[Mechanism of nerve growth factor promotes angiogenesis and skeletal muscle fiber remodeling in a mouse hindlimb ischemic model].

Objective: To explore the mechanism of nerve growth factor (NGF) in the skeletal muscle fiber remodeling in ischemic limbs during therapeutic angiogenesis. Methods: Eighteen female mice with SPF grade, 6 weeks old and 25-30 g weighed were randomly allocated to sham-operated group (n=6), blank control group (n=6) and NGF gene transfection group (n=6). The left hindlimb ischemia models were established by ligating the femoral artery in blank control group and NGF gene transfection group. Seven days after the operation, mice in the three groups were separately injected with normal saline, empty plasmids, and NGF plasmids. Gastrocnemius of left hindlimbs was harvested after the blood perfusion assessment of the ischemic limb on the 21st postoperative day. The gastrocnemius muscle specimens were stained with HE, CD31 and proliferating cell nuclear antigen (PCNA) immunohistochemistry staining, the mRNA expressions of myosin heavy chain-Ⅰ(MHC-Ⅰ), MHC-Ⅱa and MHC-Ⅱb were measured by real-time PCR, and the protein level of NGF and peroxisome proliferator-activated receptors-ß/δ (PPAR ß/δ) were detected by Western blot. The expression of cytochrome C oxidase (COX), isocitrate dehydrogenase (IDH) and adenosine triphosphate (ATP) were examined by enzyme-linked immunosorbent assay (ELISA). Results: On the 21st day after operation, the blood perfusion of the ischemic limb in NGF gene transfection group was (195.70±9.99)PU, which was lower than that in sham-operated group (312.15±17.32)PU (P=0.001), while it was higher than that in blank control group (82.11±8.55)PU (P=0.001). The degree of muscle atrophy in the NGF gene transfection group was lower than that in the blank control group. The capillary density of NGF gene transfection group (0.34±0.05) was higher than that of sham-operated group (0.11±0.03) and blank control group (0.27±0.04) (P<0.05). The endothelial cell proliferation index in NGF gene transfection group (0.39±0.19) was significantly higher than that in sham-operated group (0.18±0.01) and blank control group (0.25±0.14) (P<0.05). The expression of NGF, PPAR ß/δ, COX, IDH, ATP, and MHC-Ⅰ mRNA in NGF gene transfection group were significantly higher than those in sham-operated group and blank control group (P<0.05). Conclusions: NGF gene transfection can promote angiogenesis in the ischemic limbs of mice, increase the blood perfusion, and thus induce the remodeling of skeletal muscle fibers to type Ⅰ. This process may be related to NGF-induced PPAR ß/δ expression and promote the cellular aerobic metabolism in skeletal muscle.

Urinalysis in patients with neuromyelitis optica spectrum disorder.

BACKGROUND AND PURPOSE: Increasing evidence has demonstrated that aquaporin-4 (AQP4) immunoglobulin G causes damage to the kidney in neuromyelitis optica spectrum disorder (NMOSD). However, changes in urinalysis in NMOSD have not been investigated thus far. Our objective was to evaluate the changes in urinalysis in NMOSD patients. METHODS: Case data were collected from 44 patients with AQP4 antibody-positive NMOSD, 53 patients with multiple sclerosis (MS) and 79 age- and sex-matched healthy controls. Analyses of early morning urine and 24-h urine samples comparing NMOSD with MS patients were conducted. RESULTS: In the acute phase, urine pH levels (P < 0.001) and urine specific gravity levels (P < 0.001) from NMOSD patients were significantly higher and lower, respectively, than for MS patients. 24-h urine sodium and 24-h urine volume from NMOSD patients were significantly higher than for MS patients (both P = 0.001). A 24-h urine volume higher than 2500 ml (odds ratio 11.7, 95% confidence interval 1.863-73.066) and a 24-h urine sodium higher than 200 mmol (odds ratio 16.0, 95% confidence interval 2.122-120.648) are more likely to occur in NMOSD patients in the acute phase than in MS patients. CONCLUSIONS: The urinalysis results were significantly different between NMOSD patients and MS patients. The pathophysiological changes in AQP4 antibody-positive NMOSD patients were not limited to the central nervous system.

[The analysis of risk factors and outcomes of carotid endarterectomy for carotid artery stenosis].

Objective: To evaluate the early and long-term outcomes of carotid endarterectomy for carotid artery stenosis and analyse the risk factors for the outcomes. Methods: A retrospective review of 369 patients underwent carotid endarterectomy(CEA) in Peking Union Medical College Hospital from Oct 2006 to Nov 2012 was conducted. Clinical data including general conditions, perioperative and follow-up outcomes were collected. Results: Three hundred sixty-nine patients underwent 407 CEAs. The long-term follow-up rate (≥30 d) was 89.9% and follow-up period was 11.8-48.3 months. Among 407 CEAs, patients with symptomatic carotid artery stenosis, carotid stenosis over 70% and contralateral severe carotid stenosis occupied 78.0%(317/407), 98.4%(400/407) and 12.04%(49/407) respectively. Total early complications (<30 d) of stroke, cardiac events and death was 3.93% (16/407). Univariate analysis showed no risk factor had significant effect on early complications (P>0.05). Total long-term complications of stroke, cardiac events and death was 8.7% (32/366). Univariate analysis showed that total long-term complication rate of smoking group was higher than non-smoking group (12.1% vs 5.1%, P<0.05), contralateral carotid artery stenosis group was higher than opposite one (28.6% vs 8.0%, P<0.05). Multivariate Logistic regression showed the HR of long-term complications rate in patients aged over 65 years, smoking history, myocardial infarction and contralateral carotid stenosis were 2.59, 2.66, 2.48 and 6.06, respectively. Conclusions: CEA is safe method for the treatment of carotid stenosis. To CEA, age over 65 years, smoking history, myocardial infarction and contralateral carotid stenosis are risk factors for long-term adverse outcomes.

Phosphorylation-mediated activation of LDHA promotes cancer cell invasion and tumour metastasis.

Metastases remain the major cause of death from cancer. Recent molecular advances have highlighted the importance of metabolic alterations in cancer cells, including the Warburg effect that describes an increased glycolysis in cancer cells. However, how this altered metabolism contributes to tumour metastasis remains elusive. Here, we report that phosphorylation-induced activation of lactate dehydrogenase A (LDHA), an enzyme that catalyses the interconversion of pyruvate and lactate, promotes cancer cell invasion, anoikis resistance and tumour metastasis. We demonstrate that LDHA is phosphorylated at tyrosine 10 by upstream kinases, HER2 and Src. Targeting HER2 or Src attenuated LDH activity as well as invasive potential in head and neck cancer and breast cancer cells. Inhibition of LDH activity by small hairpin ribonucleic acid or expression of phospho-deficient LDHA Y10F sensitized the cancer cells to anoikis induction and resulted in attenuated cell invasion and elevated reactive oxygen species, whereas such phenotypes were reversed by its product lactate or antioxidant N-acetylcysteine, suggesting that Y10 phosphorylation-mediated LDHA activity promotes cancer cell invasion and anoikis resistance through redox homeostasis. In addition, LDHA knockdown or LDHA Y10F rescue expression in human cancer cells resulted in decreased tumour metastasis in xenograft mice. Furthermore, LDHA phosphorylation at Y10 positively correlated with progression of metastatic breast cancer in clinical patient tumour samples. Our findings demonstrate that LDHA phosphorylation and activation provide pro-invasive, anti-anoikis and pro-metastatic advantages to cancer cells, suggesting that Y10 phosphorylation of LDHA may represent a promising therapeutic target and a prognostic marker for metastatic human cancers.

[Prognostic factors for inability to walk independently in patients with multiple system atrophy].

Objective: To explore the prognostic factors for inability to walk independently in patients with multiple system atrophy (MSA). Methods: A total of 123 patients with clinically confirmed MSA admitted to Navy General Hospital and Dongfang Hospital affiliated to the Second Clinical Medical College of Beijing University of Chinese Medicine, from February 2013 to February 2016, were retrospectively reviewed. Clinical data and all records were collected and all subjects were followed up by a telephone call in February 2016. The second milestone of activities of daily living scale (ADL), defined as inability to walk independently, was taken as the primary outcome. Eight possible prognostic factors were investigated and the survival analysis was performed with Cox proportional hazards model regression. Results: Of all the MSA patients, 74 subjects were men and 49 were women with a sex radio of 1.51∶1(M∶F). Seventy cases were diagnosed with MSA-cerebellar type (MSA-C) and 53 with MSA-Parkinson type (MSA-P) (C∶P=1.32∶1). Mean age at the onset of first symptom was (53±8) years old. All patients had severe autonomic nervous dysfunction. At the last follow-up, 56 cases (45.5%) were unable to walk independently. The median survival time from the onset of MSA to inability to walk independently was 73 months. The age of onset ≥ 55 years (HR=1.969, 95%CI 1.095-3.542, P=0.024) and the interval time from disease onset to combined motor and autonomic involvement≤3 years (HR=2.308, 95%CI 1.158-4.600, P=0.017) were independent prognostic factors for inability to walk independently, while gender, MSA clinical subtypes, initial symptoms, alcohol intake, smoking and toxic exposure were not indicators for independent walking (P>0.05). Conclusions: The prognostic factors for inability to walk independently in patients with MSA are the age of onset ≥55 years and the interval time from disease onset to combined motor and autonomic involvement≤3 years. Although factors including gender, MSA clinical subtypes, initial symptoms, alcohol intake, smoking and toxic exposure are not the predictive factors for inability to walk independently in our MSA patients, their roles in the prognosis of MSA still need further investigation.

Contrast Functions of αA- and αB-Crystallins in Cancer Development.

α-Crystallins, initially identified as the structural proteins of the ocular lens, belong to the small heat shock protein family. They play significant roles in maintaining the lens transparency and preventing protein aggregation. α-Crystallins exist in two isoforms: αA and αB, and they display differential tissue distribution. Their mutations are implicated in several human diseases including cardiac myopathies, neurodegenerative diseases, cataracts and various types of cancers. Increased αB expression was detected in retinoblastoma, breast cancer, glioblastoma, prostate and renal cell carcinomas, indicating its role in promoting tumor growth. A complex picture emerges for αA. Although earlier studies suggest that αA may promote cancer development, recent studies from our laboratory demonstrate that αA can act as a tumor suppressor inhibiting cell transformation and retarding cell migration through modulating MAP kinase activity. In this review, we summarize the recent progress about the functions of αA and αB in cancer development.

RSK2 signals through stathmin to promote microtubule dynamics and tumor metastasis.

Metastasis is responsible for >90% of cancer-related deaths. Complex signaling in cancer cells orchestrates the progression from a primary to a metastatic cancer. However, the mechanisms of these cellular changes remain elusive. We previously demonstrated that p90 ribosomal S6 kinase 2 (RSK2) promotes tumor metastasis. Here we investigated the role of RSK2 in the regulation of microtubule dynamics and its potential implication in cancer cell invasion and tumor metastasis. Stable knockdown of RSK2 disrupted microtubule stability and decreased phosphorylation of stathmin, a microtubule-destabilizing protein, at serine 16 in metastatic human cancer cells. We found that RSK2 directly binds and phosphorylates stathmin at the leading edge of cancer cells. Phosphorylation of stathmin by RSK2 reduced stathmin-mediated microtubule depolymerization. Moreover, overexpression of phospho-mimetic mutant stathmin S16D significantly rescued the decreased invasive and metastatic potential mediated by RSK2 knockdown in vitro and in vivo. Furthermore, stathmin phosphorylation positively correlated with RSK2 expression and metastatic cancer progression in primary patient tumor samples. Our finding demonstrates that RSK2 directly phosphorylates stathmin and regulates microtubule polymerization to provide a pro-invasive and pro-metastatic advantage to cancer cells. Therefore, the RSK2-stathmin pathway represents a promising therapeutic target and a prognostic marker for metastatic human cancers.

[Efficacy and safety analysis of surgical bypass and endovascular management in the treatment of 116 Takayasu arteritis].

OBJECTIVE: To analyze the efficacy and safety of surgery and endovascular management in treating Takayasu arteritis. METHODS: The data of 116 patients (24 males and 92 females; mean age (32±12) years) with Takayasu arteritis and underwent surgery or endovascular therapy was retrospective analyzed. According to the two different surgical procedures, the patients were divided into two groups: open repair group and endovascular repair group. One hundred and fifty-four surgical procedures were done including 69 cases of open repair and 85 cases of endovascular repair. A total of 211 arterial lesions were revascularized (open repair 114; endovascular repair 97). RESULTS: Among the 154 surgical procedures, 11(7.1%) presented a complication during perioperative period including 6(8.7%) of open repair and 5(5.9%) of endovascular repair. After a median follow-up of 38.5(0.5-142.0) months, three(4.3%) cases of stroke and death were observed in open repair group, two(2.3%) cases of stroke and 4(4.7%) cases of death were observed in endovascular repair group. At 1, 3, 5 and 10 years of follow-up, primary patency rate of open repair and endovascular repair were 95.0% and 89.3%, 84.3% and 69.8%, 73.3% and 56.3%, 53.4% and 48.1%, respectively; Primary assisted patency rate were 100% and 97.5%, 90.4% and 78.2%, 79.1% and 72.8%, 60.7% and 54.0%, respectively; Secondary patency rate were 100% and 98.8%, 95.6% and 92.7%, 85.8% and 78.1%, 74.8% and 58.0%, respectively. Cumulative survival rate were 97.0% and 100%, 97.0% and 97.6%, 97.0% and 90.6%, 91.3% and 84.5%, respectively (χ(2)=0.182, P=0.669). CONCLUSIONS: Both of the surgical revascularization and endovascular management are safe and effective in the treatment of Takayasu arteritis. Although long-term patency of endovascular therapy is low, it can be performed repeatedly and can be used as a preferred approach in treating a short stenosis. Surgical repair shows excellent long-term durability, it seems to be more suitable for complex lesions and failure cases of endovascular management.

Synergistic effects of arsenic trioxide combined with ascorbic acid in human osteosarcoma MG-63 cells: a systems biology analysis.

OBJECTIVE: To further understand the synergistic mechanism of As2O3 and asscorbic acid (AA) in human osteosarcoma MG-63 cells by systems biology analysis. MATERIALS AND METHODS: Human osteosarcoma MG-63 cells were treated by As2O3 (1 µmol/L), AA (62.5 µmol/L) and combined drugs (1 µmol/L As2O3 plus 62.5 µmol/L AA). Dynamic morphological characteristics were recorded by Cell-IQ system, and growth rate was calculated. Illumina beadchip assay was used to analyze the differential expression genes in different groups. Synergic effects on differential expression genes (DEGs) were analyzed by mixture linear model and singular value decomposition model. KEGG pathway annotations and GO enrichment analysis were performed to figure out the pathways involved in the synergic effects. RESULTS: We captured 1987 differential expression genes in combined therapy MG-63 cells. FAT1 gene was significantly upregulated in all three groups, which is a promising drug target as an important tumor suppressor analogue; meanwhile, HIST1H2BD gene was markedly downregulated in the As2O3 monotherapy group and the combined therapy group, which was found to be upregulated in prostatic cancer. These two genes might play critical roles in synergetic effects of AA and As2O3, although the exact mechanism needs further investigation. KEGG pathway analysis showed many DEGs were related with tight junction, and GO analysis also indicated that DEGs in the combined therapy cells gathered in occluding junction, apical junction complex, cell junction, and tight junction. CONCLUSIONS: AA potentiates the efficacy of As2O3 in MG-63 cells. Systems biology analysis showed the synergic effect on the DEGs.

Prophylactic thyroidectomy for MEN 2-related medullary thyroid carcinoma based on predictive testing for RET proto-oncogene mutation and basal serum calcitonin in China.

INTRODUCTION: Early and normative surgery is the only curative method for multiple endocrine neoplasia type 2 (MEN 2)-related medullary thyroid carcinoma (MTC). AIMS: To study the timing of prophylactic total thyroidectomy (TT) for MEN 2-related MTC with different RET mutations in a Chinese population, and to compare the sensitivity and accuracy of fully-automated chemiluminescence immunoassay (FACLIA) and radioimmunoassay (RIA) for serum calcitonin (Ct). METHODS: We collected 24 asymptomatic individuals from 8 unrelated Chinese families with MEN 2, and analyzed RET mutation and Ct levels. Then we performed TT on 17 of the 24 individuals, including TT (2/17), TT with bilateral level VI lymph-node dissection (B-LND(VI); 12/17) and TT with B-LND(VI) + modified unilateral/bilateral/local neck dissection (3/17). RESULTS: Histopathology revealed bilateral/unilateral MTC in 15/17 (88.2%; median diameter, 1.0 cm) and bilateral C-cell hyperplasia in 2/17 (11.8%; p.V292M/R67H/R982C and p.C618Y). Lymph-node metastasis/fibro-adipose tissue invasion (p.C634R) or solely fibro-adipose tissue invasion (p.C634Y) were found in 2/17 (11.8%). Elevated pre-surgical Ct (pre-Ct) was identified by FACLIA in 17/17 (median age, 24.0), while pre-Ct by RIA was found in only 6/15 (P < 0.001). The median follow-up was 22.0 months, during which 16/17 had no abnormality (one p.C634R individual had elevated Ct), and another 7 carriers still had consistently undetectable Ct by FACLIA. CONCLUSIONS: Our study highlights the importance and feasibility of individualized prophylactic TT for MEN 2-related MTC, based on predictive integrated screening of RET and pre-Ct levels. Besides, we recommend FACLIA to measure Ct for earlier diagnosis, treatment and follow-up monitoring of MTC.

IKK-ß mediates hydrogen peroxide induced cell death through p85 S6K1.

The IκB kinase (IKK)/NF-κB pathway has been shown to be a major regulator in cell survival. However, the mechanisms through which IKK mediates cell death are not clear. In this study, we showed that IKK-ß contributed to hydrogen peroxide (H(2)O(2))-induced cell death independent of the NF-κB pathway. Our results demonstrated that the pro-death function of IKK-ß under oxidative stress was mediated by p85 S6K1 (S6 kinase 1), but not p70 S6K1 through a rapamycin-insensitive and mammalian target of rapamycin complex 1 kinase-independent mechanism. We found that IKK-ß associated with p85, but not p70 S6K1, which was required for H(2)O(2)-induced activation of p85 S6K1. IKK-ß and p85 S6K1 contributed to H(2)O(2)-induced phosphorylation of Mdm2 (S166) and p53 accumulation. p85 S6K1 is critical for IKK-ß-mediated cell death. Thus, these findings established a novel oxidative stress-responsive pathway that involves IKK-ß, p85 S6K1 and Mdm2, which is response for H(2)O(2)-induced cell death. Our results have important implications for IKK-ß and p85 S6K1 as potential targets for the prevention of diseases involved in oxidative stress-induced aberrant cell death.

Case report: a p.C618S RET proto-oncogene germline mutation in a large Chinese pedigree with familial medullary thyroid carcinoma.

We report a Chinese pedigree with familial medullary thyroid carcinoma. Direct sequencing of the entire coding sequences of Rearranged during Transfection (RET) identified a recurrent c.T1852A (p.C618S) mutation in 13 of 23 members. The polymorphisms c.A135G (p.A45A), c.A1296G (p.A432A), c.T2307G (p.L769L) and IVS19 + 15T > C were also found in 13 carriers, and c.G2073A (p.G691S) was found in 1 carrier. Of the 13 carriers, seven (mean age: 42.6 years, range: 27-64) presented MTC as the isolated clinical phenotype, with elevated basal serum calcitonin (average: 1077.9 ng/L, range: 504-2,652) and a mean diameter of thyroid nodules of 2.97 cm (range: 1.6-4.3); they underwent a total thyroidectomy with modified bilateral/unilateral neck dissection and/or level VI lymph node dissection. The other 6 carriers did not accept surgery (4 rejected, 2 awaited). These were 2 older patients (63 and 32 years) with elevated calcitonin (1359 and 41.4 ng/L) and multi-centric hypoechoic nodules (1.5 and 0.6 cm) with calcifications in both/left thyroid lobes; and Doppler ultrasound showed normal bilateral thyroids in 4 younger carriers (median age: 8.3 years, range: 4-12) but with increased calcitonin (average: 9.7 ng/L, range: 7.87-12.2) in 3 of them. The phenotype here is consistent with the clinical symptoms reported worldwide. We recommend that screening of hotspot regions of RET should be preferentially carried out, while whole-exon sequencing should be performed when clinical signs fail to reveal hotspot mutations or different phenotype discrepancies. Moreover, we strongly suggest prophylactic thyroidectomy should be performed before age 5 in carriers with p.C618S to prevent the occurrence and metastasis of MTC.

Resveratrol, an effective regulator of ovarian development and oocyte apoptosis.

Resveratrol, a phytopolyphenol compound found chiefly in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. However, little is known about the effects of resveratrol on ovarian development and oocyte apoptosis. We investigated the effects of resveratrol on ovarian development in rats with different ages [from post-natal day (PD) 1 to 15 months], as well as on oocyte apoptosis in PD1 and PD2 rat ovaries. We show that: a) ip injection of resveratrol (20 mg/kg/day) increased the percentage of unassembled follicles and the total number of oocytes in PD1 and PD2 rat ovaries. Similar results were obtained when mothers were treated with resveratrol (20 mg/kg/day) by intragastric administration from day 11, after the detection of vaginal plug, until delivery. In PD4 rat ovaries, the total number of oocytes was significantly increased in the groups treated with resveratrol. Moreover, more unassembled follicles and fewer primary follicles were present in the groups treated with resveratrol than in the controls; b) in 15-month-old rat ovaries, resveratrol increased the number of resting follicles and total oocytes, and decreased the number of developing follicles and atretic follicles; 3) the percentage of TUNEL-positive oocytes decreased in PD1 and PD2 rat ovaries after resveratrol treatment, and the number of oocytes positive for Foxo3a, Bim, and p27KIP1 in PD2 rat ovaries was lower in the resveratrol treatment group than in controls. These results suggest that resveratrol may delay oocyte nest breakdown and inhibit both the primordial-to-developing-follicle transition and apoptosis by decreasing the activation of Foxo3a, Bim, and p27KIP1, thus augmenting the resting follicle reserves, maintaining regular estrous cycles of early aged rats and delaying climacterium.

Activation of Akt and MAPK pathways enhances the tumorigenicity of CD133+ primary colon cancer cells.

Cancer stem cells (CSCs) play an important role in carcinogenesis, resistance to treatment and may lead to cancer recurrence and metastasis. However, the molecular mechanism of CSC involved in these events needs to be further elucidated. In this study, CD133(+) colon cancer cells were cultured, which showed CSC properties both in vitro and in vivo from metastatic tissue. Upstream molecules in Akt and mitogen-activated protein kinase (MAPK) pathways were preferentially expressed in these CD133(+) cells, as revealed by a global gene chip. The kinase activities of Akt and extracellular signal-regulated kinase (Erk)1/2 were also significantly upregulated in CD133(+) cells. In addition, the clonogenic growth of CD133(+) cell was reduced greatly by inhibiting the activity of Akt and Erk1/2. The results revealed the Akt and MAPK pathways were involved in the tumorigenesis of CD133(+) colon cancer cells, suggesting that molecules in these two pathways might be potential targets in the future therapy.

PUMA mediates EGFR tyrosine kinase inhibitor-induced apoptosis in head and neck cancer cells.

Overexpression of epidermal growth factor receptor (EGFR) is found in over 80% of head and neck squamous cell carcinomas (HNSCC) and associated with poor clinical outcomes. EFGR selective tyrosine kinase inhibitors (TKIs) or antibodies have recently emerged as promising treatments for solid tumors, including HNSCC, though the response rate to these agents is low. p53 upregulated modulator of apoptosis (PUMA), a BH3-only Bcl-2 family protein, is required for apoptosis induced by p53 and various chemotherapeutic agents. In this study, we show that PUMA induction is correlated with EGFR-TKI sensitivity, and is mediated through the p53 family protein p73beta and inhibition of the PI3K/AKT pathway. In some HNSCC cells, the gefitinib-induced degradation of oncogenic Delta Np63 seems to facilitate p73-mediated PUMA transcription. Inhibiting PUMA expression by small hairpin RNA (shRNA) impairs gefitinib-induced apoptosis. Furthermore, PUMA or BH3 mimetics sensitize HNSCC cells to gefitinib-induced apoptosis. Our results suggest that PUMA induction through p73 represents a new mechanism of EGFR inhibitor-induced apoptosis, and provide potential ways for enhancing and predicting the sensitivity to EGFR-targeted therapies in HNSCC.

Restoration of caveolin-1 expression suppresses growth and metastasis of head and neck squamous cell carcinoma.

Caveolin-1 (Cav-1) plays an important role in modulating cellular signalling, but its role in metastasis is not well defined. A significant reduction in Cav-1 levels was detected in lymph node metastases as compared with primary tumour of head and neck squamous cell carcinoma (HNSCC) specimens (P<0.0001), confirming the downregulation of Cav-1 observed in a highly metastatic M4 cell lines derived from our orthotopic xenograft model. To investigate the function of Cav-1 in metastasis of HNSCC, we compared stable clones of M4 cells carrying human cav-1 cDNA (CavS) with cells expressing an empty vector (EV) in vitro and in the orthotopic xenograft model. Overexpression of Cav-1 suppressed growth of the CavS tumours compared with the EV tumours. The incidence of lung metastases was significantly lower in animals carrying CavS tumours than those with EV tumours (P=0.03). In vitro, CavS cells displayed reduced cell growth, invasion, and increased anoikis compared with EV cells. In CavS cells, Cav-1 formed complex with integrin beta1 and Src. Further application of integrin beta1 neutralising antibody or Src inhibitor PP2 to EV cells illustrated similar phenotypes as CavS cells, suggesting that Cav-1 may play an inhibitory role in tumorigenesis and lung metastasis through regulating integrin beta1- and Src-mediated cell-cell and cell-matrix interactions.

The POU homeodomain protein Oct-1 binds Cis-regulatory element essential for the human GnRH upstream promoter activity in JEG-3 cells.

In previous studies, we have localized four specific nuclear protein-binding elements in the human GnRH upstream promoter. To test whether these four elements are reproductive tissue specific, we placed the four elements upstream to a thymidine kinase (TK) promoter/luciferase reporter gene, and transfected the constructs into human placental choriocarcinoma (JEG-3) cells. The 272-bp fragment (-994 to -723) containing the four elements can drive heterologous TK promoter expression in JEG-3 cells about 15 times more than that of basal TK promoter activity. Deletion of element 4 (E4, -987/-968) significantly decreased (4-fold) the luciferase activity. Further deletion of the other elements (E3 individual, -960/-940 or E3 and E2 in combination, -919/-896) only slightly decreased the luciferase activity. In contrast, deletion of element 1 (E1, -876/-851) caused a 2-fold loss of luciferase activity and elimination of E2 and E3 only lost less than 2-fold of the luciferase activity. Study performed with 5' end deletion of this region confirmed these observations. Furthermore, E4 DNA-protein complex can be supershifted by Oct-1 antibody, indicating that Oct-1 binds to E4. These results clearly demonstrated that all four elements are required to confer tissue-specific expression of the hGnRH gene in JEG-3 cells. However, the E4 is the most important for the tissue-specific expression of the hGnRH gene in JEG-3 cells. Oct-1 factor binds with E4 element and may be involved in the mediation of the human GnRH upstream promoter activity.

[Repair of humeral fracture and non-union with transfer of vascularized periosteal flap].

OBJECTIVE: To study the results of humeral fracture and non-union repaired by vascularized periosteal flap transfer. METHODS: The clinical data of humeral fracture and non-union in 23 cases were analysized retrospectively since 1995. Among them, minuted or several segmental fracture in 12 cases, non-union in 11 cases, and following injury of radial nerve in 7 cases. The operative method was open reduction, inner or external fixation with vascularized periosteal flap transfer. RESULTS: The period of follow-up was 6 months to 2 years. The repair result of all patients was excellent and good, but elbow joint motion in 2 cases of non-union was not satisfactory. The periosteal flap had good osteogenic ability. The period of bone union was 2 to 5 months in humeral fracture and non-union. And function of radial nerve was recovery. CONCLUSION: Transfer of distal humeral periosteal flap pedicled with radial collateral vessels is a better method for humeral fracture and non-union.