 cells to lungs was effectively inhibited by enzymatic removal of tumor cell surface CS or by preadministration of CS-E rich in E-units in a dose-dependent manner. In addition, immunocytochemical analysis showed that LM66-H11 rather than P29 cells expressed more strongly the CS-E epitope, which was specifically recognized by the phage display antibody GD3G7. More importantly, this antibody and a CS-E decasaccharide fraction, the minimal structure recognized by GD3G7, strongly inhibited the metastasis of LM66-H11 cells probably by modifying the proliferative and invading behavior of the metastatic tumor cells. These results suggest that the E-unit-containing epitopes are involved in the metastatic process and a potential target for the diagnosis and treatment of malignant tumors.

Gene Expression Profiling Distinguishes Essential Thrombocythemia from Polycythemia Vera Patients and Identifies a Common Expressed Set of Genes in Relation to JAK2V617F Status

Introduction. The existence of the JAK2V617F mutation in a high proportion of Myeloproliferative Disorders (MPD) BCR-ABL-negative has provided important insight into the pathogenesis of these diseases. However, much of the molecular abnormalities associated to BCR-ABL-negative MPD remain unknown, specially in those which do not display JAK2V617F. In a previous study, we performed gene expression analysis by using the microarray technique in 20 essential thrombocythemia (ET) patients (44K whole human genome oligo microarrays, Agilent Technologies) and the results were confirmed in 40 ET patients by using TaqMan® Low Density Arrays Arrays (LDA, Applied Biosystems). In our previous experience the results showed different gene expression patterns in ET and a supervised clustering of the data identified genes differentially expressed between JAK2V617F-negative and JAK2V617F-positive ET patients, and a characteristic gene expression profile for JAK2V617F-negative patients (Puigdecanet et al.,2008).Aim. Our aim was to confirm the ET gene expression profile in an extended group of patients and to explore the differences and similarities in polycythemia vera (PV) and reactive thrombocytosis (RT) patients by real-time quantitative RT-PCR (RQ-PCR) technique using the LDA platform. In addition, we wanted to analyze the relationship between gene expression data and JAK2V617F status.Patients and Methods. The following patients were included in the study: 58 ET (23 JAK2V617F-negative, 34 JAK2V617F in heterozygosity and one JAK2V617F in homozygosity) and 41 PV (7 JAK2V617F-negative, 25 JAK2V617F in heterozygosity and 9 JAK2V617F in homozygosity) patients, diagnosed according to the WHO criteria (2001) and who had never received cytoreductive treatment, and six patients with RT. Based on the previous results, we designed a new LDA platform containing 96 assays in duplicate, which included the most expressed genes in ET in relation to healthy controls and the most differentially expressed genes between JAK2V617F-negative and JAK2V617F-positive ET patients. The RQ-PCR analysis was performed in RNA from peripheral blood granulocytes and the relative gene expression quantification was achieved using GAPDH as the endogenous control and a pool of 10 healthy individuals as the calibrator.Results. ETvsPV: The majority of the genes studied presented significant higher expression in ET than in PV patients. Interestingly, FOSB was one of the most differentially expressed gene (FC= 8.3), and CISH and C13orf18 did not distinguish between the two groups.ET: We confirmed the differentially expression of the majority of the genes previously detected between JAK2V617F-negative and JAK2V617F-positive ET patients and we extended the set of genes. Among them, we highlight the differential expression of CISH, FOSB and C13orf18 genes (p<0.01).PV: Supervised analysis showed that CD44, BATF and CISH clearly distinguish JAK2V617F-negative PV patients from the JAK2V617F-positive.ET and PVvsRT: Some differentially expressed genes between ET and RT patients were detected, but the most significant gene was TNF (p<0.001), which presented a higher expression in RT (FC=5.9). The same difference was observed between PV and RT.Conclusions. We have detected a different gene expression pattern in ET and in PV patients. However, we also identified a set of genes which expression was related to JAK2V617F status, both in ET and PV patients. These findings would be interesting to identify other signal transduction pathways besides JAK-STAT involved in the pathogenesis of ET and PV.

Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig

The aim of this study was to develop a set of reliable reference genes for quantification of mRNA expression in the pig. The mRNA expression stability in pig tissues was studied for 4 genes: EEF1A1, GAPDH, HPRT1 and TOP2B. The level of expression was characterized by Ct values for each gene and each tissue. By using the geNorm algorithm, the stability of the reference genes was determined in the diaphragm, heart, kidney, liver, lungs, longissimus muscle, and spleen. On the basis of this information, suitable reference genes can be selected for mRNA expression studies in relevant pig tissues.

Haplotypes and gene expression implicate the MAPT region for Parkinson disease

Microtubule-associated protein tau (MAPT) has been associated with several neurodegenerative disorders including forms of parkinsonism and Parkinson disease (PD). We evaluated the association of the MAPT region with PD in a large cohort of familial PD cases recruited by the GenePD Study. In addition, postmortem brain samples from patients with PD and neurologically normal controls were used to evaluate whether the expression of the 3-repeat and 4-repeat isoforms of MAPT, and neighboring genes Saitohin (STH) and KIAA1267, are altered in PD cerebellum. Twenty-one single-nucleotide polymorphisms (SNPs) in the region of MAPT on chromosome 17q21 were genotyped in the GenePD Study. Single SNPs and haplotypes, including the H1 haplotype, were evaluated for association to PD. Relative quantification of gene expression was performed using real-time RT-PCR. After adjusting for multiple comparisons, SNP rs1800547 was significantly associated with PD affection. While the H1 haplotype was associated with a significantly increased risk for PD, a novel H1 subhaplotype was identified that predicted a greater increased risk for PD. The expression of 4-repeat MAPT, STH, and KIAA1267 was significantly increased in PD brains relative to controls. No difference in expression was observed for 3-repeat MAPT. This study supports a role for MAPT in the pathogenesis of familial and idiopathic Parkinson disease (PD). Interestingly, the results of the gene expression studies suggest that other genes in the vicinity of MAPT, specifically STH and KIAA1267, may also have a role in PD and suggest complex effects for the genes in this region on PD risk.

Conifer WOX-related homeodomain transcription factors, developmental consideration and expression dynamic of WOX2 during Picea abies somatic embryogenesis

In angiosperms, the WOX family of transcription factors has important functions in meristem regulation and in control of the partitioning of developing embryos into functional domains. In this study, a putative WOX2 homologous gene was isolated from Picea abies, and its expression pattern during somatic embryo development was followed using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). We used strategies of both absolute and relative quantification of gene expression, and benefits and disadvantages of the two methods are presented and discussed. During embryogenesis, PaWOX2 expression was highest at the earliest stages of development, but low levels were also detected in seedling tissues. No PaWOX2 expression was detected in a non-embryogenic cell culture, indicating that PaWOX2 plays a fundamental role during early somatic embryo development, and can be used as a possible marker for embryogenic potential. Additional results show that conifers, like angiosperms, contain a large number of WOX-related genes, many of them expressed during embryo development. In phylogenetic analysis based on the deduced homeodomain of retrieved pine and spruce EST sequences, no conifer WUS homolog was found. Neither did we find any homeodomain to cluster with WOX5. Interestingly, a clade including only conifer sequences derived from various tissues was resolved as sister to a Physcomitrella WOX-like gene, suggestive of the early origin of this gene family. Our results thus provide basic information for further studies of the evolution of this gene family and of their function in relation to meristem dynamics and specification of stem cells in gymnosperms.

Cytokine expression in the brains of Toxocara canis‐infected mice

Toxocara canis can infect a number of hosts including mice and humans. In the murine host, larvae exhibit a predilection for the central nervous system, resulting in an increasing number of parasites migrating to the brain as infection progresses. Previous studies have shown that larval burdens vary between individual outbred mice receiving the same inocula, suggesting a role for immunity in the establishment of cerebral infection. Although the systemic immune response to T. canis has been widely reported, there has been no investigation of the cerebral immune response. The aim of the present study was to characterize the cerebral immune response in two inbred strains of T. canis-infected mice (BALB/c and NIH) at several time points post-infection (p.i.). Relative quantification of gene expression in the brains of these mice showed increased expression of IL-5, IL-10, IFN-gamma and inducible nitric oxide synthase (iNOS). This response was detected as early as 3 days p.i., persisting up to 97 days p.i., and was more pronounced in BALB/c-infected mice. These results have implications for the role of these cytokines and iNOS in the cerebral establishment of T. canis, and in the cerebral pathology reported during infection.

Relative gene expression quantification in a fungal gas‐phase biofilter

Monitoring of gas-phase biofilter performance generally relies on macroscale measurements that neglect the molecular level phenomena that can control the biodegradation process. The present study was undertaken to determine whether or not quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) could detect changes in relative gene expression resulting from feed variations typically encountered in the field. Specifically, homogentisate-1,2-dioxygenase, ElHDO, expression was quantified as a function of short-term chemical feed variations and shutdown period in a biofilter seeded with a pure culture of the fungus Exophiala lecanii-corni. ElHDO was previously shown to be involved in ethylbenzene degradation in E. lecanii-corni. Overall, relative gene target expression numbers (T(N)) were consistent with gas-phase biofilter performance during each short-term experiment although no direct mathematical correlation was found between T(N) and ethylbenzene removal rate. During the chemical feed experiments, no effect on T(N) was measured in the presence of o-xylene which does not affect ElHDO expression. In the presence of phenylacetate, an inducer of ElHDO, T(N) increased once a threshold substrate concentration was exceeded. When methyl propyl ketone, a repressor of ElHDO, was introduced, T(N) decreased rapidly and acted as a leading indicator of bioreactor failure. In the transient loading experiments, ElHDO expression slowly decreased over a 24-h time period when the ethylbenzene feed was discontinued, but rapidly recovered upon its re-introduction. These results indicate that qRT-PCR reflects microbial activity changes that occur in gas-phase biofilters in response to short-term changes in feed conditions and provides a useful complement to the macroscale measurements typically collected.

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.

Molecular and functional characterization of Kv4.2 and KChIP2 expressed in the porcine left ventricle

Recent studies showed that the Ca(2+)-independent transient outward current (I (to)) is very small or even not detectable in the porcine left ventricle. We investigated whether an altered molecular expression or function of voltage-dependent potassium channels belonging to the Kv4 sub-family and their ancillary Ca(2+)-binding beta sub-unit KChIP2, which contribute to the major fraction of I (to )in other species, may underlie this lack of a significant I (to) in the porcine left ventricle. RT-PCR analysis with degenerate primers showed that both Kv4 mRNA and KChIP2 mRNA are expressed in porcine left ventricular tissue and in isolated ventricular myocytes. PCR cloning and sequence analysis predicted proteins with >98% identity to rat and human Kv4.2 and >99% identity to rat and human KChIP2. Heterologous expression of porcine Kv4.2 in Xenopus laevis oocytes gave rise to currents with characteristic properties of rat and human Kv4.2, and co-expression with its KChIP2 sub-unit increased current density (tenfold), slowed inactivation (twofold) and accelerated recovery from inactivation (tenfold). Kv4.2 immunohistochemistry in porcine left ventricular tissue revealed a predominant membrane-bound signal. Relative quantification of gene expression indicated that Kv4.2 and KChIP2 mRNA and protein are expressed at comparable ratios in porcine and rat left ventricular tissues, which displays a large I (to). Collectively, these data demonstrate that the lack of a significant I (to) in the porcine left ventricle does not result from dysfunctional or insufficiently expressed Kv4.2 and KChIP2 sub-units.

Alpha-tocopheryl succinate (α-TOS) modulates human prostate LNCaP xenograft growth and gene expression in BALB/c nude mice fed two levels of dietary soybean oil

Malignancy of the prostate constitutes a leading cause of cancer-related deaths in America and Europe. Alpha-tocopheryl succinate (alpha-TOS) has been shown to inhibit human prostate cancer growth in vitro, via several mechanisms, including inhibiting prostate-specific antigen (PSA) and vascular endothelial growth factor (VEGF) expressions. The route of alpha-TOS administration has a profound effect on its antitumor activity, and few studies have investigated its effects on prostate cancer growth in vivo. The present study tested the hypothesis that alpha-TOS wil reduce the growth of human prostate LNCaP tumors in mice fed low (7%) and high (20%) levels of dietary soybean oil, compared to the controls receiving vehicle, by modulating PSA and VEGF gene expressions in the tumor tissue. BALB/c nude mice (n = 42) were subcutaneously inoculated with 1 x 10(6) LNCaP cells and assigned to one of four dietary groups; 7% or 20% soybean oil diet with or without alpha-TOS treatment. Three weeks later, mice received daily intraperitoneal injections of alpha-TOS (100 mg/kg body weight) in sesame seed oil (SSO) for two weeks; controls received SSO injections. Tumor volumes were recorded weekly. Sera, liver, and tumor tissues were collected at seven weeks for serum PSA, testosterone and alpha-tocopherol analyses, histopathological examination, and reverse transcription and polymerase chain reaction (RT-PCR) amplification of PSA and VEGF gene fragments in tumors. Relative quantification of gene expression was performed using real-time PCR. P < or = 0.05 was considered significant. Intraperitoneal injections of alpha-TOS caused decreased tumor growth in both groups (7% and 20% fat, P < 0.05), versus controls. alpha-TOS treatment significantly reduced serum PSA and testosterone levels in comparison to the SSO-treated controls (P < 0.05). Control tumors had a greater degree of angiogenesis than alpha-TOS tumors, as demonstrated by the greater number of blood-filled vessels. PSA and VEGF mRNA expressions, were also reduced with alpha-TOS treatment (P < 0.05), revealing the possible molecular mechanisms of growth inhibition of LNCaP xenografts by alpha-TOS. Our study shows significant reduction in LNCaP xenograft growth with alpha-TOS treatment in nude mice fed a low (7%) and high (20%) fat soybean oil diets versus controls. Serum PSA and testosterone, tumor angiogenesis, and PSA and VEGF mRNA expressions were markedly reduced by alpha-TOS administration, suggesting a possible role of alpha-TOS as a chemotherapeutic agent in human prostate cancer, and warrants further investigations on the dose and delivery of alpha-TOS in humans.

P229 The Role of Inhibitor of Differentiation in the Angiogenesis of Head and Neck Cancers

termined the global gene expression profiles of primary OSCCs and identified a subset of deregulated genes that correlated with more advanced-stage tumors. A subset of these genes are involved in signal transduction genetic networks. Our objective is thus to examine the expression of these genes in OSCCs. Design: Tissue samples were obtained at the time of surgery from the Toronto General Hospital and snap frozen in liquid nitrogen for RNA extraction. Quantitative real-time reverse-transcriptase polymerase chain reaction using the ABI PRISM 7700 Sequence Detection System and the Sequence Detection System software (PE Applied Biosystems, Foster City, Calif) was used for relative quantification of gene expression. Data analysis was performed using the Delta Delta Ct method. Patients: Sixty-eight patients with OSCC who had surgery as primary treatment were included in this study. Results: Deregulated expression of -catenin, a gene involved in maintaining cell-to-cell adhesion in normal cells mediating the Wnt signal transduction pathway was detected. WISP1, WISP2, TCF4, and LEF1 genes are also associated with the Wnt pathway and were thus included in this study to further investigate whether the Wnt/b-catenin pathway is deregulated in head and neck cancer. Conclusion: Deregulated expression of genes involved the Wnt/beta-catenin pathway in OSCC.

P226 Lack of Exon 19 and 21 EGFR Mutations in Squamous Cell Head and Neck Cancer Specimens and Cell Lines

termined the global gene expression profiles of primary OSCCs and identified a subset of deregulated genes that correlated with more advanced-stage tumors. A subset of these genes are involved in signal transduction genetic networks. Our objective is thus to examine the expression of these genes in OSCCs. Design: Tissue samples were obtained at the time of surgery from the Toronto General Hospital and snap frozen in liquid nitrogen for RNA extraction. Quantitative real-time reverse-transcriptase polymerase chain reaction using the ABI PRISM 7700 Sequence Detection System and the Sequence Detection System software (PE Applied Biosystems, Foster City, Calif) was used for relative quantification of gene expression. Data analysis was performed using the Delta Delta Ct method. Patients: Sixty-eight patients with OSCC who had surgery as primary treatment were included in this study. Results: Deregulated expression of -catenin, a gene involved in maintaining cell-to-cell adhesion in normal cells mediating the Wnt signal transduction pathway was detected. WISP1, WISP2, TCF4, and LEF1 genes are also associated with the Wnt pathway and were thus included in this study to further investigate whether the Wnt/b-catenin pathway is deregulated in head and neck cancer. Conclusion: Deregulated expression of genes involved the Wnt/beta-catenin pathway in OSCC.