Suppression of FGFR3- and MYC-dependent oncogenesis by tubacin: association with HDAC6-dependent and independent activities.

Fibroblast growth factor receptor 3 (FGFR3) is amplified, translocated or mutated in a number of different human cancer types, but most commonly in bladder cancers. We previously found that the accumulation of FGFR3 is dependent on histone deacetylase 6 (HDAC6). Here we show that HDAC6 loss or inhibition reduces FGFR3 accumulation in cells made tumorigenic by ectopic expression of a mutant activated version of FGFR3 together with the MYC oncoprotein and in a bladder cancer cell line whose tumorigenicity is dependent on expression of a translocated version of FGFR3. In tumor xenoplant assays, HDAC6 deficiency or small molecule inhibition by the selective HDAC6 inhibitors tubacin or tubastatin A was found to significantly impede tumor growth. However, tubacin was more effective at inhibiting tumor growth than tubastatin A or HDAC6 deficiency. The superior anti-tumor activity of tubacin was linked to its ability to not only inhibit accumulation of mutant FGFR3, but also to cause robust downregulation of MYC and cyclin D1, and to induce a DNA damage response and apoptosis. Neither HDAC6 deficiency nor treatment with tubastatin A altered MYC or cyclin D1 levels, and neither induced a DNA damage response or apoptosis. Thus while tubacin and tubastatin A inhibit HDAC6 with similar selectivity and potency, our results reveal unique HDAC6-independent activities of tubacin that likely contribute to its potent anti-tumor activity.

HDAC6 deficiency or inhibition blocks FGFR3 accumulation and improves bone growth in a model of achondroplasia.

Mutations that cause increased and/or inappropriate activation of FGFR3 are responsible for a collection of short-limbed chondrodysplasias. These mutations can alter receptor trafficking and enhance receptor stability, leading to increased receptor accumulation and activity. Here, we show that wildtype and mutant activated forms of FGFR3 increase expression of the cytoplasmic deacetylase HDAC6 (Histone Deacetylase 6) and that FGFR3 accumulation is compromised in cells lacking HDAC6 or following treatment of fibroblasts or chondrocytes with small molecule inhibitors of HDAC6. The reduced accumulation of FGFR3 was linked to increased FGFR3 degradation that occurred through a lysosome-dependent mechanism. Using a mouse model of Thanatophoric Dysplasia Type II (TDII) we show that both HDAC6 deletion and treatment with the small molecule HDAC6 inhibitor tubacin reduced FGFR3 accumulation in the growth plate and improved endochondral bone growth. Defective endochondral growth in TDII is associated with reduced proliferation and poor hypertrophic differentiation and the improved bone growth was associated with increased chondrocyte proliferation and expansion of the differentiation compartment within the growth plate. These findings further define the mechanisms that control FGFR3 accumulation and contribute to skeletal pathology caused by mutations in FGFR3.

[Pollution Level and Health Risk Assessment of Heavy Metals in Atmospheric PM2.5 in Nanjing Before and After the Youth Olympic Games].

The influence of human activities on the atmospheric environment has attracted people's attention. This study reported the dynamic changes in PM2.5 concentration, its heavy metal compositions and health risk assessment from April to September, 2014 in Nanjing when the Youth Olympic Games ( YOG) was held. The results showed that the mass levels of PM2.5 ranged from 26.39 to 80.31 µg · m⁻³ from April to September. The mass levels of PM2.5 met the level II standard of ambient air quality in China (24 h average concentration, 75 µg · m⁻³) in months of April, May and July while met the level I standard (24 h average concentration, 35 µg · m⁻³) in August during the YOG. The average mass concentration of PM2.5 reached 76.14 µg · m⁻¹ after the YOG, showing resilience of air pollution. The variations of heavy metals were not consistent with each other throughout the observation period. Principal component analysis indicated that emission sources significantly affected the variations of PM2.5 and its heavy metals. PM2.5 and all of the heavy metals decreased to their minimum values during the YOG, indicating the effectiveness of those temporary measures for reducing atmospheric pollutant before and during the YOG. The health risks of Cd, Cu, Ni and Pb in PM2.5 via breathing and dermal contact exposure were all within the acceptable ranges, but potential carcinogenic risk existed for Cr in PM2.5. There was potential non-carcinogenic health risk for adult males via breathing of Mn and greater non-carcinogenic health risk for children via dermal contact exposures to all these 6 heavy metals.

Preparation and characterization of a novel injectable strontium-containing calcium phosphate cement with collagen.

PURPOSE: To develop a novel injectable strontium-containing calcium phosphate cement with collagen. METHODS: A novel calcium phosphate bone cement (CPC) was prepared with the addition of strontium element, collagenl, and modified starch; the injectability, solidification time, microstructure, phase composition, compressive strength, anti-collapsibility and histological properties of material were evaluated. RESULTS: The results showed that the material could be injected with an excellent performance; the modified starch significantly improved the anti-washout property of cement; with the liquid to solid ratio of 0.3, the largest compressive strength of cement was obtained (48.0 MPa ± 2.3 MPa); histological examination of repair tissue showed that the bone was repaired after 16 weeks; the degradation of cement was consistent with the new bone growth. CONCLUSION: A novel injectable collagen-strontium-containing CPC with excellent compressive strength and suitable setting time was prepared, with addition of modified starch. The CPC showed a good anti-washout property and the degradation time of the cement met with the new bone growing. This material is supposed to be used in orthopedic and maxillofacial surgery for bone defects.

A risk score for no reflow in patients with ST-segment elevation myocardial infarction after primary percutaneous coronary intervention.

BACKGROUND: Many factors are associated with no-reflow (NRF) phenomenon in ST-segment elevation myocardial infarction (STEMI), including plasma glucose, age, and pre-percutaneous coronary intervention (PCI) thrombus score. Initial clinical assessment would benefit from accurate NRF prediction. This study aimed to develop a simple scoring system to predict the risk of NRF in patients undergoing primary PCI with STEMI. METHODS: Baseline clinical and procedural variables were used for risk score development (the training dataset, n = 912) and validation (the test dataset, n = 864). Independent predictors of NRF from the multivariable model were assigned integer weights based on their coefficients and incorporated into a risk score. The discriminant ability of the score was tested by receiver operating characteristic analysis using the test dataset. RESULTS: The final model included 7 significant variables, which were age, pain-to-PCI time, neutrophil count, admission plasma glucose level, pre-PCI thrombus score, collateral circulation, and Killip class. All these variables were then used to build a risk score in terms of the prediction of NRF. Receiver operating characteristic analysis demonstrated good risk prediction with a c statistic of 0.800 (95% confidence interval: 0.772-0.826) in the test dataset. CONCLUSIONS: In patients with STEMI treated by primary PCI, incidence of NRF phenomenon may be predicted with an acceptable accuracy based on a 7-item simplified risk score.

Mutant activated FGFR3 impairs endochondral bone growth by preventing SOX9 downregulation in differentiating chondrocytes.

Fibroblast growth factor receptor 3 (FGFR3) plays a critical role in the control of endochondral ossification, and bone growth and mutations that cause hyperactivation of FGFR3 are responsible for a collection of developmental disorders that feature poor endochondral bone growth. FGFR3 is expressed in proliferating chondrocytes of the cartilaginous growth plate but also in chondrocytes that have exited the cell cycle and entered the prehypertrophic phase of chondrocyte differentiation. Achondroplasia disorders feature defects in chondrocyte proliferation and differentiation, and the defects in differentiation have generally been considered to be a secondary manifestation of altered proliferation. By initiating a mutant activated knockin allele of FGFR3 (FGFR3K650E) that causes Thanatophoric Dysplasia Type II (TDII) specifically in prehypertrophic chondrocytes, we show that mutant FGFR3 induces a differentiation block at this stage independent of any changes in proliferation. The differentiation block coincided with persistent expression of SOX9, the master regulator of chondrogenesis, and reducing SOX9 dosage allowed chondrocyte differentiation to proceed and significantly improved endochondral bone growth in TDII. These findings suggest that a proliferation-independent and SOX9-dependent differentiation block is a key driving mechanism responsible for poor endochondral bone growth in achondroplasia disorders caused by mutations in FGFR3.

Development and validation of a clinical risk score predicting the no-reflow phenomenon in patients treated with primary percutaneous coronary intervention for ST-segment elevation myocardial infarction.

OBJECTIVE: The 'no-reflow' phenomenon after a primary percutaneous coronary intervention (pPCI) in patients with acute ST-segment elevation myocardial infarction (STEMI) is a strong predictor of both short- and long-term mortality. We therefore developed and prospectively validated a risk score system in order to identify STEMI patients at high risk in terms of no-reflow after primary PCI. METHODS: The first part of our study used data from 1,615 STEMI patients who underwent primary PCI within 12 h from symptom onset. Using logistic regression, we derived a risk score to predict angiographic no-reflow using baseline clinical variables. From this score, we developed a simplified fast-track screen that can be used before reperfusion. In the second part of our study, we prospectively validated the score system using receiver-operating characteristic (ROC) curves with data from 692 STEMI patients. RESULTS: The model included six clinical items: age, neutrophil count, admission plasma glucose, ß-blocker treatment, time-to-hospital admission and Killip classes. The risk score system demonstrated a good risk prediction with a c-statistic of 0.757 (95% CI 0.732-0.781) based on ROC analysis. CONCLUSION: A simple risk score system based on clinical variables is useful to predict the risk of developing no-reflow after pPCI in patients with STEMI.

Prevalence of arterial stiffness in North China, and associations with risk factors of cardiovascular disease: a community-based study.

BACKGROUND: Brachial-ankle pulse wave velocity (baPWV), which reflects the stiffness of both central and peripheral muscular arteries, has been frequently used as a simple index for assessing arterial stiffness. The aim of the present study was to investigate the prevalence of arterial stiffness in North China based on baPWV measurements, and explore the associations between increased arterial stiffness and risk factors of cardiovascular diseases (CVD). METHODS: Twenty-three community populations were established in North China. For each participant, parameters for calculating baPWV, including blood pressures and pressure waveforms, were measured using a non-invasive automatic device. All participants were required to respond to an interviewer-led questionnaire including medical histories and demographic data, and to receive blood tests on biochemical indictors. RESULTS: A total of 2,852 participants were finally investigated. Among them, 1,201 people with low burden of CVD risk factors were chosen to be the healthy reference sample. The cut-off point of high baPWV was defined as age-specific 90th percentile of the reference sample. Thus, the prevalence of high baPWV was found to be 22.3% and 26.4% in men and women respectively. After adjusted for age, heart rate (HR), systolic blood pressure (SBP), fasting glucose level, and smoking were significantly associated with high baPWV in men; while level of serum total cholesterol (TC), HR, SBP, and diabetes were significantly associated with high baPWV in women. CONCLUSIONS: Based on the age-specific cut-off points, the middle-aged population has a higher prevalence of high baPWV in North China. There exists a difference between men and women in terms of the potential risk factors associated with arterial stiffness.

A critical role for Mnt in Myc-driven T-cell proliferation and oncogenesis.

Mnt (Max's next tango) is a Max-interacting transcriptional repressor that can antagonize both the proproliferative and proapoptotic functions of Myc in vitro. To ascertain the physiologically relevant functions of Mnt and to help define the relationship between Mnt and Myc in vivo, we generated a series of mouse strains in which Mnt was deleted in T cells in the absence of endogenous c-Myc or in the presence of ectopic c-Myc. We found that apoptosis caused by loss of Mnt did not require Myc but that ectopic Myc expression dramatically decreased the survival of both Mnt-deficient T cells in vivo and Mnt-deficient MEFs in vitro. Consequently, Myc-driven proliferative expansion of T cells in vitro and thymoma formation in vivo were prevented by the absence of Mnt. Consistent with T-cell models, mouse embryo fibroblasts (MEFs) lacking Mnt were refractory to oncogenic transformation by Myc. Tumor suppression caused by loss of Mnt was linked to increased apoptosis mediated by reactive oxygen species (ROS). Thus, although theoretically and experimentally a Myc antagonist, the dominant physiological role of Mnt appears to be suppression of apoptosis. Our results redefine the physiological relationship between Mnt and Myc and requirements for Myc-driven oncogenesis.

Disruption of a Sox9-ß-catenin circuit by mutant Fgfr3 in thanatophoric dysplasia type II.

Mutations in fibroblast growth factor (FGF) receptors are responsible for a variety of skeletal birth defects, but the underlying mechanisms responsible remain unclear. Using a mouse model of thanatophoric dysplasia type II in which FGFR3(K650E) expression was directed to the appendicular skeleton, we show that the mutant receptor caused a block in chondrocyte differentiation specifically at the prehypertrophic stage. The differentiation block led to a severe reduction in hypertrophic chondrocytes that normally produce vascular endothelial growth factor, which in turn was associated with poor vascularization of primary ossification centers and disrupted endochondral ossification. We show that the differentiation block and defects in joint formation are associated with persistent expression of the chondrogenic factor Sox9 and down-regulation of ß-catenin levels and activity in growth plate chondrocytes. Consistent with these in vivo results, FGFR3(K650E) expression was found to increase Sox9 and decrease ß-catenin levels and transcriptional activity in cultured mesenchymal cells. Coexpression of Fgfr3(K650E) and Sox9 in cells resulted in very high levels of Sox9 and cooperative suppression of ß-catenin-dependent transcription. Fgfr3(K650E) had opposing effects on Sox9 and ß-catenin protein stability with it promoting Sox9 stabilization and ß-catenin degradation. Since both Sox9 overexpression and ß-catenin deletion independently blocks hypertrophic differentiation of chondrocytes and cause chondrodysplasias similar to those caused by mutations in FGFR3, our results suggest that dysregulation of Sox9 and ß-catenin levels and activity in growth plate chondrocytes is an important underlying mechanism in skeletal diseases caused by mutations in FGFR3.

Sequential and coordinated actions of c-Myc and N-Myc control appendicular skeletal development.

BACKGROUND: During limb development, chondrocytes and osteoblasts emerge from condensations of limb bud mesenchyme. These cells then proliferate and differentiate in separate but adjacent compartments and function cooperatively to promote bone growth through the process of endochondral ossification. While many aspects of limb skeletal formation are understood, little is known about the mechanisms that link the development of undifferentiated limb bud mesenchyme with formation of the precartilaginous condensation and subsequent proliferative expansion of chondrocyte and osteoblast lineages. The aim of this study was to gain insight into these processes by examining the roles of c-Myc and N-Myc in morphogenesis of the limb skeleton. METHODOLOGY/PRINCIPAL FINDINGS: To investigate c-Myc function in skeletal development, we characterized mice in which floxed c-Myc alleles were deleted in undifferentiated limb bud mesenchyme with Prx1-Cre, in chondro-osteoprogenitors with Sox9-Cre and in osteoblasts with Osx1-Cre. We show that c-Myc promotes the proliferative expansion of both chondrocytes and osteoblasts and as a consequence controls the process of endochondral growth and ossification and determines bone size. The control of proliferation by c-Myc was related to its effects on global gene transcription, as phosphorylation of the C-Terminal Domain (pCTD) of RNA Polymerase II, a marker of general transcription initiation, was tightly coupled to cell proliferation of growth plate chondrocytes where c-Myc is expressed and severely downregulated in the absence of c-Myc. Finally, we show that combined deletion of N-Myc and c-Myc in early limb bud mesenchyme gives rise to a severely hypoplastic limb skeleton that exhibits features characteristic of individual c-Myc and N-Myc mutants. CONCLUSIONS/SIGNIFICANCE: Our results show that N-Myc and c-Myc act sequentially during limb development to coordinate the expansion of key progenitor populations responsible for forming the limb skeleton.

The role of senescence and prosurvival signaling in controlling the oncogenic activity of FGFR2 mutants associated with cancer and birth defects.

Mutations in fibroblast growth factor receptors (FGFRs) cause human birth defect syndromes and are associated with a variety of cancers. Although forced expression of mutant activated FGFRs has been shown to oncogenically transform some immortal cell types, their activity in primary cells remains unclear. Here, we show that birth defect and cancer-associated FGFR2 mutants promote DNA-damage signaling and p53-dependent senescence in primary mouse and human cells. Senescence promoted by FGFR mutants was associated with downregulation of c-Myc and forced expression of c-Myc facilitated senescence escape. Whereas c-Myc expression facilitated senescence bypass, mutant FGFR2 signaling suppressed c-Myc-dependent apoptosis and led to oncogenic transformation. Cells transformed by coexpression of a constitutively activated FGFR2 mutant plus c-Myc appeared to be become highly addicted to FGFR-dependent prosurvival activities, as small molecule inhibition of FGFR signaling resulted in robust p53-dependent apoptosis. Our data suggest that senescence-promoting activities of mutant FGFRs may normally limit their oncogenic potential and may be relevant to their ability to disrupt morphogenesis and cause birth defects. Our results also raise the possibility that cancers originating through a combination of constitutive FGFR activation and deregulated Myc expression may be particularly sensitive to small molecule inhibitors of FGF receptors.

Obesity criteria for identifying metabolic risks.

Criteria of obesity in the Chinese population with multiple metabolic risk factors remains unclear. The objective was to determine the best anthropometrical measurements with regard to the metabolic syndrome (MetS) and to propose optimal cut-off values. Between April and August, 2007, 3,704 men and 6,392 women aged 18-85 years were recruited from four community centers. Medical examinations included measurement of weight, height, waist circumference (WC), hip circumference, fasting blood triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), glucose concentrations, and blood pressure (BP). Body mass index (BMI), waist to hip ratio (WHR), WC and waist to stature ratio (WSR) were calculated. Four metabolic risk factors were examined: 1) high BP; 2) high levels of TG; 3) low levels of HDL-C; 4) impaired glucose tolerance. The relationships between studied indices and risk factors were analyzed using partial correlation analyses, analysis of variance (ANOVA), linear regression, and Receiver Operator Characteristic (ROC) curve analysis. The optimal cut-off values of each obesity index were calculated using ROC analysis respectively. All obesity indices were positively associated with metabolic risk factors. Area under curve (AUC) of WC was the largest for >or= 2 risk factors after adjustment for age in both genders. Optimal cut-off points for WC were 89 cm in men, and 80.5cm, 82.5cm, and 89.5cm in < 40-yr, 40-60-yr, and > 60-yr women respectively. Waist circumference is best associated with metabolic risk factors among the studied indices in Chinese adults. Indices of abdominal obesity for older age groups tend to be higher than younger age groups in women.

Activities of N-Myc in the developing limb link control of skeletal size with digit separation.

The developing limb serves as a paradigm for studying pattern formation and morphogenetic cell death. Here, we show that conditional deletion of N-Myc (Mycn) in the developing mouse limb leads to uniformly small skeletal elements and profound soft-tissue syndactyly. The small skeletal elements are associated with decreased proliferation of limb bud mesenchyme and small cartilaginous condensations, and syndactyly is associated with a complete absence of interdigital cell death. Although Myc family proteins have pro-apoptotic activity, N-Myc is not expressed in interdigital cells undergoing programmed cell death. We provide evidence indicating that the lack of interdigital cell death and associated syndactyly is related to an absence of interdigital cells marked by expression of Fgfr2 and Msx2. Thus, instead of directly regulating interdigital cell death, we propose that N-Myc is required for the proper generation of undifferentiated mesenchymal cells that become localized to interdigital regions and trigger digit separation when eliminated by programmed cell death. Our results provide new insight into mechanisms that control limb development and suggest that defects in the formation of N-Myc-dependent interdigital tissue may be a root cause of common syndromic forms of syndactyly.

Mnt-Max to Myc-Max complex switching regulates cell cycle entry.

The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt-Max complexes and a transient switch in the ratio of Mnt-Max to c-Myc-Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt-Max to c-Myc-Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

[An epidemiological survey of atrial fibrillation in China].

OBJECTIVE: To know the current prevalence of atrial fibrillation (AF) in China and contribute to further Chinese studies on AF in future. METHODS: We chose 14 natural populations from 14 different provinces across China. Using international standardized methods, we performed an epidemical study which was mainly on AF. RESULTS: The crude rate of prevalence of AF in China is 0.77%, which would be 0.61% after being standardized. Also, it is increasing with aging. The prevalence is higher in men than in women (0.9% vs 0.7%, P = 0.013). Among all the AF cases, valvular, nonvalvular, and lone AF were 12.9%, 65.2%, and 21.9% respectively. Ischemic stroke was the most frequent type seen among AF cases and the stroke rate among cases with AF was significantly higher than that without (12.1% vs 2.1%, P < 0.01). CONCLUSIONS: The prevalence of AF in China, whether classified by age, gender or cause, is similar to the results from other countries, especially North America and Europe. The incidence of stroke among AF cases is rather high in China. However, patients with AF would not like to take the necessary medicine. Therefore it is advisable to enforce the control of AF. We will continue to do the follow-up in these populations.

Loss of the Max-interacting protein Mnt in mice results in decreased viability, defective embryonic growth and craniofacial defects: relevance to Miller-Dieker syndrome.

The Mnt gene encodes a Mad-family bHLH transcription factor located on human 17p13.3. Mnt is one of 20 genes deleted in a heterozygous fashion in Miller-Dieker syndrome (MDS), a contiguous gene syndrome that consists of severe neuronal migration defects and craniofacial dysmorphic features. Mnt can inhibit Myc-dependent cell transformation and is hypothesized to counterbalance the effects of c-Myc on growth and proliferation in vivo by competing with Myc for binding to Max and by repressing target genes activated by Myc : Max heterodimers. Unlike the related Mad family members, Mnt is expressed ubiquitously and Mnt/Max heterodimers are found in proliferating cells that contain Myc/Max heterodimers, suggesting a unique role for Mnt during proliferation. To examine the role of Mnt in vivo, we produced mice with null (Mnt(KO)) and loxP-flanked conditional knock-out (Mnt(CKO)) alleles of Mnt. Virtually all Mnt(KO/KO) mutants in a mixed (129S6 x NIH Black Swiss) or inbred (129S6) genetic background died perinatally. Mnt-deficient embryos exhibited small size throughout development and showed reduced levels of c-Myc and N-Myc. In addition, 37% of the mixed background mutants displayed cleft palate as well as retardation of skull development, a phenotype not observed in the inbred mutants. These results demonstrate an important role for Mnt in embryonic development and survival, and suggest that Mnt may play a role in the craniofacial defects displayed by MDS patients.

Evidence of mnt-myc antagonism revealed by mnt gene deletion.

Myc proteins play a central role in promoting cell proliferation and contribute to a diverse array of cancers. My function appears completely dependent on heterodimerization with Max through related bHLHZip regions. Max interaction with Myc is required for DNA binding at so-called E-box sequences and Myc-dependent transcriptional activation. The repressor with similar DNA binding specificity raised the possibility that Mnt may serve a general role as a Myc antagonist.