Guan Wu

Parathyroid hormone-related protein: A potential autocrine growth regulator in human prostate cancer cell lines

OBJECTIVE We recently demonstrated that parathyroid hormone-related protein (PTHrP) is widely expressed by human prostate cancer tissue, suggesting that PTHrP might be involved in the growth and development of prostate cancer. To study this further, the production of PTHrP and its biologic effect were investigated using human prostate cancer cell lines. METHODS The cell lines used were one androgen-dependent cell line, LNCaP, and two androgen-independent cell lines, PC-3 and DU-145. PTHrP secreted by cancer cells was measured by radioimmunoassay. The effect of PTHrP on DNA synthesis in these cells was determined by thymidine incorporation assay. RESULTS All cell lines secreted immunodetectable levels of PTHrP in the culture-conditioned media. PC-3 cells secreted significantly higher amounts than the other two cell lines. A synthetic peptide, PTHrP(1-34), stimulated thymidine uptake in PC-3 and DU-145 cells more than threefold the control under serum-free and steroid-free conditions, whereas LNCaP was not affected. However, in the presence of dihydrotestosterone, DNA synthesis of LNCaP cells was stimulated by PTHrP in a dose-dependent manner. Additionally, this PTHrP-induced DNA synthesis was completely neutralized by a validated mouse monoclonal antibody (8B12) raised against PTHrP(1-34). CONCLUSIONS Our data suggest that PTHrP may play a significant role in the growth of prostate cancer by acting locally in an autocrine fashion.

VHL protein‐interacting deubiquitinating enzyme 2 deubiquitinates and stabilizes HIF‐1α

Hypoxia‐inducible factor (HIF)‐1α is a short‐lived protein and is ubiquitinated and degraded through the von Hippel–Lindau protein (pVHL)–E3 ubiquitin ligase pathway at normoxia. Deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination‐related processes. Here, we show that pVHL‐interacting deubiquitinating enzyme 2, VDU2, but not VDU1, interacts with HIF‐1α. VDU2 can specifically deubiquitinate and stabilize HIF‐1α and, therefore, increase expression of HIF‐1α targeted genes, such as vascular endothelial growth factor (VEGF). These findings suggest that ubiquitination of HIF‐1α is a dynamic process and that ubiquitinated HIF‐1α might be rescued from degradation by VDU2 through deubiquitination. Although pVHL functions as a master control for HIF‐1α stabilization, as pVHL–E3 ligase mediates the ubiquitination of both HIF‐1α and VDU2, the balance between the pVHL‐mediated ubiquitination and VDU2‐mediated deubiquitination of HIF‐1α provides another level of control for HIF‐1α stabilization.

Ubiquitination of a Novel Deubiquitinating Enzyme Requires Direct Binding to von Hippel-Lindau Tumor Suppressor Protein

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of theVHL gene. Recent studies suggest that VHL protein (pVHL) is a component of an E3 ubiquitin ligase, but the detailed biological function of pVHL remains to be determined. To further elucidate the biological functions of pVHL, we searched pVHL-interacting proteins using yeast two-hybrid screening. A novel protein named VHL-interacting deubiquitinating enzyme 1 (VDU1) was identified as being able to directly interact with pVHL in vitro and in vivo. We have determined the full-length cDNA of this enzyme, which includes two putative subtypes. Type I consists of 942 amino acids, and type II consists of 911 amino acids with predicted molecular masses of 107 and 103 kDa, respectively. We have also cloned a mouse homologue of this enzyme. Sequence analysis reveals that this protein is conserved between human and mouse and contains the signature motifs of the ubiquitin-specific processing protease family. Enzymatic function studies demonstrate its deubiquitinating activity. We have determined that the VDU1-interacting region in pVHL is located in its β-domain, and several naturally occurring mutations located in this domain disrupt the interaction between pVHL and VDU1 protein. Co-immunoprecipitation demonstrates that VDU1 can be recruited into the pVHL-elongin C-elongin B complex. Finally, we demonstrate that VDU1 is able to be ubiquitinated via a pVHL-dependent pathway for proteasomal degradation, and VHL mutations that disrupt the interaction between VDU1 and pVHL abrogate the ubiquitination of VDU1. Our findings indicate that VDU1, a novel ubiquitin-specific processing protease, is a downstream target for ubiquitination and degradation by pVHL E3 ligase. Targeted degradation of VDU1 by pVHL could be crucial for regulating the ubiquitin-proteasome degradation pathway.

Prostate cancer in the elderly: frequency of advanced disease at presentation and disease-specific mortality.

The objectives of this study were to determine the frequency of metastatic (M1) prostate cancer (PC) at presentation in different age groups, to examine the association of age with PC‐specific mortality, and to calculate the relative contribution of different age groups to the pool of M1 cases and PC deaths.

Prostate cancer in the elderly

The objectives of this study were to determine the frequency of metastatic (M1) prostate cancer (PC) at presentation in different age groups, to examine the association of age with PC‐specific mortality, and to calculate the relative contribution of different age groups to the pool of M1 cases and PC deaths.

Identification of a deubiquitinating enzyme subfamily as substrates of the von Hippel-Lindau tumor suppressor

The VHL protein (pVHL) is a component of an E3 ubiquitin ligase complex which is involved in the ubiquitination and degradation of the alpha subunits of HIF (hypoxia-inducible factor) in the presence of oxygen. However, it is of considerable interest to identify pVHL substrates other than HIF. In our previous studies, we have shown that VDU1 (pVHL-interacting deubiquitinating enzyme-1) can be ubiquitinated for rapid degradation in a pVHL-dependent manner. In this report we show that another uncharacterized deubiquitinating enzyme, named VDU2 (pVHL-interacting deubiquitinating enzyme-2), is a substrate of pVHL. Based on human and mouse cDNA sequences, VDU1 and VDU2 are identical in approximately 59% of the amino acids with strong homology in the N-terminus and C-terminus and a weaker similarity in the middle region. VDU2 contains the signature motifs of the ubiquitin-specific processing protease family and possesses deubiquitinating activity. Like VDU1, VDU2 interacts with pVHL beta-domain and these two proteins can compete with each other to bind to pVHL. Finally, we demonstrate that VDU2 can also be ubiquitinated and degraded in a pVHL-dependent manner. Based on their amino acid sequence homology and functional interaction with pVHL, VDU1 and VDU2 define a subfamily of ubiquitin specific processing proteases. Since deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes, VDU1 and VDU2 could be important substrates of pVHL E3 ligase complex.

Near infrared fluorescence imaging with robotic assisted laparoscopic partial nephrectomy: initial clinical experience for renal cortical tumors.

PURPOSE We evaluated the utility of near infrared fluorescence of intravenously injected indocyanine green in performing robotic assisted laparoscopic partial nephrectomy. In addition, we evaluated the initial performance of a novel near infrared fluorescence imaging system integrated into the da Vinci® Si Surgical System during robotic assisted laparoscopic nephrectomy. MATERIALS AND METHODS Fluorescence imaging for the da Vinci Si Surgical System was used for all cases. Indocyanine green was injected before near infrared imaging. Immediate imaging assessed the renal vasculature while delayed imaging differentiated renal cortical tumors from normal parenchyma. The intraoperative performance of near infrared fluorescence of intravenous indocyanine green was evaluated for tumor appearance relative to surrounding renal parenchyma as well as identification of the renal vasculature. RESULTS A total of 11 patients underwent robotic assisted laparoscopic nephrectomy with 2 converted to robotic assisted laparoscopic radical nephrectomy. Indocyanine green injections were repeated up to a total of 5 times depending on the goal of visualization. Of the 11 patients 10 demonstrated malignancy on final pathology. Of the malignant tumors 7 were hypofluorescent and 3 were isofluorescent compared to the surrounding renal parenchyma. Near infrared fluorescence imaging delineated the vascular anatomy in all cases. All surgical margins were negative on final pathology. CONCLUSIONS Intraoperative imaging of indocyanine green with near infrared fluorescence is a safe and effective method to accurately identify the renal vasculature and to differentiate renal tumors from surrounding normal parenchyma. The capacity for multimodal imaging within the surgical console further facilitates this imaging. Further study is needed to determine if this technique will help improve outcomes of robotic assisted laparoscopic nephrectomy.

Parathyroid hormone-related protein is expressed by prostatic neuroendocrine cells☆

OBJECTIVE Parathyroid hormone-related protein (PTHrP) is a regulatory peptide that has been associated with normal fetal growth and differentiation as well as the regulation of fetal calcium. In a variety of cancers, PTHrP has been implicated in the humoral hypercalcemia of malignancy. Recently, we demonstrated that all prostatic adenocarcinomas express PTHrP. In the present study, the localization of PTHrP and its mRNA in nonmalignant prostate tissue was assessed. METHODS Formalin-fixed, paraffin-embedded prostatic tissues from 23 patients were evaluated. Immunocytochemistry (ICC) was performed by the streptavidin-peroxidase enzyme conjugate method using a monoclonal antibody, 8B12, generated against fragment 1-34 of the amino-terminal end of PTHrP. Nonradioactive in situ hybridization was carried out using a digoxigenin labeled single-stranded cDNA probe complementary to the sequence coding for PTHrP(15-120). RESULTS PTHrP immunoreactivity was observed in the cytosol of a few epithelial cells. Double labeling and serial section ICC with 8B12 and a monoclonal antibody to chromogranin A (a generic neuroendocrine [NE] marker) revealed that PTHrP was present in a subpopulation of prostatic NE cells. In situ hybridization of mirror image sections demonstrated positive signals in prostatic NE cells in complete accordance with the ICC findings. CONCLUSIONS The localization and production of PTHrP in prostatic NE cells suggest that this polypeptide may act in an endocrine-paracrine fashion involved in the prostatic growth and differentiation as well as the regulation of calcium in semen and seminal fluid.

The VHL protein recruits a novel KRAB‐A domain protein to repress HIF‐1α transcriptional activity

The von Hippel‐Lindau tumor suppressor (pVHL) is a component of an E3 ubiquitin ligase and targets hypoxia‐inducible factor‐1α (HIF‐1α) for ubiquitylation and degradation under normoxic conditions. pVHL also directly inhibits HIF‐1α transactivation by recruiting histone deacetylases. Here, we report a novel pVHL‐interacting protein that functions as a negative regulator of HIF‐1α transactivation. This protein, generated from the ZnF197 locus by alternative splicing, contains a Kruppel‐associated box (KRAB)‐A domain and a SCAN domain, but lacks the 22 C2H2‐type zinc fingers present in ZnF197. Therefore, we named this protein pVHL‐associated KRAB‐A domain‐containing protein (VHLaK). We demonstrate that the KRAB‐A domain in VHLaK mediates pVHL binding and functions as a transcriptional repression module. The SCAN domain mediates VHLaK homo‐oligomerization, which enhances VHLaK repressive activity. pVHL can recruit VHLaK to repress HIF‐1α transcriptional activity and HIF‐1α‐induced VEGF expression. Finally, we demonstrate that pVHL, VHLaK and KAP1/TIF‐1β can be recruited into a single complex, indicating that KAP1/TIF‐1β may participate in pVHL‐mediated transcriptional repression of HIF‐1α. Our findings provide a novel mechanism for the modulation of HIF‐1α transactivation by pVHL.

Identification of the RNA polymerase II subunit hsRPB7 as a novel target of the von Hippel—Lindau protein

Inactivation of the von Hippel—Lindau (VHL) tumor suppressor gene is linked to the hereditary VHL disease and sporadic clear cell renal cell carcinomas (CCRCC). VHL‐associated tumors are highly vascularized, a characteristic associated with overproduction of vascular endothelial growth factor (VEGF). The VHL protein (pVHL) is a component of the ubiquitin ligase E3 complex, targeting substrate proteins for ubiquitylation and subsequent proteasomic degradation. Here, we report that the pVHL can directly bind to the human RNA polymerase II seventh subunit (hsRPB7) through its β‐domain, and naturally occurring β‐domain mutations can decrease the binding of pVHL to hsRPB7. Introducing wild‐type pVHL into human kidney tumor cell lines carrying endogenous mutant non‐functional pVHL facilitates ubiquityl ation and proteasomal degradation of hsRPB7, and decreases its nuclear accumulation. pVHL can also suppress hsRPB7‐induced VEGF promoter transactivation, mRNA expression and VEGF protein secretion. Together, our results suggest that hsRPB7 is a downstream target of the VHL ubiquitylating complex and pVHL may regulate angiogenesis by targeting hsRPB7 for degradation via the ubiquitylation pathway and preventing VEGF expression.