Nuclear receptor TLX inhibits TGF-ß signaling in glioblastoma.

TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-ß (TGF-ß) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-ß has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-ß signaling we wanted to find out if there is any interaction between TLX and TGF-ß in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-ß signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-ß receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-ß receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-ß target genes. The interaction between TLX and TGF-ß may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells.

Age-related intraneuronal accumulation of αII-spectrin breakdown product SBDP120 in the human cerebrum is enhanced in Alzheimer's disease.

Spectrins are a part of cytoskeletal platform that lines the intracellular side of plasma membrane, which can be proteolyzed by calcium-sensitive enzymes including calpains and caspases. Caspase-3 mediated αII-spectrin proteolysis results in the release of a 120kDa spectrin breakdown product (SBDP120), known to occur in conditions with cell death. In rodents, intraneuronal SBDP120 accumulation in the forebrain develops with age, which is enhanced in transgenic models of Alzheimer's disease (AD). The present study was set to explore age-related SBDP120 formation and its relevance to AD-type hallmark lesions in the human brains. SBDP120 immunoreactivity (IR) was detected in neuronal somata and dendrites in the cortex and hippocampal formation in postmortem brains from aged (n=10, mean age=84.2) and AD (n=10, mean age=84.8) subjects, but not mid-aged controls (n=10, mean age=58.2). The overall density of SBDP120 IR quantified in the temporal neocortex was increased in the aged and AD groups, more robust in the latter, relative to mid-aged control, while no regional, laminar or cellular association was found between SBDP120 accumulation and Aß deposition or phosphorylated-tau aggregation. In cultured rat retinal ganglion cells (RGC-5), SBDP120 elevation occurred with caspase-3 activation following oxygen as well as serum deprivation, suggestive of SBDP120 formation in stressful conditions with and without apparent neuronal death. These results confirm an age-related intraneuronal SBDP120 accumulation in the human cerebrum that is enhanced in AD. This neuronal change appears to occur independent of amyloid deposition, tau pathology and overt neuronal death.

The cell death and DNA damages caused by the Tet-On regulating HSV-tk/GCV suicide gene system in MCF-7 cells.

Ganciclovir (GCV) affects the molecular mechanism of cell death and DNA damage by the rAAV (recombinant adeno-associated virus)-mediated Tet-On/HSV-tk/GCV suicide gene system in human breast cancer cell line MCF-7. A rAAV/TRE/Tet-On/HSV-tk combining a Tet-On regulating system and a suicide gene HSV-tk was used to transfect human breast cancer cell line MCF-7, and therapeutic effects on this system were studied. Afterwards, we used RT-PCR, western blotting, and a modified comet-assay to explore the potential mechanism of the HSV-tk/GCV suicide gene system in breast cancer treatments. MTT assay has shown that the cell number of GCV+rAAV+Dox group was significantly decreased compared with that of other groups after treatment and flow cytometric analysis detected that there was a substantial increase of S phase cells in this group, which means the HSV-tk/GCV suicide gene system probably works on the cell cycle. RT-PCR detected the expression level of p21 increased and PCNA had an opposite trend. Western blotting detected the protein expression of p21 and p53 increased and PCNA, CDK1, cyclin B decreased in GCV+rAAV+Dox group. The modified comet-assay shown that the very small extra fragments generated by the GCV+rAAV+Dox group treatment are visible as a small cloud extending from the comet in the direction of electrophoresis. The therapeutic mechanism of the HSV-tk/GCV suicide gene system on human breast cancer cell line MCF-7 is probably by upregulating the expression of p21 through a p53-dependent DNA damage signalling pathway, leading the decrease of protein expression of PCNA, cyclin B, CDK1 in MCF-7 cells and promoting the cell cycle arrest at G1/S phase. In summary, the HSV-tk/GCV suicide gene system arouses the death of MCF-7 cells from blocking the cell cycle and DNA damage.

TLX controls angiogenesis through interaction with the von Hippel-Lindau protein.

TLX is known as the orphan nuclear receptor indispensable for maintaining neural stem cells in adult neurogenesis. We report here that neuroblastoma cell lines express high levels of TLX, which further increase in hypoxia to enhance the angiogenic capacity of these cells. The proangiogenetic activity of TLX appears to be induced by its direct binding to the von Hippel-Lindau protein (pVHL), which stabilizes TLX. In turn, TLX competes with hydroxylated hypoxia-inducible factor (HIF-α) for binding to pVHL, which contributes to the stabilization of HIF-2α in neuroblastoma during normoxia. Upon hypoxia, TLX increases in the nucleus where it binds in close proximity of the HIF-response element on the VEGF-promoter chromatin, and, together with HIF-2α, recruits RNA polymerase II to induce VEGF expression. Conversely, depletion of TLX by shRNA decreases the expression of HIF-2α and VEGF as well as the growth-promoting and colony-forming capacity of the neuroblastoma cell lines IMR-32 and SH-SY5Y. On the contrary, silencing HIF-2α will slightly increase TLX, suggesting that TLX acts to maintain a hypoxic environment when HIF-2α is decreasing. Our results demonstrate TLX to play a key role in controlling angiogenesis by regulating HIF-2α. TLX and pVHL might counterbalance each other in important fate decisions such as self-renewal and differentiation, as well as angiogenesis and anti-angiogenesis.

[Anterior operative approach for the treatment of old inferior-cervical fracture-dislocation].

OBJECTIVE: To explore the choice of operative approach for old inferior-cervical fracture-dislocation and analyze the clinical effects of anterior operative approach. METHODS: From January 2003 to May 2010, 17 patients with inferior-cervical fracture-dislocation delayed for more than 4 weeks were treated with continued closed skull traction and anterior decompression, bone graft and internal fixation with steel plate. Among the patients, 11 patients were male and 6 patients were female with an average age of 41 years (ranged from 24 to 56 years). The time between injury and operation was from 4 weeks to 3 months. According to Frankel grade, grade A was in 7 cases, B in 4, C in 2, D in 2, E in 2. Neurological function, bone fusion height of vertebral body and cervical sequence and curvature were observed. RESULTS: The incision of 17 cases obtained primary healing. There was 1 case with hoarseness, and symptoms disappeared after 1 month. The mean time of follow-up was 23 months (ranged from 4 to 47 months). The X-ray films showed satisfactory reduction and good alignment and lordosis. The Frankel grade improved obviously at final follow-up, grade A was in 5 cases, B in 5, C in 1 , D in 3, E in 3. CONCLUSION: Single anterior operative approach can successfully reduce old inferior-cervical fracture-dislocation of DF stage I , II and some stage III; anterior decompression, bone graft and internal fixation with steel plate is a safe, effective method for old inferior-cervical fracture-dislocation.

TLX activates MASH1 for induction of neuronal lineage commitment of adult hippocampal neuroprogenitors.

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs.

Recombinant AAV-mediated HSVtk gene transfer with direct intratumoral injections and Tet-On regulation for implanted human breast cancer.

BACKGROUND: HSVtk/ganciclovir (GCV) gene therapy has been extensively studied in tumors and relies largely on the gene expression of HSVtk. Most studies, however, have failed to demonstrate any significant benefit of a controlled gene expression strategy in cancer treatment. The Tet-On system is commonly used to regulate gene expression following Dox induction. We have evaluated the antitumor effect of HSVtk/ganciclovir gene therapy under Tet-On regulation by means of adeno-associated virus-2 (AAV-2)-mediated HSVtk gene transfer with direct intratumoral injections in mice bearing breast cancer tumors. METHODS: Recombinant adeno-associated virus-2 (rAAV) was constructed and transduced into MCF-7 cell line. GCV treatment to the rAAV infected MCF-7 cells was performed by MTT assay under the doxycycline (Dox) induction or without Dox induction at a vp (viral particle) number of > or =10(4)/cell. The virus was administered intratumorally to nude mice that had also received GCV intraperitoneally. The antitumor effects were evaluated by measuring tumor regression and histological analysis. RESULTS: We have demonstrated that GCV treatment to the infected MCF-7 cells under the Dox induction was of more inhibited effects than those without Dox induction at > or =10(4) vp/cell. In ex vivo experiments, tumor growth of BALB/C nude mice breast cancer was retarded after rAAV-2/HSVtk/Tet-On was injected into the tumors under the Dox induction. Infiltrating cells were also observed in tumors after Dox induction followed by GCV treatment and cells were profoundly damaged. The expression of HSVtk gene in MCF-7 cells and BALB/C nude mice tumors was up-regulated by Tet-On under Dox induction with reverse transcription-PCR (RT-PCR) analysis. CONCLUSION: The antitumor effect of rAAV-mediated HSVtk/GCV gene therapy under the Dox induction with direct intratumoral injections may be a useful treatment for breast cancer and other solid tumors.

[The construction of recombinant AAV vector expressing HSVtk gene controlled by Tet-On and the detection of its activity].

In order to investigate the application of recombinant adeno-associated virus (rAAV) vector containing Tet regulation system and HSVtk gene in cancer gene therapy, pAAV/TRE/HSVtk/Tet-On was constructed and identified with PCR and restriction enzyme digestion. Packaging cells HEK293 were cotransfected with plasmids pAAV/TRE/HSVtk/Tet-On, pAAV-RC and pAAV-helper to produce infectious rAAV, and CsCl2 densitygradient centrifugation method was performed for purification and concentration of rAAV. The viruses were then transduced into MCF-7 cells. The results of dot blot hybridization indicate that the rAAV can transfer the target gene into MCF-7 cells. MTT assay showed that GCV could kill AAV-infected MCF-7 cells under the induction of Dox. The data demonstrated that rAAV containing Tet regulation system and HSVtk gene was successfully obtained, and could be used for further investigation of in vivo and in vitro experiments.

Suicide gene therapy of human breast cancer in SCID mice model by the regulation of Tet-On.

BACKGROUND: RevTet-On gene expression system was used to deliver the suicide gene tk to human breast cancer cell line MCF-7 and control the tk gene expression level. The animal model of human breast cancer on severe combined immune deficiency (SCID) mice was set up to explore the suicide gene therapy by the regulation of Tet-On. METHODS: Herpes simplex virus-thymidine kinase (HSVtk) gene was inserted into the plasmid pRevTRE and the recombinant retroviral vector pRevTRE/HSVtk was constructed. Using modified calcium phosphate co-precipitation method, two transfections, pRevTRE/HSVtk and pRevTet-On were performed for MCF-7 cell line and selected by hygromycin B and G418. MCF-7 cell line that stably expressed Tet-regulated tk gene was established. HSVtk gene expression in the MCF/TRE/tk/Tet-On cell line was under the control of Doxycycline (Dox). Cell viability was also determined by MTT assay, whereas HSVtk gene expression was analyzed by reverse transcription-PCR (RT-PCR). RESULTS: MCF/TRE/tk/Tet-On cell survival rate was decreased from 100% to less than 20% when ganciclovir (GCV) concentration was increased from 0 to 1000 microg/ml at 1 microg/ml of Dox after 72 hours of GCV administration. At 1 microg/ml of GCV concentration, the cell numbers decreased from 7 x 10(4) cells/ml to 2 x 10(4) cells/ml when Dox concentration was increased from 0 to 1500 ng/ml after 72 hours culture. In addition, bystander effects were generated in vitro when 10% - 25% of transduced MCF-7 cells were mixed in untransduced MCF-7 cells. On the other hand, the human breast cancer models in SCID mice were set up. The tk gene was expressed with the regulated character after MCF/TRE/tk/Tet-On cells were implanted into the female SCID mice 7 days after Dox induction followed by intraperitoneally administration of GCV for 23 days. Subcutaneous tumors in SCID mice that were implanted with MCF/TRE/tk/Tet-On cells shrank remarkably after Dox and GCV administration as compared with the control. CONCLUSION: The human breast tumor cells (MCF-7) expressing HSVtk gene can be eradicated by administration of GCV and induced with tetracycline or its derivative Dox in vitro and in vivo.

[Retroviral vector-mediated HSVtk gene expression and acquisition of high titer recombinant virus].

OBJECTIVE: To explore the HSVtk gene expression mediated by the retroviral vector and to obtain high titer recombinant retroviral virus. METHODS: The recombinant vector pRevTRE/HSVtk was constructed by inserting HSVtk gene into pRevTRE. The recombinant retrovirus, which was produced from cloned PA317 cells screened by hygromycin B after "micro-pingpong" technique transferring with pRevTRE/HSVtk plasmids DNA by using modified calcium phosphate precipitation method. HSVtk gene expression was performed on target cells and virus titers were detected in different cultured temper, time and sodium butyrate concentration. RESULTS: The recombinant retroviral vector pRevTRE/HSVtk was constructed and HSVtk gene expression was detected on target cells after they were infected with the recombinant retrovirus. CONCLUSION: High titer of retroviruses could be obtained in the culture medium of PA317 cell line through "micro-pingpong" technique at 30 hours and 10 mmol/L sodium butyrate concentration followed by frozen ultrafiltration.