VHL Expression Research Articles

Neutrophils from patients with heterozygous germline mutations in the von Hippel Lindau protein (pVHL) display delayed apoptosis and enhanced bacterial phagocytosis

AbstractNeutrophils are key mediators of the innate immune response and are required to function at sites of low oxygenation. We have shown that in hypoxia neutrophils are protected from apoptosis via a mechanism dependent on prolyl hydroxylase domain/hypoxia-inducible factor 1α (PHD/HIF-1α). This response would be predicted to involve the von Hippel Lindau protein (pVHL)–dependent ubiquitination and degradation of HIF-1α. Patients with VHL disease inherit a mutation in one VHL allele, which allows us to study the effects of heterozygous VHL expression in human neutrophils. Neutrophils exhibited a striking “partial hypoxic” pheno-type, with delayed rates of apoptosis and enhanced bacterial phagocytosis under normoxic conditions and preserved responses to low levels of oxygen. This provides direct evidence that the HIF-1α/VHL pathway regulates the innate immune response in humans. It also establishes that heterozygous VHL defects are sufficient to perturb normal responses and illustrates the potential to use this to address the role of HIF and VHL in human biology.

Restriction of rRNA Synthesis by VHL Maintains Energy Equilibrium under Hypoxia

Biological evolution abides by an unbending rule obligating organisms to maintain energy equilibrium. Hypoxia reduces cellular energy supply and is thus thought to be deleterious. We report that cells have evolved pH-sensitive mechanisms to maintain energy equilibrium by lowering energy demand. We found that fermentation-induced acidosis allows hypoxic cells to maintain energy equilibrium and viability under hypoxia by restricting ribosomal biogenesis, the most energy-demanding cellular process. Acidosis triggers nucleolar condensation, decreases precursor rRNA synthesis, reduces the dynamic profile of the RNA polymerase I preinitiation factor UBF1 and its interaction with the promoter of rRNA genes (rDNA). These changes require the pH-dependent interaction of the statically detained von Hippel-Lindau tumor suppressor protein (VHL) with rDNA. This phenomenon is promoted by, but does not require, activation of the hypoxia-inducible factor (HIF), a transcription factor implicated in extracellular acidification, energy production and oxygen homeostasis. Abrogating this program by silencing VHL expression, competing rDNA-VHL interaction or preventing environmental acidification triggers energy starvation and cell death under hypoxia. Our data suggest that oxygen-starved cells maintain energy equilibrium by gauging the environmental concentration of H+ to statically detain VHL to nucleolar rDNA and restrict ribosome production. These findings also provide an explanation for the protective effect of acidosis in ischemic settings such as development, stroke, and cancer.

Primary Cilium Formation Requires von Hippel-Lindau Gene Function in Renal-Derived Cells

Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene, VHL, occurs in the majority of renal clear cell carcinomas (RCC). VHL's function, regulating the degradation of hypoxia-inducible factor alpha (HIFalpha) subunits, explains the angiogenic nature of these tumors, but not tumor initiation. Because the development of renal cysts precedes tumor formation, and because the dysfunction of primary cilium is a common pathogenic mechanism in polycystic kidney diseases, we determined whether kidney-derived VHL- cells required VHL for the generation of cilium. Ectopic expression of VHL in RCC(VHL-) cells induced increased polarization and primary cilium formation. Cilium formation correlated directly with the expression of both wild-type VHL isoforms and a VHL mutant not associated with RCC development, whereas expression of RCC-associated VHL mutants did not support ciliogenesis. Requirement of VHL for ciliogenesis was independent of HIFalpha abundance. These data indicate separable independent functions for VHL (HIFalpha degradation and differentiation) and suggest a mechanism whereby disruption of both functions is required for renal carcinogenesis.

The von Hippel‐Lindau tumor suppressor gene expression level has prognostic value in neuroblastoma

Deletions of the short arm of chromosome 3 are often observed in a specific subset of aggressive neuroblastomas (NBs) with loss of distal 11q and without MYCN amplification. The critical deleted region encompasses the locus of the von Hippel-Lindau gene (VHL, 3p25). Constitutional loss of function mutations in the VHL gene are responsible for the VHL syndrome, a dominantly inherited familial cancer syndrome predisposing to a variety of neoplasms, including pheochromocytoma. Pheochromocytomas are, like NB, derived from neural crest cells, but, unlike NB, consist of more mature chromaffin cells instead of immature neuroblasts. Further arguments for a putative role of VHL in NB are its function as oxygen sensitizer and the reported relation between hypoxia and dedifferentiation of NB cells, leading to a more aggressive phenotype. To test the possible involvement of VHL in NB, we did mRNA expression analysis and sought evidence for VHL gene inactivation. Although no evidence for a classic tumor suppressor role for VHL in NB could be obtained, a strong correlation was observed between reduced levels of VHL mRNA and low patient survival probability (p=0.013). Furthermore, VHL appears to have predictive power in NTRK1 (TRKA) positive tumor samples with presumed favorable prognosis, which makes it a potentially valuable marker for more accurate risk assessment in this subgroup of patients. The significance of the reduced VHL expression levels in relation to NB tumor biology remains unexplained, as functional analysis demonstrated no clear effect of the reduction in VHL mRNA expression on protein stability of its downstream target hypoxia-inducible factor alpha.

Dynamic HIF1A Regulation During Human Placental Development1

The human placenta is a unique organ in terms of oxygenation as it undergoes a transition from a low to a more oxygenated environment. This physiological switch in oxygen tension is a prerequisite for proper placental development and involves the hypoxia inducible factor (HIF). HIF is stable and initiates gene transcription under hypoxia, whereas in normoxia, interaction with the von Hippel-Lindau tumor suppressor protein (VHL) leads to rapid degradation of the HIF1A subunit. The degradation requires formation of a multiprotein complex (VHLCBC) and hydroxylation of HIF1A proline residues via members of the egg-laying-defective nine (EGLN) family. Herein, we have investigated the regulatory mechanisms of HIF1A expression during human placental development. Expression of HIF1A and VHL was high at 7-9 wk of gestation, when oxygen tension is low, and decreased when placental oxygen tension increases (10-12 wk of gestation). During early placentation, HIF1A localized in cytotrophoblasts, while VHL was present in syncytiotrophoblasts. At 10-12 wk, VHL appeared in cytotrophoblast cells, which coincided with the disappearance of HIF1A. At the same time the association of VHL and Cullin 2 as well as ubiquitination of HIF1A was maximal. EGLN1, EGLN2, and EGLN3 were also temporally expressed in an oxygen-dependent fashion, with greatest mRNA expression at 10-12 wk of gestation. Inhibition of EGLN activity increased HIF1A stability in villous explants and stimulated transforming growth factor beta 3 (TGFB3) expression consistent with promoter analyses showing that HIF1A transactivates TGFB3. These data demonstrate that during placental development, HIF1A is regulated by temporal and spatial changes in expression and association of molecules forming the multi-protein VHLCBC complex as well as prolyl hydroxylase activities.

Hypoxia-Inducible Factor-1-Dependent Repression of E-cadherin in von Hippel-Lindau Tumor Suppressor–Null Renal Cell Carcinoma Mediated by TCF3, ZFHX1A, and ZFHX1B

A critical event in the pathogenesis of invasive and metastatic cancer is E-cadherin loss of function. Renal clear cell carcinoma (RCC) is characterized by loss of function of the von Hippel-Lindau tumor suppressor (VHL), which negatively regulates hypoxia-inducible factor-1 (HIF-1). Loss of E-cadherin expression and decreased cell-cell adhesion in VHL-null RCC4 cells were corrected by enforced expression of VHL, a dominant-negative HIF-1alpha mutant, or a short hairpin RNA directed against HIF-1alpha. In human RCC biopsies, expression of E-cadherin and HIF-1alpha was mutually exclusive. The expression of mRNAs encoding TCF3, ZFHX1A, and ZFHX1B, which repress E-cadherin gene transcription, was increased in VHL-null RCC4 cells in a HIF-1-dependent manner. Thus, HIF-1 contributes to the epithelial-mesenchymal transition in VHL-null RCC by indirect repression of E-cadherin.

Overexpression of von Hippel-Lindau tumor suppressor protein and antisense HIF-1α eradicates gliomas

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible-factors responsible for stimulating tumor angiogenesis and glycolysis, and targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to tumorigenesis and development of sporadic renal cell carcinomas and central nervous system hemangioblastomas. In the present study, we investigated whether engineered overexpression of pVHL in C6 glioma cells, which already express endogenous pVHL, would suppress the tumorigenicity of this particular tumor cell type. C6 cells overexpressing VHL displayed a reduced growth rate (70% inhibition) compared to the parental cell line when subcutaneously implanted in athymic (nu/nu) mice. Growth inhibition was associated with a 50% reduction in the number of tumor vessels and a 60% increase in tumor cell apoptosis, due in part to downregulation of HIF-1, VEGF, and the antiapoptotic factor Bcl-2, respectively. Gene transfer of VHL suppressed the growth of established C6 gliomas, and synergized with antisense HIF-1 to completely eradicate tumors. The data suggest that VHL gene therapy and/or agents that increase VHL expression could have utility in the treatment of gliomas, particularly when combined with agents that inhibit the expression or function of HIF-1.

Chorionic gonadotropin regulates the transcript level of VHL, p53, and HIF-2alpha in human granulosa lutein cells.

The ovarian corpus luteum plays a critical role in reproduction being the primary source of circulating progesterone. After ovulation the corpus luteum is build by avascular granulosa lutein cells through rapid vascularization regulated by gonadotropic hormones. The present study was performed to investigate whether this process might be influenced by the human chorionic gonadotropin (hCG)-dependent expression of different tumor suppressor genes and hypoxia dependent transcription factors. RNA was isolated from cultured granulosa lutein cells, transcribed into cDNA, and the transcript level of following genes were determined: RB-1, VHL, NF-1, NF-2, Wt-1, p53, APC, and hypoxia inducible factor-1 (HIF-1), -2, and -3alpha. Additionally, the influence of hCG on the expression of VHL, p53, and HIf2alpha were investigated. We demonstrate that in human granulosa lutein cells the tumor suppressor genes RB-1, VHL, NF-1, NF-2, Wt-1, p53, and APC and the hypoxia dependent transcription factors HIF-1alpha, -2alpha, and -3alpha are expressed. In addition, we showed that hCG regulates the expression of p53, VHL, and HIF-2alpha. Our results indicate that hCG may determine the growth and development of the corpus luteum by mediating hypoxic and apoptotic pathways in human granulosa lutein cells.

Effect of radiation and ibuprofen on normoxic renal carcinoma cells overexpressing hypoxia-inducible factors by loss of von Hippel-Lindau tumor suppressor gene function.

Tumor hypoxia is a major limiting factor for radiation therapy. Hypoxia-inducible factors (HIFs) are overexpressed in several human cancers and are considered prognostic markers and potential targets for cancer therapy. The purpose of the present study was to investigate the impact of HIFs on radiosensitivity. Renal clear cell carcinoma (RCC) cell lines overexpressing HIFs under normoxic conditions because of inactivation of von Hippel-Lindau tumor suppressor gene function (VHL-ve) and their matched pairs in which overexpression of HIFs was abolished by expression of functional VHL (VHL+ve) were irradiated. Radiosensitivity was determined by clonogenic assay. HIF and VHL protein levels were evaluated by Western blot analysis. RCC cells were also treated with ibuprofen, a radiosensitizer and HIF inhibitor in prostate cancer cells. The effect of ibuprofen on radiosensitization and HIF and VHL proteins was compared in RCC matched-pair cell lines. The data showed only small differences in the radiosensitivity between the cells overexpressing HIFs and cells with basal HIF levels. The dose-modifying factors for C2, 786-0, and A498 RCC cells were 1.14, 1.14 and 1.15, respectively. Radiation did not alter HIF or VHL protein levels. Ibuprofen inhibited HIFs in VHL+ve cells expressing basal levels of HIFs. In VHL-ve cells overexpressing HIFs, the inhibition was very modest. Ibuprofen radiosensitized C2 RCC cells to the same extent irrespective of their HIF status. Overexpression of HIFs in RCC cells harboring VHL mutations has only a modest effect on the radiosensitivity. Radiosensitization by ibuprofen appears to be independent of HIF status.

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha.

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.

Targeting of the VHL-hypoxia-inducible factor-hypoxia-induced gene pathway for renal cell carcinoma therapy.

Treatment of advanced renal cancer has made little progress in the past 30 yr. Most clinical efforts have incorporated cytokine-based therapy. The presumption has been that the cytokines may trigger a host immune response against the renal cancer. Only IFN-alpha and high-dose IL-2 seemed to have positive effects on patient outcomes. IFN has prolonged the lives of patients by a few months, and high-dose IL-2 is capable of inducing very prolonged remissions (>5 yr) for a small number of patients. Nephrectomy in the presence of metastatic disease has been established as an effective procedure for select patients, providing palliation and prolonging survival. Finally, enthusiasm has focused on the use of nonmyeloablative allogeneic stem cell transplantation and donor leukocyte infusion for the induction of graft versus tumor effects. Early results are both provocative and promising. A number of agents that target the critical gene products downstream from pVHL and hypoxia-inducible factor-1, such as vascular endothelial growth factor, PDGF, EGF receptor, and TGF-alpha, have recently become available. The new agents are capable of inhibiting specific cellular targets, and the biologic characteristics of clear cell carcinoma of the kidney support their application. If the correct targets are carefully selected for inhibition in tumors in which the targets are present (clear cell histologic features and loss of VHL expression), then results should resemble those others have observed with targeted therapy, such as the use of STI-571 (Gleevec; Novartis Pharmaceuticals, East Hanover, NJ) for treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors or anti-HER2/neu (Herceptin; Genentech, South San Francisco, CA) for treatment of breast cancer.

Genetic and functional analysis of the von Hippel-Lindau (VHL) tumour suppressor gene promoter

The VHL gatekeeper tumour suppressor gene is inactivated in the familial cancer syndrome von Hippel-Lindau disease and in most sporadic clear cell renal cell carcinomas. Recently the VHL gene product...

Inhibiting HIF-1 Transcriptional Activity

Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that regulates genes important for cellular adaptation to low O 2 conditions. The HIF-1α subunit is regulated under normoxia conditions by its interaction with the E3 ligase von Hippel-Lindau protein (VHL), which leads to ubiquitination and degradation. Mahon et al. used the yeast two-hybrid system to identify a protein, factor-inhibiting HIF-1 (FIH-1), that interacts with the transcriptional transactivation domain of HIF-1α. FIH-1 inhibits HIF-1 reporter gene activity under low O 2 conditions, and in a test that eliminates the proline hydroxylation-dependent VHL-mediated degradation of HIF-1, inhibition under normoxia conditions could also be detected. Glutathione- S -transferase fusion proteins were used to demonstrate that FIH-1, HIF-1α, and VHL could interact through nonoverlapping regions, indicating that a ternary complex could form. The effect of expression of VHL, FIH-1, or both, on reporter gene expression stimulated by fusion proteins of the GAL4 DNA binding domain and fragments of the COOH-terminus of HIF-1α representing the binding sites for VHL, FIH-1, or both, was tested. The fragment with only the VHL-binding site was only able to inhibit transcription when both VHL and FIH-1 were expressed, suggesting that VHL recruits FIH-1 and this is required for transcriptional inhibition. Finally, VHL interacted with histone deactylases in vitro; thus, recruitment of these corepressors of transcription may be one mechanism by which the transcriptional activity of HIF-1 is inhibited. P. C. Mahon, K. Hirota, G. L. Semenza, FIH-1: A novel protein that interacts with HIF-1α and VHL to mediate repression of HIF-1 transcriptional activity. Genes Dev. 15 , 2675-2686 (2001). [Abstract] [Full Text]

Molecular genetic analysis of the von Hippel-Lindau and human peroxisome proliferator-activated receptor gamma tumor-suppressor genes in adenocarcinomas of the gastroesophageal junction.

We investigated whether 2 candidate tumor-suppressor genes, VHL at 3p25-26 and PPAR gamma at 3p24.2-25, are involved in GEJ adenocarcinogenesis. In 43 GEJ tumor samples from 40 patients, the entire coding sequence of the VHL gene and the 5' and part of the 3' UTR as well as exons 3 and 5 of the PPAR gamma gene were screened by PCR-SSCP analysis. LOH at 3p25-26 was analyzed with 2 polymorphic microsatellite markers and with the VHL exon 1 and intron 2 polymorphisms. The relationship between LOH and clinicopathologic parameters was assessed. Expression of VHL was investigated by immunohistochemistry with a VHL-specific antibody. PCR-SSCP analysis of VHL revealed 2 different aberrant patterns in 19 patients. Upon DNA sequencing, 1 pattern appeared to be a previously described exon 1 polymorphism. The other single aberrant pattern was an intron 2 polymorphism, not yet described. PCR-SSCP analysis of PPAR gamma showed no aberrant migration patterns. LOH analysis revealed 3p25-26 loss in 24/36 (67%) informative cases, but this was not significantly correlated with clinicopathologic parameters. By immunohistochemistry, all tumors showed expression of VHL protein. Despite the very frequent LOH of 3p in GEJ adenocarcinomas, mutations in VHL and PPAR gamma were not detected. Mutations outside the screened sequences, a gene dosage effect or involvement of another tumor-suppressor gene on 3p as the target of LOH should be considered.

Coexpression of Erythropoietin and Vascular Endothelial Growth Factor in Nervous System Tumors Associated With von Hippel-Lindau Tumor Suppressor Gene Loss of Function

AbstractHemangioblastomas are highly vascular tumors of the central nervous system that overexpress the hypoxia-inducible gene, vascular endothelial growth factor (VEGF), as a consequence of mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL). Previous reports showed that hemangioblastomas can also express erythropoietin (Epo), which is also hypoxia-inducible. However, Epo expression in hemangioblastomas was observed only in individual cases, and the analyses were mainly based on indirect determination of erythropoiesis-stimulating activity. Therefore, we analyzed a series of 11 hemangioblastomas for Epo, VEGF, and VHL expression by Northern blot analysis and compared the results with normal brain and glioblastomas. Surprisingly, we observed Epo mRNA expression in all hemangioblastoma specimens analyzed, but in none of four glioblastomas. In contrast, VEGF mRNA was expressed in all hemangioblastomas and all glioblastomas. In situ hybridization revealed neoplastic stromal cells as Epo- and VEGF-producing cells in hemangioblastomas. These results suggest that in the nonhypoxic microenvironment of hemangioblastoma, Epo, similar to VEGF, might be negatively regulated by the VHL gene product.© 1998 by The American Society of Hematology.

Coexpression of Erythropoietin and Vascular Endothelial Growth Factor in Nervous System Tumors Associated With von Hippel-Lindau Tumor Suppressor Gene Loss of Function

Hemangioblastomas are highly vascular tumors of the central nervous system that overexpress the hypoxia-inducible gene, vascular endothelial growth factor (VEGF), as a consequence of mutational inactivation of the von Hippel-Lindau tumor suppressor gene (VHL). Previous reports showed that hemangioblastomas can also express erythropoietin (Epo), which is also hypoxia-inducible. However, Epo expression in hemangioblastomas was observed only in individual cases, and the analyses were mainly based on indirect determination of erythropoiesis-stimulating activity. Therefore, we analyzed a series of 11 hemangioblastomas for Epo, VEGF, and VHL expression by Northern blot analysis and compared the results with normal brain and glioblastomas. Surprisingly, we observed Epo mRNA expression in all hemangioblastoma specimens analyzed, but in none of four glioblastomas. In contrast, VEGF mRNA was expressed in all hemangioblastomas and all glioblastomas. In situ hybridization revealed neoplastic stromal cells as Epo- and VEGF-producing cells in hemangioblastomas. These results suggest that in the nonhypoxic microenvironment of hemangioblastoma, Epo, similar to VEGF, might be negatively regulated by the VHL gene product.© 1998 by The American Society of Hematology.

Putative control of angiogenesis in hemangioblastomas by the von Hippel-Lindau tumor suppressor gene.

The hypoxia-inducible endothelial cell-specific mitogen vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is expressed in low amounts in adult human brain, but is highly upregulated in the perinecrotic palisading cells of glioblastomas. We observed high VEGF expression in cerebellar hemangioblastomas, which are highly vascular, nonnecrotic and presumably nonhypoxic tumors, and hypothesized that a mechanism other than hypoxia leads to VEGF upregulation. Because hemangioblastomas develop in patients with von Hippel-Lindau disease, and mutations of the von Hippel-Lindau tumor suppressor (VHL) gene have also been reported in sporadic hemangioblastomas, we investigated VHL expression in normal cerebellum and in hemangioblastomas and tested the hypothesis that mutations in the VHL gene lead to upregulation of VEGE We observed constitutive expression of VHL mRNA, but downregulation of VEGF mRNA in the postnatal cerebellum. In the adult cerebellum, VHL is predominantly expressed in neuronal cells. In hemangioblastomas, VHL expression appears to be restricted to stromal cells, suggesting that the neoplastic component is the stromal cell. VHL-deficient renal cell carcinoma cells (786-0) produced significantly higher levels of VEGF mRNA and protein compared with 786-0/ wt10 cells, which were stably transfected with the wild-type VHL gene. Our observations suggest that VHL mutations affect stromal cells in hemangioblastomas and that VEGF is upregulated in stromal cells as a consequence of mutations in the VHL gene.

Defective placental vasculogenesis causes embryonic lethality in VHL-deficient mice.

Inheritance of an inactivated form of the VHL tumor suppressor gene predisposes patients to develop von Hippel-Lindau disease, and somatic VHL inactivation is an early genetic event leading to the development of sporadic renal cell carcinoma. The VHL gene was disrupted by targeted homologous recombination in murine embryonic stem cells, and a mouse line containing an inactivated VHL allele was generated. While heterozygous VHL (+/-) mice appeared phenotypically normal, VHL -/- mice died in utero at 10.5 to 12.5 days of gestation (E10.5 to E12.5). Homozygous VHL -/- embryos appeared to develop normally until E9.5 to E10.5, when placental dysgenesis developed. Embryonic vasculogenesis of the placenta failed to occur in VHL -/- mice, and hemorrhagic lesions developed in the placenta. Subsequent hemorrhage in VHL -/- embryos caused necrosis and death. These results indicate that VHL expression is critical for normal extraembryonic vascular development.

Post-transcriptional regulation of vascular endothelial growth factor mRNA by the product of the VHL tumor suppressor gene.

The VHL tumor suppressor gene is inactivated in patients with von Hippel-Lindau disease and in most sporadic clear cell renal carcinomas. Although VHL protein function remains unclear, VHL does interact with the elongin BC subunits in vivo and regulates RNA polymerase II elongation activity in vitro by inhibiting formation of the elongin ABC complex. Expression of wild-type VHL in renal carcinoma cells with inactivated endogenous VHL resulted in unaltered in vitro cell growth and decreased vascular endothelial growth factor (VEGF) mRNA expression and responsiveness to serum deprivation. VEGF is highly expressed in many tumors, including VHL-associated and sporadic renal carcinomas, and it stimulates neoangiogenesis in growing solid tumors. Despite 5-fold differences in VEGF mRNA levels, VHL overexpression did not affect VEGF transcription initiation or elongation as would have been suggested by VHL-elongin association. These results suggest that VHL regulates VEGF expression at a post-transcriptional level and that VHL inactivation in target cells causes a loss of VEGF suppression, leading to formation of a vascular stroma.

Natural killer (NK) cell lytic dysfunction and putative NK cell receptor expression abnormality in members of a family with chromosome 3p-linked von Hippel-Lindau disease.

BACKGROUND. Using antibodies to a putative natural killer (NK) cell receptor (pNKR), we recently cloned a novel cDNA and localized this gene to the short arm of human chromosome 3, region 3p21-3p24. Individuals susceptible to or clinically manifesting von Hippel-Lindau disease (VHL) have a genetic defect telomeric to this region on chromosome 3. This defect, resulting in VHL, is manifested by a high incidence of certain tumors. Based on the location of this gene, we sought to determine if VHL patients have a defect in gene expression of pNKR. Because of the proximity of the VHL and pNKR genetic regions, the variable expression of VHL tumors, and the ability of NK cells to target tumor cells, we investigated NK cell activity and other aspects of the immunologic status in 40 members (four branches) of a family with a high incidence of VHL tumors. Individuals affected with VHL and lacking in normal surface expression of pNKR had virtually no NK cell lytic activity. Analysis of genotypes and phenotypes of all subjects revealed that the greatest difference in NK cell lytic activity (P = .0002) was seen when family members exhibited both VHL and pNKR surface expression defects, compared with normal relatives who had neither defect. Furthermore, the lack of NK cell activity strongly correlated (P = .0005) with abnormal pNKR protein surface expression. Of particular interest, individuals who lacked NK cell activity had normal numbers of NK cells. In addition, analysis of leukocyte subsets indicated normal numbers of T and B cells, monocytes, and NK cells in both affected and normal individuals. These data indicate that although all affected individuals have the cell population responsible for NK cell activity, many have cells low in expression of pNKR and lack functional NK cell activity. Overall, these results indicate that, in addition to a predisposition to the development of neoplasms, VHL patients have a defect in a specific mechanism of natural immunosurveillance that correlates with a defect in expression of a novel large granular lymphocyte pNKR protein.