Loss of VHL promotes progerin expression,leading to impaired p14/ARF function and suppression of p53 activity

Renal cell carcinomas (RCCs) are frequently occurring genitourinary malignancies in the aged population. A morphological characteristic of RCCs is an irregular nuclear shape, which is used to index cancer grades. Other features of RCCs include the genetic inactivation of the von Hippel-Lindau gene, VHL, and p53 genetic-independent inactivation. An aberrant nuclear shape or p53 suppression has not yet been demonstrated. We examined the effect of progerin (an altered splicing product of the LMNA gene linked to Hutchinson Gilford progeria syndrome; HGPS) on the nuclear deformation of RCCs in comparison to that of HGPS cells. In this study, we showed that progerin was suppressed by pVHL and was responsible for nuclear irregularities as well as p53 inactivation. Thus, progerin suppression can ameliorate nuclear abnormalities and reactivate p53 in response to genotoxic addition. Furthermore, we found that progerin was a target of pVHL E3 ligase and suppressed p53 activity by p14/ARF inhibition. Our findings indicate that the elevated expression of progerin in RCCs results from the loss of pVHL and leads to p53 inactivation through p14/ARF suppression. Interestingly, we showed that progerin was expressed in human leukemia and primary cell lines, raising the possibility that the expression of this LMNA variant may be a common event in age-related cancer progression.     

Osteoblast CFTR Inactivation Reduces Differentiation and Osteoprotegerin Expression in a Mouse Model of Cystic Fibrosis-Related Bone Disease

Low bone mass and increased fracture risk are recognized complications of cystic fibrosis (CF). CF-related bone disease (CFBD) is characterized by uncoupled bone turnover—impaired osteoblastic bone formation and enhanced osteoclastic bone resorption. Intestinal malabsorption, vitamin D deficiency and inflammatory cytokines contribute to CFBD. However, epidemiological investigations and animal models also support a direct causal link between inactivation of skeletal cystic fibrosis transmembrane regulator (CFTR), the gene that when mutated causes CF, and CFBD. The objective of this study was to examine the direct actions of CFTR on bone. Expression analyses revealed that CFTR mRNA and protein were expressed in murine osteoblasts, but not in osteoclasts. Functional studies were then performed to investigate the direct actions of CFTR on osteoblasts using a CFTR knockout (Cftr−/−) mouse model. In the murine calvarial organ culture assay, Cftr−/− calvariae displayed significantly less bone formation and osteoblast numbers than calvariae harvested from wildtype (Cftr+/+) littermates. CFTR inactivation also reduced alkaline phosphatase expression in cultured murine calvarial osteoblasts. Although CFTR was not expressed in murine osteoclasts, significantly more osteoclasts formed in Cftr−/− compared to Cftr+/+ bone marrow cultures. Indirect regulation of osteoclastogenesis by the osteoblast through RANK/RANKL/OPG signaling was next examined. Although no difference in receptor activator of NF-κB ligand (Rankl) mRNA was detected, significantly less osteoprotegerin (Opg) was expressed in Cftr−/− compared to Cftr+/+ osteoblasts. Together, the Rankl:Opg ratio was significantly higher in Cftr−/− murine calvarial osteoblasts contributing to a higher osteoclastogenesis potential. The combined findings of reduced osteoblast differentiation and lower Opg expression suggested a possible defect in canonical Wnt signaling. In fact, Wnt3a and PTH-stimulated canonical Wnt signaling was defective in Cftr−/− murine calvarial osteoblasts. These results support that genetic inactivation of CFTR in osteoblasts contributes to low bone mass and that targeting osteoblasts may represent an effective strategy to treat CFBD.     

2-Methoxyestradiol Induces Mitotic Arrest,Apoptosis, and Synergistic Cytotoxicity with Arsenic Trioxide in Human Urothelial Carcinoma Cells

2-Methoxyestradiol (2-ME), an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC) cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24). After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting), with annexin V-FITC staining and propidium iodide (PI) labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose) polymerase (PARP) cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155) and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10) were activated. In combination with arsenic trioxide (As₂O₃), 2-ME elicited synergistic cytotoxicity (combination index <1) in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As₂O₃ provides a novel implication in clinical treatment of UC.     

Expressed Sequence Tags for Bovine Muscle Satellite Cells,Myotube Formed-Cells and Adipocyte-Like Cells

Background

Muscle satellite cells (MSCs) represent a devoted stem cell population that is responsible for postnatal muscle growth and skeletal muscle regeneration. An important characteristic of MSCs is that they encompass multi potential mesenchymal stem cell activity and are able to differentiate into myocytes and adipocytes. To achieve a global view of the genes differentially expressed in MSCs, myotube formed-cells (MFCs) and adipocyte-like cells (ALCs), we performed large-scale EST sequencing of normalized cDNA libraries developed from bovine MSCs.

Results

A total of 24,192 clones were assembled into 3,333 clusters, 5,517 singletons and 3,842contigs. Functional annotation of these unigenes revealed that a large portion of the differentially expressed genes are involved in cellular and signaling processes. Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis of three subsets of highly expressed gene lists (MSC233, MFC258, and ALC248) highlighted some common and unique biological processes among MSC, MFC and ALC. Additionally, genes that may be specific to MSC, MFC and ALC are reported here, and the role of dimethylarginine dimethylaminohydrolase2 (DDAH2) during myogenesis and hemoglobin subunit alpha2 (HBA2) during transdifferentiation in C2C12 were assayed as a case study. DDAH2 was up-regulated during myognesis and knockdown of DDAH2 by siRNA significantly decreased myogenin (MYOG) expression corresponding with the slight change in cell morphology. In contrast, HBA2 was up-regulated during ALC formation and resulted in decreased intracellular lipid accumulation and CD36 mRNA expression upon knockdown assay.

Conclusion

In this study, a large number of EST sequences were generated from the MSC, MFC and ALC. Overall, the collection of ESTs generated in this study provides a starting point for the identification of novel genes involved in MFC and ALC formation, which in turn offers a fundamental resource to enable better understanding of the mechanism of muscle differentiation and transdifferentiation.     

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.     

Tumor-infiltrating PD1-Positive Lymphocytes and FoxP3-Positive Regulatory T Cells Predict Distant Metastatic Relapse and Survival of Clear Cell Renal Cell Carcinoma

BACKGROUND: Clear cell renal cell carcinoma (CRCC) is the most common malignant tumor of the kidney, and the clinical outcome of CRCC is related with the metastatic potential of CRCC. A significant proportion of metastatic CRCC remains incurable. Recently, immunotherapy against specific targets such as programmed death 1 (PD1) has been adapted for fatal cases of CRCC. MATERIALS AND METHODS: In this study, we aimed to evaluate the potential of tumor-infiltrating PD1-positive lymphocytes or FoxP3-positive regulatory T cells (Tregs) as predictors of the metastatic potential or prognosis of CRCC and investigate possible correlations with Epstein-Barr virus (EBV) infection in 199 cases of CRCC. RESULTS: PD1 positivity, high Treg number, and EBV infection all predicted poor overall survival (OS) by univariate analysis. PD1 positivity and high Treg numbers were also significantly correlated with more distant metastatic relapse (DMR) and poor relapse-free survival (RFS) by univariate analysis. PD1 positivity and high Treg number were independent prognostic indicators for OS. In addition, PD1 positivity was an independent predictor of RFS and DMR. EBV infection was an independent predictor of OS of CRCC. CONCLUSION: This study demonstrates that intratumoral infiltration of PD1-positive or FoxP3-positive lymphocytes can be used as significant prognostic indicators of CRCC and PD1 positivity could be very helpful in the prediction of latent distant metastasis of CRCCs. Therefore, evaluation of the infiltration of PD-positive cells or Tregs in CRCC may be useful diagnostic tools for the selection of patients who could benefit from PD1- or Treg-based immunotherapy.     

Expression of DBC1 and Androgen Receptor Predict Poor Prognosis in Diffuse Large B Cell Lymphoma

BACKGROUND: Recently, deleted in breast cancer 1 (DBC1) has been suggested as a poor prognostic indicator of various human cancers and may possibly have a role as a coactivator of androgen receptor (AR). However, their roles in lymphoma are still unknown. MATERIALS AND METHODS: We investigated the effect of the expression of DBC1 and AR in diffuse large B cell lymphoma (DLBCL). Immunohistochemical expression of DBC1 and AR were evaluated in 101 DLBCL samples by tissue microarray. RESULTS: Positive expression of DBC1 and AR was seen in 73% and 70% of DLBCL, respectively. In total DLBCL patients, DBC1 and AR expression were significantly associated with high clinical stage, elevated serum lactate dehydrogenase levels, and high international prognostic index scores, and they predicted shorter overall survival (OS) and relapse-free survival (RFS) by univariate analysis. DBC1 expression was also an independent prognostic indicator by multivariate analysis (OS, P = .017; RFS, P = .004). Especially, both DBC1 and AR expression significantly correlated with shorter OS and RFS in non-germinal center B cell (non-GCB)-type DLBCL by univariate analysis. In multivariate analysis, DBC1 expression was an independent prognostic predictor for OS (P = .035) and AR expression significantly correlated with RFS (P = .005). CONCLUSION: We demonstrate that the expression of DBC1 and AR are significant prognostic indicators for DLBCL patients, especially for unfavorable non-GCB-type DLBCL.