Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek-Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)'s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs.

KRAS mutations in primary tumours and post-FOLFOX metastatic lesions in cases of colorectal cancer.

BACKGROUND: KRAS mutations are predictive markers for the efficacy of anti-EGFR antibody therapies in patients with metastatic colorectal cancer. Although the mutational status of KRAS is reportedly highly concordant between primary and metastatic lesions, it is not yet clear whether genotoxic chemotherapies might induce additional mutations. METHODS: A total of 63 lesions (23 baseline primary, 18 metastatic and 24 post-treatment metastatic) from 21 patients who were treated with FOLFOX as adjuvant therapy for stage III/IV colorectal cancer following curative resection were examined. The DNA samples were obtained from formalin-fixed paraffin-embedded specimens, and KRAS, NRAS, BRAF and PIK3CA mutations were evaluated. RESULTS: The numbers of primary lesions with wild-type and mutant KRAS codons 12 and 13 were 8 and 13, respectively. The mutational status of KRAS remained concordant between the primary tumours and the post-FOLFOX metastatic lesions, irrespective of patient background, treatment duration and disease-free survival. Furthermore, the mutational statuses of the other genes evaluated were also concordant between the primary and metastatic lesions. CONCLUSION: Because the mutational statuses of predictive biomarker genes were not altered by FOLFOX therapy, specimens from both primary tumours and post-FOLFOX tumour metastases might serve as valid sources of DNA for known genomic biomarker testing.

KRAS mutations detected by the amplification refractory mutation system-Scorpion assays strongly correlate with therapeutic effect of cetuximab.

BACKGROUND: We aimed to compare the sensitive and quality-controlled KRAS testing with direct sequencing and to assess the impact on decision making of treatment. PATIENTS AND METHODS: We analysed genomic DNA isolated from macrodissected formalin-fixed paraffin-embedded specimens by direct sequencing and an amplification refractory mutation system-Scorpion assay (ARMS/S) method. Cetuximab was administered to patients identified as having wild-type (WT) KRAS using direct sequencing. Therapeutic effects were evaluated according to their KRAS status as determined by ARMS/S. RESULTS: Among the 159 patients, the overall mutation rate was determined to be 37.0% by direct sequencing and 44.0% by ARMS/S. For the patients diagnosed as WT by direct sequencing and treated with cetuximab (n=47), a response rate of 16.0% was observed for 38 ARMS/S WT patients, whereas 9 ARMS/S mutant (MUT) patients failed to respond. The ARMS/S WT patients showed significant improvement in progression-free survival (PFS) and overall survival (OS) compared with ARMS/S MUT patients (PFS median 5.0 vs 1.7 months, hazards ratio (HR)=0.29, P=0.001; OS median 12.1 vs 3.8 months, HR=0.26, P=0.001). CONCLUSION: Sensitive and quality-controlled KRAS testing may provide improved predictive power to determine the efficacy of anti-epidermal growth factor antibodies.

[Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 mRNA and its alternative splice variants in rats with experimental diabetes mellitus].

We studied the expression mRNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-2 (PFKFB-2) in the rat lung and kidney in experimental diabetes mellitus. For investigation we select two isoforms of PFKFB-2 with different C-terminus. The level of the expression of both PFKFB-2 mRNA isoforms is decreased in the kidney and lung in rats with experimental diabetes mellitus respect to the control animals. Moreover, four new alternative splice variants of PFKFB-2 mRNA were identified in the rat kidney. These splice variants of PFKFB-2 mRNA have different inserts and/or deletions in 6-phosphofructo-2-kinase as well as in fructose-2,6-bisphosphatase part of PFKFB-2. Three alternative splice variants cannot encode active 6-phosphofructo-2-kinase as a result of deletion of two catalytic domains (E and F). They encode fructose-2,6-bisphosphatase. It was shown that these alternative splice variants express in the kidney and lung and that this expression changes in rats with experimental diabetes mellitus with respect to the control animals. The results of this investigation clearly demonstrated that diabetes mellitus significantly affects the expression and alternative splicing of PFKFB-2 in the kidney and lungs and showed the complexity of regulatory mechanisms of glucose metabolism in this disease.

Expression of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-3 and VEGF mRNA in rat liver, lung and heart: effect of methyl tertbutyl ether.

The main goal of this work was investigation of the effect of methyl tertbutyl ether, ecologically dangerous chemical compound, on the expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) and vascular endothelial growth factor (VEGF) mRNA in different rat organs. Expression of PFKFB-3 and VEGF is a hypoxia inducible factor (HIF)-dependent process which significantly increases under hypoxia, in malignant tumors and other pathology. In this study we have shown that PFKFB-3 and VEGF mRNA expression in the liver, lung, and heart changes in rats, treated with methyl tertbutyl ether for two months, in organ-specific manner. Expression of alternative splice variants of PFKFB-3 mRNA as well as VEGF mRNA also changes in organ-specific manner in rats, treated with methyl tertbutyl ether. The effect of methyl tertbutyl ether on the expression of PFKFB-3 and VEGF mRNA and its alternative splice variants is dose-dependent. Results of this investigation clearly demonstrated that methyl tertbutyl ether affects the expression of PFKFB-3, a key regulatory enzyme of glycolysis, as well as VEGF, very important factor of angiogenesis, in an organ-specific and dose-dependent manner.

Expression of mouse 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 mRNA alternative splice variants in hypoxia.

Expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB-3) mRNA alternative splice variants was studied in different mouse tissues in hypoxic conditions in vivo. Significant increase of the expression of PFKFB-3 mRNA was observed in the mouse lungs, testes and brain in hypoxia. Several new alternative splice variants of PFKFB-3 mRNA were identified in the lung, testis, brain and skeletal muscle. They have different length and amino acid sequence of C-terminal regulatory part. However, 6-phosphofructo-2-kinase and fructose-2,6-bisphosphatase catalytic domains were identical. Moreover, the expression of different alternative splice variants of PFKFB-3 mRNA has shown tissue specificity and different levels of induction in hypoxic conditions in vivo. Results of this investigation indicate a possible role of PFKFB-3 splice isoform in cell adaptation to hypoxic conditions.

Alternative splice variants of rat 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase-4 mRNA.

Expression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase-4 (PFKFB-4) mRNA was studied in different rat organs. Several new unique alternative splice variants of PFKFB-4 mRNA were identified. They have deletions or inserts in fructose-2,6-bisphosphatase region as well as different length and amino acid sequence of C-terminal part. However, 6-phosphofructo-2-kinase catalytic domains were identical in all variants. Moreover, the expression of different alternative splice variants of PFKFB-4 mRNA has shown tissue specificity. Expression of both alternative splice variants of PFKFB-4 mRNA significantly changed in rats treated by methyl tretbutyl ether, ecologically dangerous chemical compound. Results of this investigation indicate a possible role of PFKFB-4 splice isoforms in cell-specific regulation of glycolysis and demonstrate the sensitivity of the regulation of alternative splicing to the action of toxic chemical compounds, in particular methyl tertbutyl ether.

[HIF-1alpha, HIF-2alpha and VHL mRNA expression in different cell lines during hypoxia].

Hypoxia inducible factor-1alpha (HIF-1alpha) mRNA expression is significantly decreased under hypoxia in different cell lines exposed directly to hypoxia or treated with dimethyloxalylglycine which mimics hypoxic effects under normoxic conditions. However, the decreased expression of HIF-1alpha mRNA is accompanied by an increase of HIF-1alpha protein (pHIF-1alpha) level as well as by overexpression of known HIF-dependent genes (VEGF, Glut1, PFKFB-3 and PFKFB-4) under hypoxic conditions or with the use of dimethyloxalylglycine. Expression of HIF-1alpha mRNA also depends on iron because desferrioxamine and cobalt chloride produce similar to hypoxia effects on the levels of this mRNA. It was shown that HIF-1alpha mRNA expression did not change significantly in some cell lines (SKBR3, MDA-MB468 and BT549) under hypoxia. However, in these cell lines hypoxia decreases expression of HIF-2alpha mRNA, another member of HIF-alpha gene family, as a result of cell specific regulation of HIF-alpha genes under hypoxia. Moreover, hypoxia slightly induces expression of PFKFB-4 mRNA in SKBR3, MDA-MB468 and BT549 as compared to other cell lines where this effect of hypoxia was much stronger and adaptation to hypoxia is controlled by HIF-1alpha. Hypoxia slightly reduces expression of tumor suppressor VHL which targets HIF-1alpha for ubiquitination. Thus, our results clearly demonstrated down regulation of HIF-1alpha or HIF-2alpha in different cell lines by hypoxia.

Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 in the human breast and colon malignant tumors.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is a bifunctional enzyme, which is responsible for maintaining the cellular level of fructose-2,6-bisphosphate, a powerful allosteric activator of glycolysis. We describe herein the overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-4 (PFKFB-4) isozyme in the human breast and colon malignant tumors as compared to corresponding non-malignant tissue counterparts. We have shown also that breast malignant cell line MCF7 constitutively express PFKFB-4 mRNA and that the expression of this gene is highly induced by hypoxia. Overexpression of PFKFB-4 transcript levels in breast and colon malignant tumors correlates with enhanced expression of PFKFB-3, hypoxia-inducible factor (HIF)-1alpha and known HIF-1 dependent genes glucose transporter 1 (Glut1) and vascular endothelial growth factor (VEGF). Thus, our data clearly demonstrates overexpression of PFKFB-4 mRNA and protein in the breast and colon malignant tumors.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene family overexpression in human lung tumor.

The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB) is a family of bifunctional enzymes which is responsible for maintaining the cellular levels of fructose-2,6-bisphosphate, a powerful allosteric activator of glycolysis. Here we report the overexpression of PFKFB-1, -2, -3 and -4 mRNA in the human lung cancers when compared with corresponding normal tissues counterparts as well as PFKFB-4 and -3 protein levels. The lung carcinoma cell line A549, under conditions of normal oxygen tension, has also shown increased transcript levels of PFKFB-2, -3 and -4 when compared to normal tissues. Moreover, hypoxia highly induced the expression of PFKFB-2, PFKFB-3 and especially PFKFB-4 isozymes are highly induced in the lung carcinoma cells. Thus, our results' clearly demonstrated overexpression of PFKFB gene family isozymes in the lung cancers and they possible role in the Warburg effect.

Strong association of ARK5 with tumor invasion and metastasis.

We recently identified a novel human AMPK family member, ARK5, and discovered that is a major factor in Akt-dependent cancer cell survival and migration activity through activation of MT1-MMPs in vitro. The mRNA expression of other AMPK family members and ARK5 was measured using RT-PCR in human colorectal carcinoma cell lines DLD-1, WiDr, HCT-15, SW620, LoVo, SW480, and mRNA expression of AMPK-alpha1, SNARK, MELK and ARK5, but not AMPK-alpha2, was detected in every line. Quantitative-PCR (Q-PCR) to estimate the amount of ARK5 mRNA expression in the cell lines showed that there is a variety of ARK5 expressions among the cell lines and high expression was observed in a cell line derived from the metastatic lesion, LoVo. To determine the effect of ARK5 overexpression on metastasis in vivo, we established human pancreas cancer cell line PANC-1 stably transfected with ARK5 full-length expression vector (P/ARK) and DLD-1 stably transfected with the same vector (D/ARK). Migration assay showed a remarkable increase in the activity both in P/ARK and D/ARK, and an in vivo metastasis assay showed a marked increase of P/ARK in liver metastasis. Based on these observations, it is suggested that ARK5 expression is involved in cancer invasion and metastasis.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of xenobiotics II: Studies with isolated perfused rat liver.

1. To elucidate the determining factors for elimination pathways of sulfate and glucuronide metabolites of xenobiotics, a single-pass perfusion of 4-methylumbelliferone (4MU) or p-nitrophenol (pNP) was performed with an isolated rat liver preparation. 2. Without bovine serum albumin in the perfusion system, clearance calculated based on the unbound concentration in the liver clearly showed that the net efflux clearances (CLeff) of sulfates from the sinusoidal membrane were much higher than those of glucuronides and that the biliary excretion clearances (CLb) of glucuronides were approximately two times larger than those of sulfates. 3. The ratios of CLeff to CLb were much higher for sulfates than those for glucuronides. The bile-oriented elimination of glucuronides or sinusoidal efflux-oriented elimination of sulfates was observed even using the perfusate including 3% bovine serum albumin, but the sinusoidal efflux of sulfates was extensively enhanced by bovine serum albumin in the perfusate. The mechanisms behind this stimulatory effect remain to be elucidated. 4. For both compounds, CLb of glucuronide was comparable with CLb of sulfate, meaning that CLb is not responsible for the biliary excretion of glucuronides at extensively higher rate than sulfates. 5. Higher concentration of glucuronides in the liver, partly caused by much lower CLeff of glucuronides than that of sulfates, is likely responsible for the bile-oriented excretion of glucuronides. The extensive sinusoidal efflux of sulfates, leading to the urine-oriented excretion, is attributed to the substantially higher CLeff than CLb. 6. In conclusion, the sinusoidal efflux is an important factor for determining elimination pathways of both sulfates and glucuronides, although further studies are needed to clarify the mechanisms of the sinusoidal efflux.

Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells.

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5' splice site (5'ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5' free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5'ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells.

Pharmacokinetic analysis of factors determining elimination pathways for sulfate and glucuronide metabolites of drugs. I: studies by in vivo constant infusion.

1. The hepatic and renal handling of glucuronides and sulphates of three phenolic compounds, 4-methylumbelliferone (4-MU), p-nitrophenol (pNP) and acetaminophen (APAP), were evaluated pharmacokinetically by in vivo constant infusion experiments in rat. It was shown that the urinary excretion rate at steady-state was larger than the biliary excretion rate for both glucuronides and sulfates, and sulfates, in particular, were extensively excreted into the urine. 2. For each glucuronide, however, biliary excretion clearances (CL(b)) calculated based on the total concentration and unbound concentration in the liver were much larger than the corresponding renal excretion clearances (CL(r)). Even in the case of sulfates, there was not any large difference between CL(r) and CL(b) based on the total and unbound concentration in tissues, which could not explain their extensive urinary excretion. From these results, these excretion clearances were recognized not to reflect necessarily the actual excretion rate obtained. 3. On the other hand, the tissue-to-plasma concentration ratio (K(p)) of both glucuronides and sulfates for every phenolic compound was much higher in the kidney than that in the liver. The results suggested that one of the most important determinants for the preferential excretion of these conjugates into the bile or urine is the extent of disposition of each compound to the liver or kidney. 4. In addition, K(p) of both glucuronides and sulfates in the liver, where these conjugates are mainly formed, was small. The K(p) of sulfates was quite low, suggesting that sulfates generated in the liver were subject to extensive sinusoidal efflux.

Loss of heterozygosity on chromosomes 9q and 16p in atypical adenomatous hyperplasia concomitant with adenocarcinoma of the lung.

Atypical adenomatous hyperplasia (AAH) has recently been implicated as a precursor to lung adenocarcinoma. We previously reported loss of heterozygosity (LOH) in tuberous sclerosis (TSC) gene-associated regions to frequently be observed in lung adenocarcinoma with multiple AAHs. In this study, we analyzed LOH in four microsatellite loci on 9q, including the TSC1 gene-associated region, and four loci on 16p, including the TSC2 gene-associated region, in both 18 AAHs and 17 concomitant lung adenocarcinomas from 11 patients. Seven of 18 (39%) AAHs and 9 of 17 (53%) adenocarcinomas displayed LOH on 9q. Five (28%) AAHs and seven (41%) adenocarcinomas harbored LOH at loci adjacent to the TSC1 gene. Four of 18 (22%) AAHs and 6 of 17 (35%) adenocarcinomas displayed LOH on 16p. One (6%) AAH and five (29%) adenocarcinomas harbored LOH at loci adjacent to the TSC2 gene. These findings may indicate a causal relationship of LOH on 9q and 16p in a fraction of AAH lesions and adenocarcinomas of the lung. Especially, the frequencies of LOH on 9q and at the TSC1 gene-associated region were high. The TSC1 gene or another neighboring tumor suppressor gene on 9q might be involved in an early stage of the pathogenesis of lung adenocarcinoma.

p53 gene alteration in atypical epithelial lesions and carcinoma in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is well known to be associated with lung cancer. Several atypical epithelial lesions are frequently observed in the fibrotic area in IPF patients, and they have been suspected to be related to lung carcinogenesis. Several studies have suggested that p53 protein accumulation and mutation occur in the early pathogenesis of squamous cell carcinoma of the lung, suggesting some abnormality of the p53 tumor-suppressor gene in interstitial lung diseases. To examine the cause of the high frequency of lung cancer in IPF, we examined the p53 changes in atypical epithelial lesions and carcinoma in patients with IPF by immunohistochemistry and mutational analysis. We examined 19 lung cancer patients with IPF who underwent surgical resection for lung cancer in our institute. Paraffin-embedded tissues were treated by microwave and stained with an anti-p53 antibody (RSP53) by the avidin-biotin-peroxidase complex method. Mutations in exons 5 through 8 of the p53 gene were also examined by polymerase chain reaction mediated single-strand conformation polymorphism (polymerase chain reaction-single-strand conformation polymorphism) analysis and DNA sequencing. p53 protein was immunohistochemically detected in 13 (62%) of 21 squamous cell carcinomas, 3 (60%) of 5 squamous metaplasia with atypia, 16 (54%) of 30 squamous metaplasia, and 1 (4%) of 26 other hyperplastic lesions. p53 mutation was detected in 12 (57%) of 21 squamous cell carcinomas, 2 (40%) of 5 squamous metaplasia with atypia, 7 (23%) of 30 squamous metaplasia, and 0 (0%) of 26 other hyperplastic lesions. In conclusion, there are frequent p53 gene alterations in squamous metaplasia, which is distributed in the peripheral zone of the fibrotic area in patients with IPF. The present findings might provide a clue to the molecular mechanisms underlying the high incidence of lung cancer, especially peripheral-type squamous cell carcinoma in IPF patients, and suggest that p53 gene alterations play an important role in the early stages of lung carcinogenesis in patients with IPF.

Cell cycle regulation via p53 phosphorylation by a 5'-AMP activated protein kinase activator, 5-aminoimidazole- 4-carboxamide-1-beta-D-ribofuranoside, in a human hepatocellular carcinoma cell line.

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) is an activator of AMP activated protein kinase (AMPK) and a regulator of de novo purine synthesis. There are several earlier reports indicating that AICAR treatment suppresses cell growth via regulation of AMPK or de novo purine synthesis. We found cell growth to be suppressed by AICAR treatment in HepG2 because of p53 accumulation, which was associated with p53-Ser15 phosphorylation. Moreover, a motif very similar to the consensus motif of AMPK phosphorylation was found around p53-Ser15, and Ser15 phosphorylation was detected in AICAR treated HepG2 as was in vitro phosphorylation by AMPK. Our results suggest that AICAR may regulate cell growth via p53 phosphorylation, and also indicate the possibility of p53 phosphorylation.

A novel method for detecting single glandular intestinal metaplasia in the mucosal surface of the fixed stomach using methylene blue.

A close association between intestinal metaplasia of the stomach and the well-differentiated type of gastric cancer is well recognized. The etiological relationship and how intestinal metaplasia contributes to gastric carcinogenesis are, however, still unclear. In order to answer this question, precise mapping and identification of the smallest lesion of intestinal metaplasia are desired. Establishment of an accurate and easy method for detecting intestinal metaplasia was the goal of this study. Surgical specimens of stomachs resected for gastric cancer were used. The specimens were stained with methylene blue, an oxidation-reduction marker, in whole mount, after fixation with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), and observed under a stereo-microscope. Normal gastric mucosa was stained blue, whereas intestinal metaplasia mucosa was not stained and had white or sky-blue island-like features. Intestinal metaplasia of complete type was unstained and showed white island-like features, while intestinal metaplasia of incomplete type showed sky-blue staining. With this method, we were able to detect even intestinal metaplasia composed of a single gland, when the intestinal metaplasia was of complete type. When stomach samples were stained in the presence of diphenyleneiodonium chloride (DPI), an inhibitor of nicotineamide adenine dinucleotide phosphate reduced form (NADPH) reductase, all the samples were homogeneously stained blue. Loss of the color of methylene blue was caused by the reductase activity of NADPH reductase, which is strongly and specifically expressed in intestinal metaplasia. A novel method for detecting intestinal metaplasia, even a single gland, was established.

Insulin up-regulates vascular endothelial growth factor and stabilizes its messengers in endometrial adenocarcinoma cells.

Angiogenesis is crucial for tumor growth and dissemination. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that promotes vascular growth and therefore tumoral growth and metastasis. Overweight, frequently associated with hyperinsulinemia, constitutes the major risk factor for endometrial carcinoma. Thus, elevated insulin levels may partly explain the increased risk of endometrial cancer observed in obese postmenopausal women. The aim of the present work was to test the role of insulin in the control of VEGF expression in endometrial carcinoma cells (HEC-1A). We have shown that insulin induced a biphasic expression of VEGF messenger ribonucleic acid, with an early, but low, induction (4 h of stimulation) and a delayed, but high, induction (24 h). The delayed effect of insulin on VEGF expression involved transcriptional and posttranscriptional regulation, as evidenced by the increased rate of VEGF transcription and the prolonged half-life of VEGF messenger ribonucleic acid. Simultaneously we observed higher levels of VEGF protein in the conditioned medium of stimulated cells compared with unstimulated ones. Therefore, insulin could contribute to the increased risk of endometrial carcinoma due to its ability to induce VEGF expression and thus participate in the maintenance of an angiogenic phenotype.

Identification of hypoxia-inducible factor 1 ancillary sequence and its function in vascular endothelial growth factor gene induction by hypoxia and nitric oxide.

Transcription of hypoxia-inducible genes is regulated by hypoxia response elements (HREs) located in either the promoter or enhancer regions. Analysis of these elements reveals the presence of one or more binding sites for hypoxia-inducible factor 1 (HIF-1). Hypoxia-inducible genes include vascular endothelial growth factor (VEGF), erythropoietin, and glycolytic enzyme genes. Site-directed mutational analysis of the VEGF gene promoter revealed that an HIF-1 binding site (HBS) and its downstream HIF-1 ancillary sequence (HAS) within the HRE are required as cis-elements for the transcriptional activation of VEGF by either hypoxia or nitric oxide (NO). The core sequences of the HBS and the HAS were determined as TACGTG and CAGGT, respectively. These elements form an imperfect inverted repeat, and the spacing between these motifs is crucial for activity of the promoter. Gel shift assays demonstrate that as yet unknown protein complexes constitutively bind to the HAS regardless of the presence of these stimuli in several cell lines, in contrast with hypoxia- or NO-induced activation of HIF-1 binding to the HBS. A common structure of the HRE, which consists of the HBS and the HAS, is seen among several hypoxia-inducible genes, suggesting the presence of a novel mechanism mediated by the HAS for the regulation of these genes.