Von Hippel-Lindau Expression Research Articles

Expression of VHL and HIF-1α and Their Clinicopathologic Significance in Benign and Malignant Lesions of the Gallbladder

The Von Hippel-Lindau (VHL) gene is a tumor-suppressor gene. Recent studies have shown that low expression of VHL has a close relationship with tumor formation, progression, and prognosis. Hypoxia-inducible factor (HIF)-1α is a transcription factor, which exists universally in mammals including humans under states of hypoxia. Some studies have also shown that expression of HIF-1α closely correlates with tumor progression, angiogenesis, metastasis, and invasion. This molecule is also an important cytokine, which may be used to evaluate the prognosis of some malignant tumors. In this study, we studied the expression of VHL and HIF-1α and evaluated their clinicopathologic significance and relationship in benign and malignant lesions of the gallbladder. EnVision immunohistochemistry was used for detecting the expression level of VHL and HIF-1α in routinely paraffin-embedded sections from specimens of gallbladder adenocarcinoma (n=108), peritumoral tissues (n=46), adenomatous polyps (n=15), and chronic cholecystitis (n=35). The frequency of positive VHL expression was significantly lower in adenocarcinoma of gallbladder (48.1%) than that in peritumoral tissues (80.4%), adenomatous polyps (80.0%), and chronic cholecystitis (88.6%) (P<0.05 or P<0.01). In contrast, the positive HIF-1α expression was significantly higher in adenocarcinoma (53.7%) than that in peritumoral tissues (34.8%), adenomatous polyps (26.7%), and chronic cholecystitis (14.3%) (P<0.05 or P<0.01). The benign lesions with positive VHL and/or negative HIF-1α expression showed atypical hyperplasia in gallbladder epithelium. The positive expression of VHL and negative expression of HIF-1α were significantly associated with differentiation, tumor mass, lymph node metastasis, and invasion of adenocarcinoma (P<0.05 or P<0.01). The highly inconsistent expression of VHL and HIF-1α in gallbladder adenocarcinoma was found (P<0.01). Univariate Kaplan-Meier analysis showed that elevated expression of VHL (P=0.023) or lowered expression of HIF-1α (P=0.020) was closely associated with decreased overall survival. Multivariate Cox regression analysis showed that positive expression of VHL (P=0.013) and/or negative expression of HIF-1α (P=0.005) was an independent poor-prognostic predictor in gallbladder adenocarcinoma. The lowered expression of HIF-1α and elevated expression of VHL in gallbladder adenocarcinoma are important markers for the progression, clinical biological behavior, and prognosis. Measurement of VHL and HIF-1α expression could be a tool for early detection of gallbladder cancer in benign lesions and in population screening. The highly inconsistent expression of VHL and HIF-1α in gallbladder may require further study to see whether they are intrinsically related.

Meta-analysis of Clear Cell Renal Cell Carcinoma Gene Expression Defines a Variant Subgroup and Identifies Gender Influences on Tumor Biology

Background Clear cell renal cell carcinoma (ccRCC) displays molecular and histologic heterogeneity. Previously described subsets of this disease, ccA and ccB, were defined based on multigene expression profiles, but it is unclear whether these subgroupings reflect the full spectrum of disease or how these molecular subtypes relate to histologic descriptions or gender. Objective Determine whether additional subtypes of ccRCC exist and whether these subtypes are related to von Hippel-Lindau (VHL) inactivation, hypoxia-inducible factor (HIF) 1 and 2 expression, tumor histology, or gender. Design, setting, and participants Six large, publicly available ccRCC gene expression databases were identified that cumulatively provided data for 480 tumors for meta-analysis via meta-array compilation. Measurements Unsupervised consensus clustering was performed on the meta-arrays. Tumors were examined for the relationship of multigene-defined consensus subtypes and expression signatures of VHL mutation and HIF status, tumor histology, and gender. Results and limitations Two dominant subsets of ccRCC were observed. However, a minor third cluster was revealed that correlated strongly with a wild type (WT) VHL expression profile and indications of variant histologies. When variant histologies were removed, ccA tumors naturally divided by gender. This technique is limited by the potential for persistent batch effect, tumor sampling bias, and restrictions of annotated information. Conclusions The ccA and ccB subsets of ccRCC are robust in meta-analysis among histologically conventional ccRCC tumors. A third group of tumors was identified that may represent a new variant of ccRCC. Within definitively clear cell tumors, gender may delineate tumors in such a way that it could have implications regarding current treatments and future drug development.

Reduced Expression of von Hippel-Lindau Protein Correlates with Decreased Apoptosis and High Chondrosarcoma Grade

Mutations and loss of the von Hippel-Lindau (VHL) tumor suppressor gene are associated with most renal cancers as well as several other types of human tumors, but the potential role of the VHL protein (pVHL) in patients with chondrosarcoma has not been characterized. The purpose of the present study was to investigate the expression profiles of pVHL in chondrosarcoma and its association with clinicopathologic parameters, Bax expression, the apoptosis index, and overall survival of patients with chondrosarcoma. The messenger RNA (mRNA) and protein levels of VHL in fresh specimens from eight chondrosarcomas were studied with use of real-time polymerase chain reaction and Western blot, respectively. The protein expression of VHL and Bax was investigated by means of immunohistochemical analysis of paraffin-embedded clinical specimens from seventeen benign cartilage tumors and thirty-four chondrosarcomas. The apoptosis index in chondrosarcoma was examined by means of the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. Curves for overall survival were drawn according to the Kaplan-Meier method, and differences were analyzed with the log-rank test. The association of pVHL expression with the clinicopathologic parameters, Bax expression, apoptosis index, and overall survival for patients with chondrosarcoma was also analyzed. Levels of VHL protein (p = 0.005) and mRNA (p = 0.008) were significantly reduced in chondrosarcoma tissues as compared with the paired adjacent normal tissues. Immunohistochemical analysis showed decreased pVHL in a significantly higher proportion of chondrosarcomas (64.7%) than benign cartilage tumors (29.4%). pVHL expression was positively correlated with Bax expression and the apoptosis index in chondrosarcoma. Longitudinal studies of a cohort of patients with chondrosarcomas showed that decreased pVHL expression significantly correlated with increased tumor grade (p = 0.026) but was not independently predictive of overall survival. Reduced pVHL expression was associated with decreased apoptosis and increasing chondrosarcoma grade, but the relationship between these findings and chondrosarcoma pathogenesis requires further study.

Cancer cells promote survival through depletion of the von Hippel–Lindau tumor suppressor by protein crosslinking

Nuclear factor-κB (NF-κB) and insulin-like growth factor-1 (IGF-1)-mediated signaling is associated with different tumors including renal cell carcinoma. NF-κB- and IGF-1-mediated signaling is found to be inhibited in the presence of wild-type von Hippel-Lindau (VHL) tumor suppresser gene. Therefore, negative regulator of VHL may be a good target for regulating NF-κB and IGF-1R. In this study, we found that VHL, a tumor suppressor protein that downregulates the NF-κB activity and the stability of IGF-1R was depleted by TGase 2 through polymerization via crosslinking and proteasomal degradation in kidney, breast and ovary cancer cell lines. We also found that TGase 2 knockdown promotes hypoxia-inducible factor 1α (HIF-1α) degradation, and thereby decrease HIF-1α transcriptional activity. Importantly, VHL expression was decreased in vivo in TGase-2-transgenic mice, and this was associated with increased NF-κB activity and the levels of expression of IGF-1R, HIF-1α and erythropoietin in kidney tissue. These results suggest a novel mechanism of regulation of the VHL tumor suppressor by TGase 2 that appears to be independent of the known cancer regulatory mechanisms.

LW6, a novel HIF-1 inhibitor, promotes proteasomal degradation of HIF-1α via upregulation of VHL in a colon cancer cell line

Hypoxia-inducible factor HIF-1 is responsible for radiation resistance and poor prognosis in cancer therapy. As part of our drug discovery program, a novel HIF inhibitor, LW6, was identified as a small compound that inhibits the accumulation of HIF-1α. We found that LW6 decreased HIF-1α protein expression without affecting HIF-1β expression. MG132, a proteasome inhibitor, protected HIF-1α from LW6-induced proteasomal degradation, indicating that LW6 affects the stability of the HIF-1α protein. We found that LW6 promoted the degradation of wild type HIF-1α, but not of a DM-HIF-1α with modifications of P402A and P564A, at hydroxylation sites in the oxygen-dependent degradation domain (ODDD). LW6 did not affect the activity of prolyl hydroxylase (PHD), but induced the expression of von Hippel-Lindau (VHL), which interacts with prolyl-hydroxylated HIF-1α for proteasomal degradation. In the presence of LW6, knockdown of VHL did not abolish HIF-1α protein accumulation, indicating that LW6 degraded HIF-1α via regulation of VHL expression. In mice carrying xenografts of human colon cancer HCT116 cells, LW6 demonstrated strong anti-tumor efficacy in vivo and caused a decrease in HIF-1α expression in frozen-tissue immunohistochemical staining. These data suggest that LW6 may be valuable in the development of a HIF-1α inhibitor for cancer treatment.

Von Hippel-Lindau tumor suppressor gene-dependent mRNA stabilization of the survival factor parathyroid hormone-related protein in human renal cell carcinoma by the RNA-binding protein HuR

We have shown that parathyroid hormone-related protein (PTHrP) is a survival factor for human renal cell carcinoma (RCC) and that its expression is negatively regulated by the von Hippel-Lindau (VHL) tumor suppressor gene at the level of messenger RNA (mRNA) stability, as observed for tumor growth factors (TGFs). Our goals were to analyze the alternative splicing of PTHrP mRNA in human RCC and from these results to identify VHL/hypoxia-induced factor (HIF) system-regulated mRNA-binding proteins involved in PTHrP mRNA stability. We used: (i) a panel of human RCC cells expressing or not VHL; (ii) VHL-deficient 786-0 cells transfected with active or inactive VHL and (iii) human RCC samples and corresponding normal tissues. By quantitative real-time reverse transcription-polymerase chain reaction analysis, the 141 PTHrP mRNA isoform was found to be predominant in all cells and tumors (80%). In cells transfected with VHL, the expressions of all isoforms were decreased by 50%. Eight proteins with molecular weights ranging from 20 to 75 kDa were found to bind to biotinylated transcripts spanning the 141 PTHrP mRNA AU-rich 3'-untranslated region whose abundancy was dependent on VHL expression. The protein having an apparent molecular weight of 30 kDa was identified by western blot as HuR, a RNA-binding protein with stabilizing functions on various mRNA coding for proteins important in malignant transformation including vascular endothelial growth factor and TGF-beta. PTHrP expression studies confirmed the involvement of HuR in PTHrP upregulation in this disease. Common mRNA-binding proteins regulated by the VHL/HIF system may constitute new therapeutic opportunities against human RCC that remains refractory to therapies.

Overexpression of von Hippel-Lindau protein in skeletal muscles of patients with chronic obstructive pulmonary disease

Background:A significant number of patients with chronic obstructive pulmonary disease (COPD) exhibit skeletal muscle wasting and decreased capillary area formation, which correlate with increased mortality.Aim:To determine the molecular mechanisms mediating...

Sorafenib therapy and CNS hemangioblastomas in individuals with Von Hippel Lindau syndrome

2065 Background: Germline mutation of the tumor suppressor gene (3p25-p26) in the familial Von Hippel Lindau (VHL) syndrome predisposes to tumor induction and progression. Loss of VHL expression re...

Targeting the Nuclear Factor-κB Rescue Pathway Has Promising Future in Human Renal Cell Carcinoma Therapy

Metastatic renal cell carcinoma (RCC) remains refractory to therapies. The nuclear factor-kappaB (NF-kappaB) transcription factor is involved in cell growth, cell motility, and vascularization. We evaluated whether targeting NF-kappaB could be of therapeutic and prognostic values in human RCC. The activation of the NF-kappaB pathway in human RCC cells and tumors was investigated by Western blot. In vitro, the effects of BAY 11-7085 and sulfasalazine, two NF-kappaB inhibitors, on tumor cell growth were investigated by cell counting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and fluorescence-activated cell sorting. Their specificity toward NF-kappaB was analyzed by Western blot, confocal microscopy, NF-kappaB small interfering RNA, and NF-kappaB transcription assay. In vivo, the effects of BAY 11-7085 on the growth of human RCC tumors were investigated in nude mice. A tissue microarray (TMA) containing 241 cases of human RCC with 12 to 22 years of clinical follow-up and corresponding normal tissues was built up to assess prognostic significance of activated NF-kappaB. NF-kappaB is constitutively activated in cultured cells expressing or not the von Hippel-Lindau (VHL) tumor suppressor gene as a consequence of Akt kinase activation and in tumors. In vitro and in vivo NF-kappaB inhibition blocked tumor cell growth by inducing cell apoptosis. On the TMA, NF-kappaB activation was correlated with tumor dimension but was not found to be an independent prognostic factor for patient survival. This report provides strong evidence that the mechanisms responsible for the intrinsic resistance of RCC cells to apoptosis converge on NF-kappaB independently of VHL expression and that targeting this pathway has great anticancer potential.

The expression of the von Hippel-Lindau gene product and its impact on invasiveness of human breast cancer cells

The von Hippel-Lindau (VHL) gene is located on the short arm of chromosome 3, the mutations of which lead to the development of von Hippel-Lindau disease. The VHL gene is a putative tumour suppressor gene in VHL and a few other conditions, possibly by negative regulation of hypoxia- inducible factor-1 (HIF-1) and the stromal-derived factor-1 (SDF-1) receptor, CXCR4, via which the VHL protein negates angiogenesis and tumour cell migration. The current study investigated the expression of VHL at the mRNA and protein levels in clinical breast tumours and evaluated the impact of VHL on the invasion of human breast cancer cells in vitro. Primary breast cancer samples (n=124), adjacent non-cancerous breast tissues obtained from patients in cohort (n=33) and a panel of human breast cancer cells (n=12) were used. Tissue distribution of VHL protein in human breast cancer tissues was assessed using immunohistochemical analysis, and VHL transcript was determined using quantitative reverse transcription PCR. Breast cancer cell line MDA-MB-231 was transfected with a human VHL expression construct (pCR3-GFP/VHL) to allow forced overexpression of VHL in the cells. Invasiveness and migration of cancer cells were assessed using the Matrigel invasion and Cytodex-2 migration assays. Statistical analysis was performed using the Student's t-test. Our results showed that breast cancer cell lines MCF-7 and ZR-75-1 expressed very high levels of VHL transcripts, but the highly aggressive MDA-MB-231, MDA-MB-435 and MDA-MB-453 expressed either no VHL or a low level. The levels of VHL transcripts were significantly lower in grade 2 and grade 3 tumours (mean +/- SD, 1.36+/-0.55 and 0.9+/-0.37), compared with grade 1 tumours (12.3+/-7.6, p<0.002). Node-positive tumours had lower levels of VHL than node-negative tumours. Although tumours from patients with metastasis and from those who died of breast cancer had low levels of VHL, the most significant reduction in VHL was seen in tumours which developed local recurrence (p=0.03). The staining of VHL protein was most abundant in mammary epithelial cells and moderate in endothelial cells. Tumour cells in breast tissues had low to moderate VHL staining. pCR3-GFP/VHL-transfected MDA-MB-231 (MDA-MB-231VHL+) exhibited a reduced spontaneous in vitro invasiveness (14.8+/-2.7) compared with the control cells (18.4+/-1.4). MDA-MB-231VHL+ cells also lost their invasion response to HGF/SF, an invasion-inducing cytokine. The MDA-MB-231VHL+ cells had substantially reduced motility compared with that of the controls (14.8+/-0.7 for MDA-MB-231VHL+ and 20.7+/-1.2 for the control; p<0.001). Thus, VHL exerts inhibitory effects on the invasive and migratory capacity of breast cancer cells in vitro. Low levels of VHL occur in most aggressive breast tumours. Taken together, VHL is a powerful putative tumour suppressor gene in human breast cancer.

Expression of hypoxia-inducible factor 1 α, hypoxia-inducible factor 2 α, and von Hippel–Lindau protein in epithelial ovarian neoplasms and allelic loss of von Hippel-Lindau gene: nuclear expression of hypoxia-inducible factor 1 α is an independent prognostic factor in ovarian carcinoma

The hypoxia-inducible factor (HIF) is a transcriptional factor with important roles in tumor biology. To clarify the possible involvement of the HIF-alpha subunit and von Hippel-Lindau (VHL) protein in the development and progression of ovarian carcinoma, we analyzed the immunohistochemical expressions of HIF-1alpha, HIF-2alpha, and VHL in 107 cases of epithelial ovarian tumors. In addition, we examined loss of heterozygosity (LOH) at VHL gene loci. The frequency of the cytoplasmic expression of HIF-2alpha in carcinomas was higher than that in benign and borderline tumors (P < .0001). Furthermore, the nuclear expression of HIF-1alpha and the cytoplasmic expression of HIF-2alpha were significantly higher in tumors of FIGO (International Federation of Gynecology and Obstetrics) stages III and IV than in those of stages I and II. On the other hand, the cytoplasmic expression of HIF-1alpha did not show differences among histological malignancies. There was a positive correlation between nuclear HIF-1alpha expression and vascular endothelial growth factor (rho = 0.320, P < .001). Although LOH at the VHL gene locus was frequent in ovarian carcinomas (24%), there is no significant correlation between LOH and loss of VHL expression. In 22 clear cell carcinomas, VHL expression showed a significantly negative correlation with the nuclear expression of HIF-1alpha (rho = -0.529, P = .0153). The log-rank test showed that nuclear positive immunostaining for HIF-1alpha (P = .002) and cytoplasmic positive immunostaining for HIF-2alpha (P = .0112) in tumor cells are associated with poor prognosis of patients with ovarian carcinoma. Multivariate analysis also showed that the nuclear expression of HIF-1alpha is an independent prognostic factor. These results show that the HIF-alpha subunit represents an important biomarker in the evaluation of the prognosis of patients with ovarian carcinoma.

Over-expression of hypoxia-inducible factor 1 alpha in ovarian clear cell carcinoma

Objective. Unlike other histological types of epithelial ovarian carcinoma, ovarian clear cell carcinoma is known to have very poor response to therapy even when discovered in its early stages. Since tumor hypoxia has been shown to be strongly associated with poor prognosis, deregulation of the representative factor of tissue hypoxia; hypoxia-inducible factor 1 alpha (HIF-1α) and related protein; Von Hippel-Lindau (VHL) may be associated with poor prognosis of ovarian clear cell carcinoma. Methods. Immunolocalization of both HIF-1α and VHL was performed on 56 cases of paraffin-embedded tissue sections of four different histological types of epithelial ovarian carcinoma and 5 cases of benign ovarian tumors as a control. Quantitative RT-PCR analysis of both HIF1A and VHL was performed on RNA isolated from 61 microdissected frozen tissues of four different histological types of epithelial ovarian carcinoma and 6 cases of normal ovarian epithelial cells. Expression levels of HIF-1α and VHL in different histological types and correlation between HIF-1α and VHL were determined by nonparametric analysis by Kruskal–Wallis and Spearman’s test. Results. HIF-1α expression levels were significantly higher in ovarian clear cell carcinoma than in other histological types ( P = 0.001). We found no correlation between mRNA and protein expression level in any type of carcinoma specimens. Among endometrioid, serous, and mucinous carcinoma, there were no differences in HIF-1α expression ( P = 0.643). There was a negative correlation between HIF-1α and VHL in serous ( r = − 0.661, P = 0.027) and in endometrioid carcinoma ( r = − 0.657 P = 0.039), but no correlation was found between HIF-1α and VHL expression levels in ovarian clear cell carcinoma ( P = 0.60). Conclusions. The results suggest that the role of hypoxia may change according to the histological type of ovarian carcinoma. High expression of HIF-1α and its independence from VHL in ovarian clear cell carcinoma may confer chemoresistance in this histological type.

Modification in the von Hippel-Lindau Protein Is Involved in the Progression of Experimentally Induced Rat Glomerulonephritis

Background/Aims: We previously demonstrated that angiotensin II (AII) combined with Habu snake venom (HV) induces glomerulonephritis (GN) in rats, with lesions being restricted to the glomeruli 2 days after the administration of both reagents, but the mechanisms inducing GN are unclear. We also indicated a role for hypoxia-inducible factor (HIF)-1α in attenuating the progression of GN. However, a role of the von Hippel-Lindau (VHL) protein in GN and mechanisms by which HV regulates the pathogenesis of GN remains unclear. Methods and Results: Immunohistochemical analysis revealed that VHL is weakly expressed in the renal tubules alone; however, HV caused elevated VHL expression in the injured glomeruli including endothelial cells and partially podocytes. Western blot analysis revealed that VHL expression was increased in HV-treated kidney compared with AII-treated or normal kidney. An in vitro study also showed HV-induced elevation in VHL expression. To investigate whether VHL pre-induction causes GN aggravation, we utilized thrombin, an inducer of VHL. Thrombin alone did not cause renal injuries; however, thrombin pre-treatment accelerated the development of GN even 1 day after treatment. Conclusion: We suggest that VHL pre-induction by thrombin aggravates GN, and that the increase in VHL expression due to HV might be involved in accelerating onset of GN.

The von Hippel-Lindau tumor suppressor gene product represses oncogenic beta-catenin signaling in renal carcinoma cells.

Loss of von Hippel-Lindau (VHL) tumor suppressor gene function occurs in familial and most sporadic clear cell renal cell carcinoma (RCC), resulting in the aberrant expression of genes that control cell proliferation, invasion, and angiogenesis. The molecular mechanisms by which VHL loss leads to tumorigenesis are not yet fully defined. VHL loss has been shown to allow robust RCC cell motility, invasiveness, and morphogenesis in response to hepatocyte growth factor (HGF) stimulation, processes that are known to contribute to tumor invasiveness and metastatic potential. Among the most likely intracellular mediators of these HGF-driven activities is beta-catenin, a structural link between cadherens and the actin cytoskeleton, as well as a gene transactivator. We show that reconstitution of VHL expression in RCC cells repressed HGF-stimulated beta-catenin tyrosyl phosphorylation, adherens junction disruption, cytoplasmic beta-catenin accumulation, and reporter gene transactivation in RCC cells. Ectopic expression of a ubiquitination-resistant beta-catenin mutant specifically restored HGF-stimulated invasion and morphogenesis in VHL-transfected RCC cells. VHL gene silencing in non-RCC renal epithelial cells phenotypically mimicked VHL loss in RCC, and HGF-driven invasiveness was blocked by the expression of a dominant-negative mutant of Tcf. We conclude that, unlike many other cancers, where HGF pathway activation contributes to malignancy through the acquisition of autocrine signaling, receptor overexpression, or mutation, in RCC cells VHL loss enables HGF-driven oncogenic beta-catenin signaling. These findings identify beta-catenin as a potential target in biomarker and drug development for RCC.

Current status and future directions of molecular markers in renal cell carcinoma

To describe the existing data regarding the prognostic and/or predictive value of molecular markers in metastatic renal cell carcinoma. Several molecular markers have been investigated in metastatic renal cell carcinoma for prognostic and/or predictive potential, many of which relate to pathogenic molecular alterations that are now therapeutic targets. Carbonic anhydrase IX is a von Hippel-Lindau (VHL)-mediated enzyme expressed in the majority of renal cell carcinoma tumors. High (>85%) expression of carbonic anhydrase IX in renal cell carcinoma tumors is a favorable prognostic marker and may predict response to interleukin-2 therapy. B7-H1 is a glycoprotein with inhibitory effects on T cells. B7-H1 expression in renal cell carcinoma tumor cells and/or lymphocytes may confer a worse survival in renal cell carcinoma patients, perhaps through impairment of inherent host of antitumor immunity. VHL pathway elements, including VHL gene status and expression of vascular endothelial growth factor protein and receptor, represent a promising area of investigation to predict responses to vascular endothelial growth factor-targeted agents. Prognostication schemas are beginning to incorporate molecular markers with clinical variables to refine the prediction of patient outcome. Several molecular markers appear promising to refine prognosis and prediction in metastatic renal cell carcinoma. Further prospective evaluation is needed.

Interplay between VHL/HIF1α and Wnt/β-catenin pathways during colorectal tumorigenesis

Activation of the Wnt signaling pathway initiates the transformation of colorectal epithelial cells, although the transition to metastatic cancer requires angiogenesis. We have investigated the expression of the von Hippel-Lindau (VHL) tumor suppressor in the intestines from humans and mice. Here, we show that VHL expression is regulated by TCF4 and is restricted to the proliferative compartment at the bottom of intestinal crypts. Accordingly, VHL is completely absent from the proliferative intestinal pockets of Tcf4(-/-) perinatal mice. We observed complementary staining of the hypoxia-inducible factor (HIF) 1alpha to VHL in normal intestinal epithelium as well as in all stages of colorectal cancer (CRC). To the best of our knowledge, this is the first report demonstrating the presence of nuclear HIF1alpha in normoxic healthy adult tissue. Although we observed upregulated levels of VHL in very early CRC lesions from sporadic and familial adenomatous polyposis patients - presumably due to activated Wnt signaling - a clear reduction of VHL expression is observed in later stages of CRC progression, coinciding with stabilization of HIF1alpha. As loss of VHL in later stages of CRC progression results in stabilization of HIF, these data provide evidence that selection for VHL downregulation provides a proangiogenic impulse for CRC progression.

Analysis of the hypoxia-sensing pathway in Drosophila melanogaster

The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1alpha (hypoxia-inducible factor-1 alpha subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1alpha to the ubiquitin/proteasome pathway. HIF-1alpha thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1alpha and Similar, the Drosophila homologue of HIF-1alpha. The enzyme promotes human and Drosophila [(35)S]VHL binding to GST (glutathione S-transferase)-ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD-GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD-GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.

Expression of von Hippel Lindau (pVHL) Protein in Placentae from Normal Pregnant Women and Women with Preeclampsia

The hypoxia inducible transcription factors, HIF-1alpha and -2alpha proteins, are overexpressed in placentae from women with preeclampsia (Biol Reprod 2001;64:499-506; Biol Reprod 2001;64:1019-1020). Normally, these proteins are regulated in an oxygen-dependent manner being rapidly degraded by the ubiquitin-mediated proteasomal pathway. Recent studies have shown that the tumor suppressor protein, von Hippel Lindau (VHL), targets HIF for ubiquitinylation under nonhypoxic conditions. The objectives of the present work were: (1) to investigate VHL protein expression in normal pregnant (NP), preeclamptic (PE), and preterm (without PE) placentae, (2) to test whether VHL protein is hypoxia inducible in term and first trimester placental villous explants, and (3) to analyze the ontogeny of VHL protein expression in the human placenta. To begin evaluating the potential contribution of VHL to HIF overexpression in preeclamptic placentae, we analyzed the levels of the VHL protein in both normal and preeclamptic placentae (n=7 each). We hypothesized a deficiency of VHL protein in preeclamptic placentae. Eight biopsy sites were tested in each placenta and protein extracts were made. Western analysis was performed using VHL specific antibodies. Human renal adenocarcinoma (ACHN) cell extracts and extracts from COS-7 cells transfected with a VHL expression vector were used as positive controls. In a total of 112 biopsy sites that were analyzed (56 each for normal and preeclamptic placentae), the composite densitometry ratios (PE/NP) for the long (28 kDa) and short (19 kDa) forms of VHL were 1.09+/-0.2 and 1.16+/-0.11, respectively (both p=NS vs 1.0). A ratio of 1.0 indicates equal expression by preeclamptic and normal placentae. The same placentae exhibited composite densitometry (PE/NP) ratios of 1.97+/-0.23 and 1.68+/-0.20 for HIF-1alpha and -2alpha proteins, respectively (both p<0.05 vs 1.0). In a separate analysis, the protein expression of the short form of VHL was also comparable among NP, PE and preterm (n=6) placentae. VHL immunoreactivity was localized to cells within the basal plate and the syncytiotrophoblast. Despite induction of HIF proteins by hypoxia in first and term villous explants, there was no significant upregulation of VHL proteins. Finally, the expression of both the short and long forms of VHL protein decreased with gestational age (both p<0.05 by ANOVA), and in villous tissue from first trimester placentae VHL immunoreactivity was predominantly localized to the cytotrophoblast. These results suggest that (1) deficiency of VHL protein does not account for HIF-alpha overexpression in preeclamptic placentae, (2) VHL protein is not regulated by hypoxia in either first trimester or term placental villous explants, and (3) VHL protein expression in the placenta decreases as a function of gestational age.

Activation of HIF-prolyl Hydroxylases by R59949, an Inhibitor of the Diacylglycerol Kinase

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

Gene transfer of von Hippel-Lindau inhibits the growth of transplanted solid tumors

To explore the effect of von Hippel-Lindau(VHL) gene on growth of EL-4 solid tumors in vivo. C57BL/6 mice model of solid tumors was established by subcutaneous injection of EL-4 lymphoma cells. Mice were randomly divided into two groups as treatment group (n=6) and control group (n=6) when tumor diameter increased to 0.1 cm and 0.4 cm respectively. Plasmid pcDNA3-VHL was injected into solid tumor in treatment group, empty pcDNA3 vector in control group. The growth of tumor was observed. Immunohistochemistry and Western blot analysis were used to examine the transgenic expression of VHL, hypoxia inducible factor-1alpha (HIF)-1alpha, bcl-2 and VEGF. Microvessel density (MVD) and apoptosis index (AI) of tumors were also detected. VHL gene transfer eradicated tumors with small size (0.1 cm diameter), but it only retarded the growth of large tumors (0.4 cm diameter). VHL was overexpressed, the expression levels of VEGF, HIF-1alpha and bcl-2 were reduced in treatment group compared with those in the control group. The level of MVD was significantly lower in treatment group (P< 0.05), but AI was higher in treatment group compared with those in the control group (P< 0.01). VHL gene therapy can inhibit the growth of EL-4 solid tumor in vivo.