Carcinoma, Renal Cell ; genetics ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Kidney Neoplasms ; genetics ; metabolism ; pathology ; Loss of Heterozygosity ; Mutation ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; metabolism ; von Hippel-Lindau Disease ; genetics ; metabolism ; pathology

OBJECTIVETo explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell.

METHODSAfter whole genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing.

RESULTSThe rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67%(8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%.

CONCLUSIONWGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.

Base Sequence ; DNA Mutational Analysis ; Female ; Genotype ; Humans ; Lymphocytes ; metabolism ; pathology ; Middle Aged ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Preimplantation Diagnosis ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; blood ; genetics

Hypoxia-inducible factor 1alpha (HIF-1alpha) is rapidly degraded by the ubiquitin-proteasome pathway under normoxic conditions. Ubiquitination of HIF-1alpha is mediated by interaction with von Hippel-Lindau tumor suppressor protein (pVHL). In our previous report, we found that hypoxia-induced active signal transducer and activator of transcription3 (STAT3) accelerated the accumulation of HIF-1alpha protein and prolonged its half-life in solid tumor cells. However, its specific mechanisms are not fully understood. Thus, we examined the role of STAT3 in the mechanism of pVHL-mediated HIF-1alpha stability. We found that STAT3 interacts with C-terminal domain of HIF-1alpha and stabilizes HIF-1alpha by inhibition of pVHL binding to HIF-1alpha. The binding between HIF-1alpha and pVHL, negative regulator of HIF-1alpha stability, was interfered dose-dependently by overexpressed constitutive active STAT3. Moreover, we found that the enhanced HIF-1alpha protein levels by active STAT3 are due to decrease of poly-ubiquitination of HIF-1alpha protein via inhibition of interaction between pVHL and HIF-1alpha. Taken together, our results suggest that STAT3 decreases the pVHL-mediated ubiquitination of HIF-1alpha through competition with pVHL for binding to HIF-1alpha, and then stabilizes HIF-1alpha protein levels.
Animals ; COS Cells ; Cell Line, Tumor ; Cercopithecus aethiops ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/*metabolism ; Immunoblotting ; Immunoprecipitation ; Protein Binding ; STAT3 Transcription Factor/genetics/*metabolism ; Signal Transduction/genetics/physiology ; Transfection ; Ubiquitination ; Von Hippel-Lindau Tumor Suppressor Protein/genetics/*metabolism

Adenoma ; metabolism ; pathology ; Adult ; Carcinoma, Renal Cell ; metabolism ; pathology ; Diagnosis, Differential ; Ear Neoplasms ; complications ; genetics ; metabolism ; pathology ; surgery ; Endolymphatic Sac ; Female ; Follow-Up Studies ; Humans ; Kidney Neoplasms ; metabolism ; pathology ; Middle Aged ; Paraganglioma ; metabolism ; pathology ; Point Mutation ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; complications ; genetics ; metabolism

OBJECTIVETo investigate clinicopathological features, molecular genetic characteristics, differential diagnoses and prognosis of renal cell carcinoma in teenagers.

METHODSMicroscopic and immunohistochemical features of 46 cases of renal cell carcinomas in teenagers were reviewed along with the clinical follow-up data. Loss of heterozygosity (LOH), analysis of von Hippel-Lindau (VHL) gene and screening for VHL gene mutations were performed in all of the tumors.

RESULTSThere were 19 Xp11.2 translocations/TFE3 gene fusions renal clear cell carcinomas (Xp11 RCCs), 9 chromophobe renal cell carcinomas (CCRCCs), 17 papillary renal cell carcinomas (PRCCs), and 1 unclassified renal cell carcinoma (RCC). All of the 19 Xp11.2 translocation RCCs showed a moderate to strong immunoreactivity for TFE, however, no TFEB expression was obtained. There were 4 histological patterns in the Xp11 RCC cases including: 8 tumors possessing a nested to papillary architecture resembling to the t(X;17) ASPL-TFE3 phenotype; 6 tumors possessing a morphologic feature like the t(X;1) PRCC-TFE3 phenotype; 4 cases morphologically resembling to clear cell RCC; and 1 Xp11 RCC case, with a special morphologic feature not searched yet in the literature, including a ground glass appearance of the nuclei accompanying occasionally with grooves on the nuclear surface; nucleoli inconspicuous with accumulation of abundant mucin-like substance in the stroma. VHL gene analysis revealed deletions at 3p25-26 in one clear cell RCC and one papillary type 2 RCC. The papillary type 2 RCC had also a family history of VHL disease, with a germline G→C mutation at a splicing site of position 553+5. There were no VHL mutations detected in the remaining 45 RCCs. Statistical analysis of tumor stage and outcome revealed that TFE+ RCCs of teen-agers were more frequently associated with a higher pT3/pT4 stage and a poorer outcome than that of the TFE-RCCs (P < 0.05).

CONCLUSIONSRCCs of the teenagers have a different morphologic spectrum and genetic background from the RCCs seen in adults. Among RCCs of the teen-agers, Xp11.2 translocation tumors are the most common RCCs and have a poorer prognosis than that of the TFE-RCCs.

Adolescent ; Adult ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; metabolism ; Carcinoma, Papillary ; genetics ; metabolism ; pathology ; Carcinoma, Renal Cell ; genetics ; metabolism ; pathology ; Child ; Child, Preschool ; Chromosomes, Human, Pair 11 ; Chromosomes, Human, X ; Diagnosis, Differential ; Female ; Follow-Up Studies ; Gene Fusion ; Humans ; Kidney Neoplasms ; genetics ; metabolism ; pathology ; Loss of Heterozygosity ; Male ; Neoplasm Staging ; Neprilysin ; metabolism ; Phenotype ; Survival Rate ; Translocation, Genetic ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; Young Adult ; von Hippel-Lindau Disease ; genetics

OBJECTIVETo evaluate the significance of somatic mutations of VHL gene and hypoxia-inducible factor-1alpha (HIF-1alpha) expression in primary renal clear cell carcinoma (RCC).

METHODSMutation of VHL gene and HIF-1alpha expression were detected by means of PCR, denaturing high-performance liquid chromatography (DHPLC), direct sequencing and immunohistochemistry in 32 samples from primary renal clear cell carcinoma patients.

RESULTSIn 32 RCC samples, 17 samples (53.1%) had and 32 samples of adjacent nonmalignant renal tissue had not mutations of VHL gene expression. Twelve RCC samples (70.6%) which had mutations of VHL gene expressed HIF-1alpha, and it had significant difference to 4 RCC (26.7%) samples which didn't have mutations of VHL gene (P < 0.05).

CONCLUSIONMutations of VHL gene may play a significant role in the tumorigenesis of RCC, and HIF-1alpha expression correlates with it.

Adenocarcinoma, Clear Cell ; genetics ; pathology ; Adult ; Aged ; Carcinoma, Renal Cell ; genetics ; pathology ; Chromatography, Liquid ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Immunohistochemistry ; Kidney ; chemistry ; metabolism ; pathology ; Kidney Neoplasms ; genetics ; pathology ; Male ; Middle Aged ; Mutation ; genetics ; Polymerase Chain Reaction ; Transcription Factors ; analysis ; genetics ; Tumor Suppressor Proteins ; analysis ; genetics ; Ubiquitin-Protein Ligases ; analysis ; genetics ; Von Hippel-Lindau Tumor Suppressor Protein

In present study, we investigated the mechanism of regulating HIF-1alpha expression by hydroxysafflor yellow A (HSYA) in Eahy 926 cell line under 1% O2 hypoxia. Eahy 926 cells were incubated with HSYA (100, 10 and 1 micromol x L(-1)) under hypoxia for the indicated time after treatment. Cell proliferation rate was detected using MTT assays. VHL and p53 location and protein expression were analyzed by immunocytochemical stain. HIF-1alpha, VHL and p53 mRNA expression were detected by RT-PCR. Protein expression of HIF-1alpha, VHL and p53 were assayed by Western blotting method. HSYA at 100 micromol x L(-1) increased Eahy 926 cells proliferation rate under hypoxia. HIF-1alpha mRNA and protein expression were up-regulated in the presence of HSYA. VHL, p53 mRNA and protein expression decreased significantly after 8 hours of treatment under hypoxia. HSYA protected Eahy 926 cells from hypoxia, and up-regulated HIF-1alpha expression partially via its inhibition of VHL and p53 expression.
Carthamus tinctorius ; chemistry ; Cell Hypoxia ; Cell Line ; Cell Proliferation ; drug effects ; Chalcone ; analogs & derivatives ; isolation & purification ; pharmacology ; Endothelial Cells ; cytology ; metabolism ; Flowers ; chemistry ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Plants, Medicinal ; chemistry ; Quinones ; isolation & purification ; pharmacology ; RNA, Messenger ; metabolism ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Umbilical Veins ; cytology ; Up-Regulation ; Von Hippel-Lindau Tumor Suppressor Protein ; biosynthesis ; genetics

In this study, we investigated the role of Nur77, an orphan nuclear receptor, in HIF-alpha transcriptional activity. We found that Nur77 associates and stabilizes HIF-1alpha via indirect interaction. Nur77 was found to interact with pVHL in vivo via the alpha-domain of pVHL. By binding to pVHL, Nur77 competed with elongin C for pVHL binding. Moreover, Nur77-binding to pVHL inhibited the pVHL-mediated ubiquitination of HIF-1alpha and ultimately increased the stability and transcriptional activity of HIF-1alpha. The ligand-binding domain of Nur77 was found to interact with pVHL and the expression of this ligand-binding domain was sufficient to stabilize and transactivate HIF-1alpha. Under the conditions that cobalt chloride was treated or pVHL was knocked down, Nur77 could not stabilize HIF-alpha. Moreover, Nur77 could not further stabilize HIF-2alpha in A498/VHL stable cells, which is consistent with our finding that Nur77 indirectly stabilizes HIF-alpha by binding to pVHL. Thus, our results suggest that an orphan nuclear receptor Nur77 binds to pVHL, thereby stabilizes and increases HIF-alpha transcriptional activity under the non- hypoxic conditions.
Animals ; DNA-Binding Proteins/chemistry/*metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Models, Biological ; PC12 Cells ; Protein Binding ; *Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Rats ; Receptors, Cytoplasmic and Nuclear/chemistry/*metabolism ; Receptors, Steroid/chemistry/*metabolism ; Thermodynamics ; Transcription Factors/chemistry/*metabolism ; Transcriptional Activation/genetics ; Ubiquitination ; Up-Regulation/*genetics ; Von Hippel-Lindau Tumor Suppressor Protein/*antagonists & inhibitors/chemistry/*metabolism

OBJECTIVETo evaluate the expression of hypoxia inducible factor (HIF)-1alpha, 2alpha in sporadic clear cell renal cell carcinoma and their relationships to the mutations of von Hippel-Lindau (VHL) gene.

METHODSMutations of VHL gene, expression of HIF-1alpha and 2alpha were detected by polymerase chain reaction (PCR), direct DNA sequencing and immunohistochemistry in 77 cases of Chinese sporadic clear cell renal cell carcinoma (CCRCC). The stage was pT(1)N(0)M(0)in 55 patients (71%), pT(2)N(0)M(0) in 7 patients (9%), pT(3)N(0)M(0) in 14 patients (18%), and pT(4)N(0)M(0) in 1 patient (1%). The classification according to the tumor nuclear grading system showed 15 carcinomas (19%) of tumor nuclear grade 1, 56 (73%) of tumor nuclear grade 2 and 6 (8%) of tumor nuclear grade 3.

RESULTSNone of the VHL gene mutations were found in all the normal tissue specimens. VHL gene mutations were detected in 40 (52%) cases of CCRCC. The positive rate of HIF-2alpha (81%) was higher than that of HIF-1alpha (66%) (chi(2) = 23.310, P < 0.01); The positive rate of HIF-1alpha and HIF-2alpha in the cases of mutations (98% and 93% respectively) was higher than that of them in non-mutations (32% and 68% respectively) (chi(2) = 36.386, 7.617, P < 0.01); The correlation between HIF-1alpha and VHL gene mutations was closer than that between HIF-2alpha and VHL gene mutations (partial correlation coefficiency was 4.481 and 2.027 respectively, P < 0.01). The expression of HIF-1alpha and 2alpha in different pathological grade and stage of CCRCC showed no significant difference (P > 0.05).

CONCLUSIONSOur study suggests that VHL gene mutations are frequent in sporadic CCRCC, and the high expression of HIF-1alpha and 2alpha are found in the group of VHL mutations. However, we have not found significant correlation between the expression of HIF-1alpha and 2alpha and pathological grade and stage of CCRCC in our study.

Adult ; Aged ; Aged, 80 and over ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Carcinoma, Renal Cell ; genetics ; metabolism ; pathology ; Female ; Humans ; Hypoxia-Inducible Factor 1 ; metabolism ; Immunohistochemistry ; Kidney Neoplasms ; genetics ; metabolism ; pathology ; Male ; Middle Aged ; Mutation ; Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics