Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

Astrocyte elevated gene-1 mediates glycolysis and tumorigenesis in colorectal carcinoma cells via AMPK signaling.

To investigate the role of AEG-1 in glycolysis and tumorigenesis, we construct myc-AEG-1 expression vector and demonstrate a novel mechanism that AEG-1 may increase the activity of AMPK by Thr172 phosphorylation. The higher expression levels of AEG-1 in colorectal carcinoma cells were found but showed significant difference in different cell lines. To study the role of AEG-1 in colorectal cells, myc-AEG-1 vector was constructed and transfected into NCM460 colonic epithelial cells. We observed consistent increasing of glucose consumption and lactate production, typical features of anaerobic glycolysis, suggesting that AEG-1 may promote anaerobic glycolysis. Moreover, we noted that AMPK phosphorylation at Thr172 as well as pPFK2 (Ser466) was increased in NCM460 cells overexpressing AEG-1. Compound C may block AMPK and PFK2 phosphorylation in both control and AEG-1-overexpressed cells and decrease the glucose consumption and lactate production. The present findings indicated that reduced AEG-1 protein levels by RNAi may decrease the glucose consumption and lactate production in HCT116 colorectal carcinoma cells. The present identified AEG-1/AMPK/PFK2 glycolysis cascade may be essential to cell proliferation and tumor growth. The present results may provide us with a mechanistic insight into novel targets controlled by AEG-1, and the components in the AEG-1/AMPK/PFK2 glycolysis process may be targeted for the clinical treatment of cancer.

Role of the calcium-sensing receptor in cardiomyocyte apoptosis via the sarcoplasmic reticulum and mitochondrial death pathway in cardiac hypertrophy and heart failure.

AIMS: Alterations in calcium homeostasis in the intracellular endo/sarcoplasmic reticulum (ER/SR) and mitochondria of cardiomyocytes cause cell death via the SR and mitochondrial apoptotic pathway, contributing to ventricular dysfunction. However, the role of the calcium-sensing receptor (CaR) in cardiac hypertrophy and heart failure has not been studied. This study examined the possible involvement of CaR in the SR and mitochondrial apoptotic pathway in an experimental model of heart failure. METHODS AND RESULTS: In Wistar rats, cardiac hypertrophy and heart failure were induced by subcutaneous injection of isoproterenol (Iso). Calindol, an activator of CaR, and calhex231, an inhibitor of CaR, were administered by caudal vein injection. Cardiac remodeling and left ventricular function were then analyzed in these rats. After 2, 4, 6 and 8 weeks after the administration of Iso, the rats developed cardiac hypertrophy and failure. The cardiac expression of ER chaperones and related apoptotic proteins was significantly increased in the failing hearts. Furthermore, the expression of ER chaperones and the apoptotic rate were also increased with the administration of calindol, whereas the expression of these proteins was reduced with the treatment of calhex231. We also induced cardiac hypertrophy and failure via thoracic aorta constriction (TAC) in mice. After 2 and 4 weeks of TAC, the expression of ER chaperones and apoptotic proteins were increased in the mouse hearts. Furthermore, Iso induced ER stress and apoptosis in cultured cardiomyocytes, while pretreatment with calhex231 prevented ER stress and protected the myocytes against apoptosis. To further investigate the effect of CaR on the concentration of intracellular calcium, the calcium concentration in the SR and mitochondria was determined with Fluo-5N and x-rhod-1 and the mitochondrial membrane potential was examined with JC-1 using laser confocal microscopy. After treatment with Iso for 48 hours, activation of CaR reduced [Ca(2+)]SR, increased [Ca(2+)]m, decreased the mitochondrial membrane potential, increased the expression of ER stress chaperones and related apoptotic proteins, and induced the release of cytochrome c from the mitochondria. CONCLUSIONS: Our results demonstrated that CaR activation caused Ca(2+) release from the SR into the mitochondria and induced cardiomyocyte apoptosis through the SR and mitochondrial apoptotic pathway in failing hearts.

[Overexpression of p-Stat3 and Mcl-1, and their correlation with differentiation and apoptotic resistance in esophageal squamous cell carcinoma].

OBJECTIVE: To detect the expression of phosphorylated-signal transducer and activator of transcription 3 (p-Stat3) and myeloid leukemia-1 (Mcl-1) as well as their correlation, and to investigate the functional role of Stat3 and Mcl-1 in the pathogenesis of esophageal squamous cell carcinoma (ESCC). METHODS: Stat3 activity in ESCC cells was inhibited with JAK/Stat3 inhibitors (AG490 or JSI-124). Specific siRNA was used to inhibit the Stat3 expression. Cell apoptosis was detected by flow cytometry. Expression of Mcl-1 protein was determined by Western blotting. Expression of phospho-Stat3 (Tyr705) and myeloid leukemia-1 (Mcl-1) proteins in ESCC tissues was detected by tissue microarray and immunohistochemistry. The relationship between p-Stat3 or Mcl-1 aberrant expression and clinicopatholohical features of ESCC was analyzed. The correlation of their expression was also analyzed. RESULTS: Suppression of the Stat3 signaling activation in ESCC cells led to marked apoptosis, and dramatic reduction of Mcl-1 protein. The positive rate of phospho-Stat3 (Tyr705) expression was 45.0% in 50/111 of the ESCC tissue samples. The lower the degree of tumor differentiation, the higher the positive rate of phospho-Stat3 (Tyr705), showing a significant difference (P = 0.018). The positive rate of Mcl-1 protein expression was 72.1% (80/111), and the lower the degree of tumor differentiation was, the higher there was the positive rate of Mcl-1, with a significant difference (P = 0.026). There was a positive correlation between the expressions of p-Stat3 and Mcl-1 proteins (P = 0.012). CONCLUSIONS: In a subset of ESCC tissues, p-Stat3 (Tyr705) and Mcl-1 are overexpressed and positively correlated with each other, and both are correlated with tumor differentiation. Persistent activation of Stat3 contributes to apoptotic resistance in ESCC cells, and may be at least partly mediated through upregulation of Mcl-1.

Cdk5 mediates vimentin Ser56 phosphorylation during GTP-induced secretion by neutrophils.

Secretion by neutrophils contributes to acute inflammation following injury or infection. Vimentin has been shown to be important for secretion by neutrophils but little is known about its dynamics during secretion, which is regulated by cyclin-dependent kinase 5 (Cdk5). In this study, we sought to examine the vimentin dynamics and its potential regulation by Cdk5 during neutrophil secretion. We show that vimentin is a Cdk5 substrate that is specifically phosphorylated at Ser56. In response to neutrophil stimulation with GTP, vimentin Ser56 was phosphorylated and colocalized with Cdk5 in the cytoplasmic compartment. Vimentin pSer56 and Cdk5 colocalization was consistent with coimmunoprecipitation from stimulated cells. Vimentin Ser56 phosphorylation occurred immediately after stimulation, and a remarkable increase in phosphorylation was noted later in the secretory process. Decreased GTP-induced vimentin Ser56 phosphorylation and secretion resulted from inhibition of Cdk5 activity by roscovitine or olomoucine or by depletion of Cdk5 by siRNA, suggesting that GTP-induced Cdk5-mediated vimentin Ser56 phosphorylation may be related to GTP-induced Cdk5-mediated secretion by neutrophils. Indeed, inhibition of vimentin Ser56 phosphorylation led to a corresponding inhibition of GTP-induced secretion, indicating a link between these two events. While fMLP also induced vimentin Ser56 phosphorylation, such phosphorylation was unaffected by roscovitine, which nonetheless, inhibited secretion, suggesting that Cdk5 regulates fMLP-induced secretion via a mechanism independent of Cdk5-mediated vimentin Ser56 phosphorylation. These findings demonstrate the distinct involvement of Cdk5 in GTP- and fMLP-induced secretion by neutrophils, and support the notion that specific targeting of Cdk5 may serve to inhibit the neutrophil secretory process.

Genetic polymorphisms in DNA repair genes OGG1, APE1, XRCC1, and XPD and the risk of age-related cataract.

PURPOSE: To analyze the association of the polymorphisms in 8-oxoguanine glycosylase-1 (OGG1), X-ray repair cross-complementing-1 (XRCC1), and AP endonuclease-1 (APE1) genes in the base excision repair pathway and xeroderma pigmentosum complementation group D (XPD) in the nucleotide excision repair pathway with the risk of cataract in a Chinese population. DESIGN: Case-control study. PARTICIPANTS: Subjects with cataract (n = 415) or no cataract (n = 386) in the Age Related Eye Disease Ancillary Study. METHODS: The study included 415 cataract patients and 386 controls. Genotyping was carried out by the polymerase chain reaction-restriction fragment length polymorphism method. Differences in the frequencies were estimated by the chi-square test, and risk was estimated by using unconditional logistic regression after adjusting for age and gender. MAIN OUTCOME MEASURES: Association of single nucleotide polymorphisms in OGG1-Ser326Cys with the development of age-related cataract. RESULTS: The OGG1 Cys/Cys genotype frequency was significantly higher in cataract patients (P = 0.014; odds ratio [OR], 2.06; 95% confidence interval [CI], 1.171-3.624). The OGG1 Ser/Ser genotype (P = 0.003; OR, 0.647; 95% CI, 0.487-0.860) seems to have a protective role against cataract, and the Cys allele (P<0.001; OR, 1.517; 95% CI, 1.204-1.911) seems to have a deleterious role in the development of cataract. The genotype frequency of the Ser/Ser of OGG1-Ser326Cys was significantly different in the cortical and mixed-type cataract group (P = 0.014; OR, 0.591; 95% CI, 0.391-0.893; and P = 0.035; OR, 0.639; 95% CI, 0.425-0.960; respectively), and the Cys/Cys genotype of OGG1-Ser326Cys was significantly different in the mixed-type cataract group (P = 0.012; OR, 2.610; 95% CI, 1.284-5.306) compared with that of healthy controls. In XRCC1-Arg399Gln, APE1-Asp148Glu, and XPD-Lys751Gln polymorphisms, there were no significant differences in frequencies of the variant homozygous in patients compared with controls. CONCLUSIONS: Results suggest that the Cys/Cys genotype of the OGG1-Ser326Cys polymorphism may be associated with increased risk of age-related cataract. However, in XRCC1-Arg399Gln, APE1-Asp148Glu, and XPD-Lys751Gln polymorphisms, there were no significant differences in frequencies of the variant homozygous in patients compared with controls.

Characterization of genetic rearrangements in esophageal squamous carcinoma cell lines by a combination of M-FISH and array-CGH: further confirmation of some split genomic regions in primary tumors.

BACKGROUND: Chromosomal and genomic aberrations are common features of human cancers. However, chromosomal numerical and structural aberrations, breakpoints and disrupted genes have yet to be identified in esophageal squamous cell carcinoma (ESCC). METHODS: Using multiplex-fluorescence in situ hybridization (M-FISH) and oligo array-based comparative hybridization (array-CGH), we identified aberrations and breakpoints in six ESCC cell lines. Furthermore, we detected recurrent breakpoints in primary tumors by dual-color FISH. RESULTS: M-FISH and array-CGH results revealed complex numerical and structural aberrations. Frequent gains occurred at 3q26.33-qter, 5p14.1-p11, 7pter-p12.3, 8q24.13-q24.21, 9q31.1-qter, 11p13-p11, 11q11-q13.4, 17q23.3-qter, 18pter-p11, 19 and 20q13.32-qter. Losses were frequent at 18q21.1-qter. Breakpoints that clustered within 1 or 2 Mb were identified, including 9p21.3, 11q13.3-q13.4, 15q25.3 and 3q28. By dual-color FISH, we observed that several recurrent breakpoint regions in cell lines were also present in ESCC tumors. In particular, breakpoints clustered at 11q13.3-q13.4 were identified in 43.3% (58/134) of ESCC tumors. Both 11q13.3-q13.4 splitting and amplification were significantly correlated with lymph node metastasis (LNM) (P = 0.004 and 0.022) and advanced stages (P = 0.004 and 0.039). Multivariate logistic regression analysis revealed that only 11q13.3-q13.4 splitting was an independent predictor for LNM (P = 0.026). CONCLUSIONS: The combination of M-FISH and array-CGH helps produce more accurate karyotypes. Our data provide significant, detailed information for appropriate uses of these ESCC cell lines for cytogenetic and molecular biological studies. The aberrations and breakpoints detected in both the cell lines and primary tumors will contribute to identify affected genes involved in the development and progression of ESCC.

The Raf-1 inhibitor GW5074 and the ERK1/2 pathway inhibitor U0126 ameliorate PC12 cells apoptosis induced by 6-hydroxydopamine.

6-Hydroxydopamine (6-OHDA) is a widely used dopaminergic neurotoxin that leads to cell apoptosis in vivo and in vitro, and is a widely accepted experimental model of neurodegeneration in Parkinson's disease. However, the molecular mechanisms responsible for 6-OHDA-induced cell apoptosis are unclear. We found that the treatment of PC12 cells with 6-OHDA resulted in a significant decrease in cell viability and elevated apoptosis as detected by MTT assay, Hoechst 33258 staining, and flow cytometry. In addition, 6-OHDA induced a time-dependent phosphorylation of ERK1/2 at Thr-202/Tyr-204 and of Raf-1 at Ser-338, but a decreased level of Raf-1 phosphorylation at Ser-259. Phosphorylation of ERK1/2 at Thr-202/Tyr-204 and Raf-1 at Ser-338 were inhibited by the Raf-1 inhibitor GW5074, while the ERK1/2 pathway inhibitor U0126 decreased phosphorylation of ERK1/2. Furthermore, 6-OHDA-induced PC12 cells apoptosis was suppressed by GW5074 and U0126. Our results suggest that GW5074 and U0126 act as neuroprotants against 6-OHDA toxicity in PC12 cells by modulating Raf-1/ERK1/2 signaling systems.

Comparison of the antiangiogenic activity of modified RGDRGD-endostatin to endostatin delivered by gene transfer in vivo rabbit neovascularization model.

PURPOSE: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo. METHODS: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 µg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days. RESULTS: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed. CONCLUSIONS: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.

Down-regulation of ING4 is associated with initiation and progression of lung cancer.

AIMS: Tumour suppressor ING4 is one of ING family genes, which are involved in cell cycle arrest, gene transcription regulation, DNA repair and apoptosis. ING4 inhibition has been reported in various tumours, including gliomas, breast tumours, and stomach adenocarcinoma. The aim was to evaluate ING4 expression in lung cancers. METHOD AND RESULTS: By immunohistochemistry of 246 lung tumour tissues, reduced ING4 nuclear and cytoplasmic expression were both revealed in lung cancer and associated with tumour grade. Interestingly, compared with normal tissues, we found more tumours with ING4 expression in the cytoplasm higher than in the nucleus. Nuclear ING4 inhibition correlated with the tumour stage and lymph node metastasis. Consistent with these findings, semiquantitative reverse transcriptase-polymerase chain reaction and Western blotting demonstrated decreased ING4 mRNA and expression in 100% (50/50) tumour tissues. Furthermore, ING4 expression was lower in grade III than in grades I-II tumours. Reduced ING4 mRNA correlated with lymph node metastasis. CONCLUSIONS: Our results indicate that overall inhibition of ING4 expression and ING4 expression higher in cytoplasm than in nucleus of tumour cells may be involved in the initiation and progression of lung cancers, and thus, analysis for ING4 expression may be useful as a clinical diagnostic and prognostic tool for lung cancer.

Reduced expression and novel splice variants of ING4 in human gastric adenocarcinoma.

ING4, a new member of the ING (inhibitor of growth) family of tumour suppressor genes, has been found to be deleted or down-regulated in gliomas, breast tumours, and head and neck squamous cell carcinomas. The goal of the present study was to investigate whether the expression and alternative splicing of ING4 transcripts are involved in the initiation and progression of stomach adenocarcinoma. ING4 mRNA and protein expression was examined in gastric adenocarcinoma tissues and human gastric adenocarcinoma cell lines by RT-PCR, real-time RT-PCR, tissue microarray immunohistochemistry, and western blot analysis. Alterations in ING4 transcripts were determined through sequence analysis of ING4 cDNA. Our data showed that ING4 mRNA and protein were dramatically reduced in stomach adenocarcinoma cell lines and tissues, and significantly less in female than in male patients. We also found that reduced ING4 mRNA expression correlated with the stage of the tumour. Interestingly, by sequence analysis, we discovered five novel aberrantly spliced variant forms of ING4_v1 and ING4_v2. These variants cause a codon frame-shift and, eventually, deletion of the NLS or PHD domain contributing to the mislocalization of p53 and/or HAT/HDAC complexes and, subsequently, altered gene expression in gastric adenocarcinoma. These results suggest that attenuated and aberrant ING4 expression may be involved in the initiation and progression of stomach adenocarcinoma.

RUNX3 inhibits cell proliferation and induces apoptosis by TGF-beta-dependent and -independent mechanisms in human colon carcinoma cells.

BACKGROUND: Genes involved in the TGF-beta signaling pathway are often altered in several types of cancers. The TGF-beta-resistant human colon cancer cell line HT-29 has inactivated TbetaRII and deficient expression of RUNX3 and Smad4, which are involved in the TGF-beta signaling pathway. METHODS: Western blot and immunocytochemistry were performed to confirm gene expression, the MTT assay to detect cell growth, flow cytometry to investigate the cell cycle and the TUNEL to detect cell apoptosis. RESULTS: In the absence of TGF-beta, Bim was upregulated, cell growth was inhibited and apoptosis was induced. TGF-beta treatment did not affect RUNX3 expression; however, the increase in Bim expression was significant and time dependent. Interestingly, Smad4 but not Smad2/3 was also upregulated upon exposure to TGF-beta. This was not the case after TGF-beta treatment of parent HT-29 cells. As expected, TGF-beta further inhibited cell growth and induced apoptosis in HT-29/RUNX3+ cells. CONCLUSION: Our data demonstrate that RUNX3 is involved in TGF-beta-dependent and -independent cell growth inhibition and apoptosis induction pathways.

Anti-apoptotic effects of a calpain inhibitor on cardiomyocytes in a canine rapid atrial fibrillation model.

PURPOSE: This study was designed to evaluate the effects of a calpain inhibitor on cardiac muscle apoptosis in rapid pacing canine atrial fibrillation (AF) models. METHODS: Twenty one dogs were divided into three groups: a sham operation group, a control AF group and a calpain inhibitor group. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute. N-Acetyl-Leu-Leu-Met (1.0 mg/kg/day) was administered in the calpain inhibitor group for three weeks. The activity of calpain I and cardiomyocyte apoptosis were measured by fluorometry and TUNEL assay, respectively. Protein expression of caspase-3 was detected by Western blot. The localizations of caspase-3, caspase-8, bcl-2 and ARC were assessed by immunohistochemistry. RESULTS: In comparison to the sham operation group, the activity of calpain I was significantly increased in the control AF group (2.3 fold, p < 0.001), and decreased in the calpain inhibitor group (1.1 fold, p < 0.005). The calpain activity correlated with the apoptosis index (r = 0.9, p < 0.05). The apoptosis index was 1.0 +/- 0.2%, 11.8 +/- 6.8% and 3.5 +/- 2.1% in the sham operation group, control AF group and calpain inhibitor group, respectively. In the sham operation group, control AF group and calpain inhibitor group, the expressions of caspase-3 (13.0 +/- 1.9%, 52.8 +/- 4.3% and 33.6 +/- 3.7%), caspase-8 (40.1 +/- 5.3%, 92.6 +/- 6.5% and 55.3 +/- 5.9%), bcl-2 (65.8 +/- 6.1%, 52.0 +/- 5.7% and 69.9 +/- 5.3%) and ARC (70.2 +/- 8.6%, 68.8 +/- 7.3% and 81.5 +/- 8.8%) were calculated as immunohistochemical indexes, respectively. CONCLUSIONS: The calpain inhibitor N-Acetyl-Leu-Leu-Met attenuated apoptosis through a complicated network of apoptosis-related proteins, which may result in improvement of structural remodeling in atrial fibrillation.

[Functional significance of TGF-beta1 signal transduction pathway in oral squamous cell carcinoma].

OBJECTIVE: The aim of this study was to investigate the expression of transforming growth factor-beta1 (TGF-beta1) and its signaling pathway molecules in oral squamous cell carcinoma (OSCC) and analyze the association between these factors and genesis and metastasis of OSCC. METHODS: The express of TGF-beta1, TbetaRI, TbetaRII and Smad4, a pivotal downstream molecule of its signaling, in 10 normal oral mucosa tissues and 108 OSCC was detected by SP immunohistochemistry, and thier correlation with genesis and metastasis of OSCC were assessed. RESULTS: The expressions of TbetaRII and Smad4 were lower in the tumors (34.3%, 38.9%) than those in the normal oral epithelium (80.0%, 100.0%, P < 0.05). The positive expression rates of TGF-beta1 and TbetaRI in the normal oral epithelium and OSCC were not significantly different (P > 0.05). There was an inverse correlation between TGF-beta1, Smad4, TbetaRII, TbetaRI expression and clinical stages (P < 0.01). The expression of TGF-beta1 was related with histological differentiation and tumor localization (P < 0.05). There was a relationship beteween Smad4 expression and histological differentiation and lymph node metastasis (P < 0.05). The expression of TbetaRII in the samples with lymph node metastasis was less than that in the ones without lymph node metastasis (P < 0.01), although there was no association between expression of TbetaRII and lymph node metastasis status. CONCLUSION: There is an important relationship between the abnormal TGF-beta1/Smad4 signal pathway and genesis and development of OSCC, while the low expressed Smad4 and TbetaRII may promote the metastasis of OSCC.

[Effects of arsenic trioxide on human coronary smooth muscle cells: experiment in vitro].

OBJECTIVE: To study the apoptotic effects of arsenic trioxide on human coronary smooth muscle cells (HCSMCs). METHODS: HCSMCs were cultured and randomly divided into 5 groups to be treated by arsenic trioxide of the concentrations 1.0, 2.0, 3.0, 4.0, and 5.0 micromol/L for 48 h. Cell growth curve was drawn by MTT method. DNA electrophoresis was used to observe the apoptosis. Western blotting was conducted to examine the protein expression of Bax, an apoptosis-promoting gene, and Bcl-2, an apoptosis-inhibiting gene. Other HCSMCs were cultured with 4.0 micromol/L arsenic trioxide for 48 h, then transmission electron microscopy was used to observe the ultra-structure and TUNEL was used to detect the percentage of apoptotic cells. HCSMCs not treated with arsenic trioxide were used as control group. RESULTS: Arsenic trioxide of different concentrations inhibited the proliferation of HCSMCs dose and time-dependently. When the concentration of arsenic trioxide was 5.0 micromol/L the number of living cells was (4.41 +/- 0.10) x 10(5)/ml, significantly lower than that of the control group [(30.11 +/- 0.93) x 10(5)/ml, P < 0.05]. Apoptosis bodies were observed under the transmission electron microscope. DNA electrophoresis showed hazy ladders, especially when the concentrations were 3.0 - 4.0 micromol/L. When the concentration was 5.0 micromol/L the number of necrotic cells increased remarkably and apoptosis became less significant. TUNEL showed that the apoptotic cells increased from 16.0% +/- 3.1% to 38.7% +/- 2.7% (P < 0.05). MTT test showed that arsenic trioxide decreased the absorbance of HCSMCs dose and time-dependently. Western blotting showed that the Bcl-2 expression was decreased and the expression of Bax increased after treatment of arsenic trioxide. CONCLUSION: Arsenic trioxide exerts an apoptotic effect on HCSMCs.

[Gene mapping and mutation detection in a family with congenital nuclear cataract].

OBJECTIVE: To map and detect the gene responsible for congenital nuclear cataract in a Chinese family. METHODS: Genomic DNA was extracted from the peripheral blood samples of members of the pedigree. Gene scan was performed using approximately 400 microsatellite markers spaced at about 10 cM intervals (ABI). Linkage analysis was carried out using a Linkage software package. Additional microsatellite markers for the positive region were selected for precise targeting, and haplotype data were processed using Cyrillic software to define the region of the disease gene. Mutation detection was carried out by sequencing candidate genes. RESULTS: Suggestive evidence of linkage was detected at marker D2S325 (LOD score [Z] = 2.29, recombination fraction [theta] = 0.00). Precise targeting and haplotype analysis traced the disease gene to a 19.04 cM region bounded by D2S117 and D2S2382 on chromosome 2q32.3-q35. Direct sequencing of the candidate gene cluster revealed a G-->A transversion in exon 3 of CRYGC. CONCLUSIONS: The present study has identified a novel nonsense mutation in CRYGC associated with congenital nuclear cataracts in a Chinese family.

Gastric cancer cell lines induced by trichostatin A.

AIM: To explore the effect of trichostatin A (TSA) on apoptosis and acetylated histone H3 levels in gastric cancer cell lines BGC-823 and SGC-7901. METHODS: The effect of TSA on growth inhibition and apoptosis was examined by MTT, fluorescence microscopy and PI single-labeled flow cytometry. The acetylated histone H3 level was detected by Western blot. RESULTS: TSA induced apoptosis in gastric cancer cell lines BGC-823 and SGC-7901 was in a dose and time-dependent manner. Apoptotic cells varied significantly between TSA treated groups (37.5 ng/mL 72 h for BGC-823 cell line and 75 ng/mL 72 h for SGC-7901 cell line) and control group (0.85+/-0.14 vs 1.14+/-0.07, P=0.02; 0.94+/-0.07 vs 1.15+/-0.06, P=0.02). Morphologic changes of apoptosis, including nuclear chromatin condensation and fluorescence strength, were observed under fluorescence microscopy. TSA treatment in BGC-823 and SGC-7901 cell lines obviously induced cell apoptosis, which was demonstrated by the increased percentage of sub-G1 phase cells, the reduction of G1-phase cells and the increase of apoptosis rates in flow cytometric analysis. The result of Western blot showed that the expression of acetylated histone H3 increased in BGC-823 and SGC-7901 TSA treatment groups as compared with the control group. CONCLUSION: TSA can induce cell apoptosis in BGC-823 and SGC-7901 cell lines. The expression of acetylated histone H3 might be correlated with apoptosis.

[Inhibitory effect of recombinant anti-angiogenic peptide of tumstatin on growth and metastasis of human ovarian cancer transplanted in nude mice].

OBJECTIVE: To evaluate the anti-angiogenic activity of peptide 21 obtained by modification of tumstatin, and its inhibitory effect on the growth and metastasis of human ovarian cancer transplanted in nude mice. METHODS: The peptide 21 was purified by affinity chromatography. Human ovarian cancer SKOV3 cells were inoculated in nude mice and the transplanted tumor was treated with the peptide 21 to observe the tumor growth and metastasis. The microvessel density (MVD) and immunohistochemical staining index of PCNA, VEGF and MMP-2 and TIMP-2 were performed to assess the inhibitory effect of the peptide 21. RESULTS: In the nude mice at 21 days after peptide 21 treatment, the inhibition rate of tumor growth was 53.17%, the tumor microvessel density was significantly reduced (P <0.05), the expression of PCNA, VEGF and MMP-2 were significantly lower (P <0.01), and TIMP-2 expression was significantly higher (P <0.01) in comparison with that of control group. CONCLUSION: The peptide 21 generated in this study has a significant anti-angiogenetic activity, showing significant inhibitory effect on the growth of human ovarian cancer transplanted in nude mice. The mechanism of its inhibitory action on ovarian cancer growth may be mediated by reduction of neovascularization and reduction of expression of angiogenetic factors.

[Genetic polymorphism of 9 STR loci in Korean population of Mudanjiang].

OBJECTIVE: To investigate the genetic polymorphism of 9 short tandem repeats (STR) gene loci, namely CSFIPO, TPOX, TH01, D16S539, D7S820, D13S317, F13A01, FESFPS and vWA in Chinese Korean population in Mudajiang area. METHODS: Amplified fragment length polymorphism (Amp-FLP) method was used to get the allele frequency distribution. RESULTS: The genotype distributions of the 9 STR loci are conformed to Hardy-Weinberg equilibrium by chi(2) test analysis. The total accord frequency, the accumulated total discrimination power and the the accumulative excluding probability of paternity were calculated. CONCLUSION: The result suggested that all 9 gene loci have high power of excluding probability of paternity and individual identification. They can be used in paternity testing and individual identification for forensic medicine. The gene frequencies of CSFIPO, TPOX and TP01 gene loci have significant differences between the Korean population in Mudanjiang area and those in Yanji area, but there is no difference in gene loci of D7S820, D17S317 and vWA.

[Effects of chronic trimetazidine treatment on atrial energy metabolism in a canine model of chronic atrial fibrillation].

OBJECTIVE: Aim of the present study was to investigate the effect of chronic trimetazidine treatment on atrial energy metabolism and endothelial function in a canine model of chronic atrial fibrillation (AF). METHODS: Eighteen canines were randomly divided into sham-operated group (n = 6), atrial pacing group (n = 6), and trimetazidine group (n = 6). In atrial pacing group and trimetazidine group, dogs were atrial paced at 400 beats per minutes for 6 weeks. Trimetazidine at 5 mgxkg(-1)xd(-1) was given one day before rapid atrial pacing for 6 weeks. Creatine phosphate (CrP), adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) in atrial tissue were analyzed by high-performance liquid chromatography. Total adenosine (TAN) was calculated. The expression of endothelial nitric oxide synthase (eNOS) in atrial tissue was determined by Western blot and immunohistochemical staining. In addition, plasma levels of von Willebrand factor (vWF) was quantified with enzyme-linked immunoadsorbent assay and NO(2)(-)/NO(3)(-) (NOx) was determined by nitrate reductase method. RESULTS: Atrial CrP (P < 0.01) and CrP/ATP were significantly decreased in paced atrium compared to atrium from sham-operated group (P < 0.05) while ATP, ADP, AMP and TAN remained unchanged (all P > 0.05). Plasma vWF was significantly increased and plasma NOx significantly decreased in paced animals compared to sham-operated animals. Atrial expression of eNOS was also significantly reduced in paced animals (P < 0.01). Trimetazidine treatment did not alter the contents of CrP, ATP, ADP, AMP and TAN, but significantly increased atrial eNOS expression (P < 0.05), decreased plasma vWF (P < 0.01) and increased plasma NOx concentration. CONCLUSION: Trimetazidine treatment affect chronic AF induced disturbance in energy metabolism but may improve endothelial function through a NOx depended manner.