Somatic mutational analysis of FAK in breast cancer: a novel gain-of-function mutation due to deletion of exon 33.

Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

Erg11 mutations associated with azole resistance in clinical isolates of Candida albicans.

The widespread use of azoles has led to increasing azole resistance among Candida albicans strains. One mechanism of azole resistance involves point mutations in the ERG11 gene, which encodes the target enzyme (cytochrome P450 lanosterol 14α-demethylase). In the present study, we amplified and sequenced the ERG11 gene of 23 C. albicans clinical isolates. Seventeen mutations encoding distinct amino acid substitutions were found, of which seven (K143Q, Y205E, A255V, E260V, N435V, G472R, and D502E) were novel. We further verified the contribution of the amino acid substitutions to azole resistance using site-directed mutagenesis of the ERG11 gene to recreate these mutations for heterologous expression in Saccharomyces cerevisiae. We observed that substitutions A114S, Y132H, Y132F, K143R, Y257H, and a new K143Q substitution contributed to significant increases (≧fourfold) in fluconazole and voriconazole resistance; changes in itraconazole resistance were not significant (≦twofold).

[Establishment of experimental angiogenic models with applications of quantitative digital image analysis].

OBJECTIVE: To establish experimental models for tumor neovascularization and to apply quantitative digital imaging analysis in the study. METHODS: An endothelial tube formation model was established by human umbilical vein endothelial cells (HUVECs). A vasculogenic mimicry model was established by SGC-7901 gastric cancer cell line. Fertilized eggs were used to establish a chorioallantoic membrane angiogenesis model. Using gene transfection experiment, IRX1 tumor suppressor gene was chosen as a therapeutic target. Image Pro Plus (IPP) analysis software was used for digital vascular images analysis with parameters including points, lines, angles and integral absorbance (IA) for the tubular formation or vasculogenic mimicry. RESULTS: Digital image analysis by IPP showed that HUVEC tubular formation was significantly inhibited in IRX1 transfectant, compared with controls. The tubular numbers in three groups were 12.80 +/- 3.83, 29.00 +/- 5.34 and 28.20 +/- 4.32 (P<0.01). The connection points of tubules in three groups were 13.20 +/- 2.59, 25.00 +/- 2.24 and 24.60 +/- 3.21 (P<0.01). The tubular lengths of three groups were (821.5 +/- 12.5), (930.9 +/- 13.5) and (948.4 +/- 18.1) microm (P=0.022). The IA values of PAS stain in three groups were 3606 +/- 363, 14 200 +/- 1251 and 15 043 +/- 1220 (P<0.01). In chick chorioallantoic membrane model, the angular numbers of tubules in three groups were 6.41 +/- 2.60, 10.27 +/- 2.65 and 9.18 +/- 1.99 (P<0.01). CONCLUSIONS: The endothelial tube formation model, vasculogenic mimicry model and chorioallantoic membrane angiogenesis model are useful for gene therapy and drug screening with targeting neoplastic vascularization. Professional image analysis software may greatly facilitate the quantitative analysis of tumor neovascularization.

Rab5a overexpression promoting ovarian cancer cell proliferation may be associated with APPL1-related epidermal growth factor signaling pathway.

Rab5a is a regulatory guanosine triphosphatase that is associated with the transport and fusion of endocytic vesicles, and participates in regulation of intracellular signaling pathways embraced by cells to adapt to the specific environment. Rab5a is also correlated with lung, stomach, and hepatocellular carcinomas. Here, we detected Rab5a in paraffin-embedded samples of 20 ovarian cysts, 20 benign cystadenomas, and 39 ovarian cancers by immunohistochemistry, and observed that Rab5a expression was significantly higher in ovarian cancer (P = 0.0001). By setting up stable HO-8910 cell lines expressing Rab5a or dominant negative Rab5a (Rab5a:S34N), we found that Rab5a overexpression enhanced the cell growth by promoting G1 into S phase. In contrast, Rab5a:S34N inhibited this process. Additionally, APPL1 (adaptor protein containing PH domain, PTB domain, and Leucine zipper motif), a downstream effector of Rab5a, was also involved in promoting HO-8910 cell cycle progress. But this function was blocked by Rab5a:S34N. Laser scanning confocal microscopy represented the colocalization of APPL1 and Rab5a in the plasmolemma, which changed with the time of epidermal growth factor (EGF) stimulation. We also found APPL1 could transfer from the membranes into the nucleus where it interacted with NuRD/MeCP1 (the nucleosome remodeling and histone deacetylase multiprotein complex). NuRD is reported to be involved in the deacetylation of histone H3 and H4 to regulate nuclear transcription. So Rab5a promoted proliferation of ovarian cancer cells, which may be associated with the APPL1-related epidermal growth factor signaling pathway.

[The transcription levels of linker for activation of T cell and its upstream regulatory factors in T cells of asthmatic patients].

OBJECTIVE: To examine the mRNA expression of the linker for activation of T cell (LAT) and its upstream regulatory factors (Syk, Lck and ZAP-70) in the peripheral blood T cells of asthmatic patients. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA expression of LAT and its upstream regulatory factors (Syk, Lck and ZAP-70) in 20 asthmatic patients and 20 nonallergic subjects. The results of LAT transcription level were confirmed by real-time RT-PCR Results were expressed as x +/- s. Differences between groups were assessed for significance by t test. RESULTS: Compared with nonallergic subjects, a significant decrease of mRNA expression of LAT gene was observed in T cells of asthmatic patients (0.54 +/- 0.14 vs 0.72 +/- 0.17, t = 3.11, P <0.01), which was verified by real-time RT-PCR (0.0065 +/- 0.0066 vs 0.0124 +/- 0.0045, t =0.0022, P <0.01). The Lck and ZAP-70 gene transcription levels were significantly up-regulated (Lck: 0.71 +/- 0.16 vs 0.53 +/- 0.17, t = 3.18, P<0.01; ZAP-70: 1.05 +/- 0.41 vs 0.82 +/- 0.27, t = 2.10, P < 0.05). CONCLUSION: A significant decrease of mRNA expression of LAT gene in T cells of asthmatic patients may be due to the up-regulation of its upstream regulatory factors (Lck and ZAP-70). The abnormal mRNA expression of LAT, Lck and ZAP-70 genes may be involved in the pathogenesis of asthma.

[The linkage disequilibrium of four loci in hepatic lipase promoter gene of cerebral infarction patients in Shanghai].

OBJECTIVE: To investigate the single nucleotide polymorphism (SNP), the distribution of their haplotypes and linkage disequilibrium of hepatic lipase (HL) gene promoter 250G/A, 514C/T, 710T/C and 763A/G in cerebral infarction patients of Shanghai. METHODS: Peripheral blood sample were collected from 133 patients with cerebral infarction and 112 healthy controls in Shanghai. The HL gene polymorphism was analyzed by polymerase chain reaction- restriction fragment length polymorphism. RESULTS: There were statistically significant differences in genotype and allele frequencies between the healthy controls and the patients with cerebral infarction in -250G/A and -514C/T genotypes and allele frequencies (all P < 0.05). However, there were no significant differences in genotype and allele frequencies in -710T/C and -763A/G between the healthy controls and the patients with cerebral infarction (all P > 0.05). Besides, there was a strong linkage disequilibrium between -250G/A and -514C/T, -710T/C, and -763A/G respectively, between -514C/T and -710T/C and -763A/G respectively, and between -710T/C and -763A/G. When the haplotypes were -250G/-514C, -250G/-710C, -250G/-763G, -514C/-710C, and 514C/-763G respectively, the frequencies in the cerebral infarction group were significantly lower than that in the healthy controls. When the haplotype was -250A/-514T, -250A/-710T, -250A/-710C, -250A/-763G, -514T/-710C, -514T/-763G, and -710T/-763G respectively, the frequencies in the cerebral infarction group were significantly higher than those in the healthy controls. CONCLUSION: There are significant haplotypes and linkage disequilibrium among the four SNPs of HL gene in the cerebral infarction patients of Shanghai. The haplotypes GC, GG, and CC lower the incidence rate of cerebral infarction, while the haplotypes AT, AC, AG, TC, and TG increase the incidence rate of cerebral infarction.

[Activation related genes of memory CD(4)(+) T cells in asthma patients].

OBJECTIVE: To screen and identify the genes of activated memory CD(4)(+) T cells in asthma. METHODS: Differential display polymerase chain reaction (DDPCR) was utilized to identify genes of memory CD(4)(+) T cells after activation from asthmatic patients and normal individuals, and the mRNA levels of the differentially expressed genes were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Nineteen differentially expressed cDNA fragments were isolated. The homology analysis indicated that two fragments possessed a high degree of homology with interleukin-7 (IL-7) mRNA and MM-1 mRNA. RT-PCR confirmed that IL-7 mRNA and MM-1 mRNA levels were increased in memory CD(4)(+) T cells after activation in asthmatic patients (0.390 +/- 0.029, 0.629 +/- 0.047, F = 983, 1264 respectively, all P < 0.05). CONCLUSION: The up-regulated expression of IL-7 and MM-1 genes in memory CD(4)(+) T cells after activation may be a molecular mechanism that differentiates the response to allergens of asthma patients from normal individuals.

Low expression of XIAP-associated factor 1 in human colorectal cancers.

OBJECTIVE: Eight cellular homologs of the inhibitors-of-apoptosis proteins (IAP) have been identified in humans and of them, the X-linked IAP (XIAP) is the most potent. XIAP-associated factor 1 (XAF1) is a newly discovered XIAP-binding protein that negatively regulates the caspase-inhibiting activity of XIAP. It is either not expressed or present at extremely low levels in many cancer cell lines. The aims of the present study were: (i) to investigate the expression of XAF1 in human colorectal cancers (CRC) both in vitro and in vivo, and (ii) to evaluate the possibility of XAF1 as a new tumor marker. METHODS: The expression of XAF1 in four human colon cancer cell lines (Colo205, Colo320, SW1116, LoVo) and in samples from 70 patients with CRC was analyzed by reverse transcriptase-polymerase chain reaction. XAF1 concentrations were also detected in the peripheral circulation of the 70 patients, as well as three traditional circulating cancer-associated antigens. RESULTS: A low concentration of XAF1 mRNA was detectable in the three colon cancer cell lines other than Colo205, which showed the strongest expression of XAF1. The expression of XAF1 in tissue was relatively lower in primary CRC compared with a relatively higher level in benign colorectal tumors (P < 0.01). Although the XAF1 expression in circulation of those with CRC was also lower than in those with benign tumors, there was no statistical significance (P > 0.05). CONCLUSIONS: The present results suggest that the low expression of XAF1 in tumor tissue coincides with a similar level in the peripheral circulation, which contributes at least part to the malignant behavior of CRC. Integrating the XAF1 relative expression value with the other three traditional tumor biomarkers created a four-parameter assay that significantly improved the rate of diagnosis of CRC.

[The relationship between the five beta-fibrinogen gene polymorphisms and cerebral infarction].

OBJECTIVE: To investigate the relationship among beta-fibrinogen (Fg) concentration, related gene polymorphisms (including -148C/T, -249C/T, -455G/A, 448G/A, 1689T/G) and cerebral infarction. METHODS: Fg level and its five gene polymorphisms were analyzed with by polymerase chain reaction-restriction fragment length polymorphism in 132 patients with cerebral infarction, 79 patients with other neurological diseases and 92 healthy elders. RESULTS: The plasma Fg level in cerebral infarction patients was significantly higher than that in the patients with other neurological diseases or healthy elders (P < 0.05). In the three groups, the plasma Fg levels in individuals with T-148 and A-455 alleles were higher than those in individuals without T-148 and A-455 alleles (P < 0.05). However, there were no statistically significant differences in the genotype and allele frequencies in the five mutation gene polymorphisms among the three groups (P > 0.05). CONCLUSIONS: Cerebral infarction is a multifactorial disease and an increased Fg level is a risk factor for cerebral infarction. T-148 and A-455 allele can lead to elevated Fg concentration.

Focal adhesion kinase regulates the phosphorylation protein tyrosine phosphatase-α at Tyr789 in breast cancer cells.

Protein tyrosine phosphatase (PTP)­α regulates the phosphorylation of focal adhesion kinase (FAK), which is important in cellular signal transduction and integration of proteins. It has been demonstrated that a FAK­Del33 mutation (deletion of exon 33; KF437463) in breast cancer tissues regulates cell migration through FAK/Src signaling activation. However, the detailed pathway for Src activation with FAK­Del33 remains to be elucidated. The present study used a retroviral expression system to examine changes in PTPα phosphorylation affected by the FAK­Del33 protein in breast cancer cells. Small interfering (si)RNA targeting PTPα interfered with the phosphorylation of Src. Wound­healing and migration assays were performed to identify cell morphology and quantitative analysis was performed by examining band color depth in western blot analysis. Significant differences were observed in the phosphorylation level of PTPα at Tyr789 between the FAK­Del33 and the wild­type breast cancer cells, suggesting that FAK regulated the phosphorylation level of PTPα at Tyr789 in breast cancer mutant FAK­Del33 cells. The gene expression profile with FAK siRNA did not alter the levels of phosphorylation in other mutants, including autophosphorylation disability (Y397F), ATP kinase dominant negative (K454R) and protein 4.1, ezrin, radixin, moesin domain attenuate (Δ375). FAK RNAi inhibited the activity of the FAK­Del33 at the Src site and rescued the elevated cell migration and invasion. The present study demonstrated for the first time, to the best of our knowledge, an increase in the phosphorylation level of PTPα­Tyr789 by its upstream activator, FAK­Del33, leading to Src activation in certain breast cancer cells, which has significant implications for metastatic potential.

Effect of N-desulfated heparin on hepatic/renal ischemia reperfusion injury in rats.

AIM: To investigate the effect of N-desulfated heparin on hepatic/renal ischemia and reperfusion injury in rats. METHODS: Using rat models of 60 minutes hepatic or renal ischemia followed by 1 h,3 h,6 h and 24 h reperfusion,animals were randomly divided into following groups,the sham operated controls,ischemic group receiving only normal saline,and treated group receiving N-desulfated heparin at a dose of 12 mg/kg at 5 minutes before reperfusion. P-selectin expression was detected in hepatic/renal tissues with immunohistochemistry method. RESULTS: P-selectin expression, serum ALT, AST, BUN and Cr levels were significantly increased during 60 minute ischemia and 1 h, 3 h, 6 h and 24 h reperfusion,while the increment was significantly inhibited,and hepatic/renal pathology observed by light microscopy was remarkably improved by treatment with the N-desulfated heparin. Furthermore,the heparin was found no effects on PT and KPTT. CONCLUSION: P-selectin might mediate neutrophil infiltration and contribute to hepatic/renal ischemia and reperfusion. The N-desulfated heparin might prevent hepatic/renal damage induced by ischemia and reperfusion injury without significant anticoagulant activity.

[Cloning, expression of the lectin-EGF domain of P-selectin, and preparation of its monoclonal antibody].

To prepare monoclonal antibody specific to P-selectin lectin-EGF domain, the gene for lectin-EGF domain of P-selectin L-EGF was amplified from normal human platelets by RT-PCR, then was cloned into prokaryotic vector pET42b(+). The recombinant plasmid was transformed into E. coli DH5 alpha strain for further screening and characterization, and was expressed in E. coli BL21 strain. Expressed protein was purified by chromatography on a Ni(2+)-NTA superflow agarose column and eluted with pH 8.0-4.5 urea gradient. Then the mAb anti-lectin-EGF was prepared with classical hybridoma technique, and 3 hybridoma cell lines (B10, F3 and H5) were obtained with Ig subclasses of these mAbs were IgG(2), IgG(1), and IgG(3) respectively, and their light chains were all kappa chain. Immuofluorescence and FACS assays demonstrated that mAbs could specifically recognize P-selectin expressed on ECV (endothelial cell line) stimulated by LPS. Meanwhile, the role of mAbs to P-selectin lectin-EGF domain was studied, and it was proved that the mAbs markedly inhibited adhesion between platelets and neutrophils in vitro. These monoclonal antibodies can specifically recognize the natural P-selectin and markedly inhibit adhesion between platelets and neutrophils in vitro.

[Association of susceptibility to chronic rhinosinusitis with genetic polymorphisms of IL-4 and IL-10].

OBJECTIVE: To investigate the relationship between the promoter polymorphism of IL-4 and IL-6 and chronic rhinosinusitis (CRS). METHODS: One hundred and twenty-three patients with CRS and 239 healthy controls in Shanghai region were chosen in this study. The genotype of IL-4 gene -33T>C and -590C>T were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and the genotype of IL-10 gene -1082A>G was determined using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method. Statistical calculations were performed using SAS 8.2 software. RESULTS: Significant differences were found in genotype distribution of -33T>C and -590C>T between the CRS group and the control group (χ2=6.6013, P=0.0102, χ2=6.6013, P=0.0304), and -33T>C remained significant following application of the Bonferroni correction (P<0.025). The relative risks of CRS with -33T>C and -590C>T were 1.818(P=0.0236, 95%CI 1.084-3.050) and 1.838 (P=0.0147, 95%CI 1.127-2.997). There was linkage disequilibrium (LD) between the -33T>C and -590C>T. The coefficient of linkage disequilibrium (D') was 0.77 and the related coefficient (r2) was 0.54. The -33T/-590T haplotype was associated with CRS and the relative risk was 1.653 (P=0.0130, 95%CI 1.107-2.469). There were only two genotypes of IL-10 gene-1082A>G and the frequencies of the AA and AG genotypes were not different between the CRS and control groups. CONCLUSION: The promoter polymorphism of IL-4 -33T>C and -590C>T were associated with the susceptibility of CRS and the -33T/-590T haplotype was a risk factor for CRS, but there were no association between the -1082A>G and CRS.