Promotion of rs3746804 (p. L267P) polymorphism to intracellular SLC52A3a trafficking and riboflavin transportation in esophageal cancer cells.

Riboflavin is an essential micronutrient for normal cellular growth and function. Lack of dietary riboflavin is associated with an increased risk for esophageal squamous cell carcinoma (ESCC). Previous studies have identified that the human riboflavin transporter SLC52A3a isoform (encoded by SLC52A3) plays a prominent role in esophageal cancer cell riboflavin transportation. Furthermore, SLC52A3 gene single nucleotide polymorphisms rs3746804 (T>C, L267P) and rs3746803 (C >T, T278M) are associated with ESCC risk. However, whether SLC52A3a (p.L267P) and (p.T278M) act in riboflavin transportation in esophageal cancer cell remains inconclusive. Here, we constructed the full-length SLC52A3a protein fused to green fluorescent protein (GFP-SLC52A3a-WT and mutants L267P, T278M, and L267P/T278M). It was confirmed by immunofluorescence-based confocal microscopy that SLC52A3a-WT, L267P, T278M, and L267P/T278M expressed in cell membrane, as well as in a variety of intracellular punctate structures. The live cell confocal imaging showed that SLC52A3a-L267P and L267P/T278M increased the intracellular trafficking of SLC52A3a in ESCC cells. Fluorescence recovery after photobleaching of GFP-tagged SLC52A3a meant that intracellular trafficking of SLC52A3a-L267P and L267P/T278M was rapid dynamics process, leading to its stronger ability to transport riboflavin. Taken together, the above results indicated that the rs3746804 (p.L267P) polymorphism promoted intracellular trafficking of SLC52A3a and riboflavin transportation in ESCC cells.

Promotion of Lung Cancer Metastasis by SIRT2-Mediated Extracellular Protein Deacetylation.

Acetylation of extracellular proteins has been observed in many independent studies where particular attention has been given to the dynamic change of the microenvironmental protein post-translational modifications. While extracellular proteins can be acetylated within the cells prior to their micro-environmental distribution, their deacetylation in a tumor microenvironment remains elusive. Here it is described that multiple acetyl-vWA domain-carrying proteins including integrin ß3 (ITGB3) and collagen 6A (COL6A) are deacetylated by Sirtuin family member SIRT2 in extracellular space. SIRT2 is secreted by macrophages following toll-like receptor (TLR) family member TLR4 or TLR2 activation. TLR-activated SIRT2 undergoes autophagosome translocation. TNF receptor associated factor 6 (TRAF6)-mediated autophagy flux in response to TLR2/4 activation can then pump SIRT2 into the microenvironment to function as extracellular SIRT2 (eSIRT2). In the extracellular space, eSIRT2 deacetylates ITGB3 on aK416 involved in cell attachment and migration, leading to a promotion of cancer cell metastasis. In lung cancer patients, significantly increased serum eSIRT2 level correlates with dramatically decreased ITGB3-K416 acetylation in cancer cells. Thus, the extracellular space is a subcellular organelle-like arena where eSIRT2 promotes cancer cell metastasis via catalyzing extracellular protein deacetylation.

Quantitative assessment of the association between MTHFR C677T polymorphism and colorectal cancer risk in East Asians.

A great number of studies regarding the association between MTHFR C677T polymorphism and risk of colorectal cancer (CRC) in East Asians were published, but the results were inconsistent. Thus, a meta-analysis was performed to investigate the association. PubMed, Embase, and CBM databases were searched for eligible publications. Pooled odds ratios (ORs) with 95 % confidence intervals (95 % CIs) were calculated using random or fixed effect models. Finally, 24 case-control studies with a total of 7,230 CRC cases and 9,285 controls were included. Meta-analyses of a total of 24 studies showed there was a statistically significant association between MTHFR C677T polymorphism and decreased CRC risk in East Asians under four genetic models (T versus C, OR = 0.92, 95 % CI 0.85-0.99; TT versus CC, OR = 0.80, 95 % CI 0.69-0.94; TT versus CT/CC, OR = 0.82, 95 % CI 0.71-0.95; TT/CT versus CC, OR = 0.92, 95 % CI 0.86-0.98). The cumulative meta-analyses for the allele contrast (T versus C), homozygote (TT versus CC), dominant (TT/CT versus CC), and recessive (TT versus CT/CC) models all showed a trend of more obvious association as information accumulated by year. Subgroup analyses by country further identified this association in Korea and Japan. This meta-analysis suggests that MTHFR C677T polymorphism is associated with decreased risk of colorectal cancer in East Asians, and MTHFR 677T variant has a protective effect on colorectal cancer.

LOXL2-dependent deacetylation of aldolase A induces metabolic reprogramming and tumor progression.

Lysyl-oxidase like-2 (LOXL2) regulates extracellular matrix remodeling and promotes tumor invasion and metastasis. Altered metabolism is a core hallmark of cancer, however, it remains unclear whether and how LOXL2 contributes to tumor metabolism. Here, we found that LOXL2 and its catalytically inactive L2Δ13 splice variant boost glucose metabolism of esophageal tumor cells, facilitate tumor cell proliferation and promote tumor development in vivo. Consistently, integrated transcriptomic and metabolomic analysis of a knock-in mouse model expressing L2Δ13 gene revealed that LOXL2/L2Δ13 overexpression perturbs glucose and lipid metabolism. Mechanistically, we identified aldolase A, glyceraldehyde-3-phosphate dehydrogenase and enolase as glycolytic proteins that interact physically with LOXL2 and L2Δ13. In the case of aldolase A, LOXL2/L2Δ13 stimulated its mobilization from the actin cytoskeleton to enhance aldolase activity during malignant transformation. Using stable isotope labeling of amino acids in cell culture (SILAC) followed by proteomic analysis, we identified LOXL2 and L2Δ13 as novel deacetylases that trigger metabolic reprogramming. Both LOXL2 and L2Δ13 directly catalyzed the deacetylation of aldolase A at K13, resulting in enhanced glycolysis which subsequently reprogramed tumor metabolism and promoted tumor progression. High level expression of LOXL2/L2Δ13 combined with decreased acetylation of aldolase-K13 predicted poor clinical outcome in patients with esophageal cancer. In summary, we have characterized a novel molecular mechanism that mediates the pro-tumorigenic activity of LOXL2 independently of its classical amine oxidase activity. These findings may enable the future development of therapeutic agents targeting the metabolic machinery via LOXL2 or L2Δ13. HIGHLIGHT OF THE STUDY: LOXL2 and its catalytically inactive isoform L2Δ13 function as new deacetylases to promote metabolic reprogramming and tumor progression in esophageal cancer by directly activating glycolytic enzymes such as aldolase A.

STAT3ß disrupted mitochondrial electron transport chain enhances chemosensitivity by inducing pyroptosis in esophageal squamous cell carcinoma.

The clinical efficacy of cisplatin in the treatment of esophageal squamous cell carcinoma (ESCC) is undesirable. Signal transducer and activator of transcription 3ß (STAT3ß), a splice variant of STAT3, restrains STAT3α activity and enhances chemosensitivity in ESCC. However, the underlying molecular mechanisms remain poorly understood. Here, we found that high expression of STAT3ß contributes to cisplatin sensitivity and enhances Gasdermin E (GSDME) dependent pyroptosis in ESCC cells after exposure to cisplatin. Mechanistically, STAT3ß was located into the mitochondria and its high expression disrupts the activity of the electron transport chain, resulting in an increase of ROS in cisplatin treatment cells. While high levels of ROS caused activation of caspase-3 and GSDME, and induced cell pyroptosis. STAT3ß blocked the phosphorylation of STAT3α S727 in mitochondria by interacting with ERK1/2 following cisplatin treatment, disrupting electron transport chain and inducing activation of GSDME. Clinically, high expression of both STAT3ß and GSDME was strongly associated with better overall survival and disease-free survival of ESCC patients. Overall, our study reveals that STAT3ß sensitizes ESCC cells to cisplatin by disrupting mitochondrial electron transport chain and enhancing pyroptosis, which demonstrates the prognostic significance of STAT3ß in ESCC therapy.

P300/CBP-associated factor (PCAF)-mediated acetylation of Fascin at lysine 471 inhibits its actin-bundling activity and tumor metastasis in esophageal cancer.

BACKGROUND: Fascin is crucial for cancer cell filopodium formation and tumor metastasis, and is functionally regulated by post-translational modifications. However, whether and how Fascin is regulated by acetylation remains unclear. This study explored the regulation of Fascin acetylation and its corresponding roles in filopodium formation and tumor metastasis. METHODS: Immunoprecipitation and glutathione-S-transferase pull-down assays were performed to examine the interaction between Fascin and acetyltransferase P300/CBP-associated factor (PCAF), and immunofluorescence was used to investigate their colocalization. An in vitro acetylation assay was performed to identify Fascin acetylation sites by using mass spectrometry. A specific antibody against acetylated Fascin was generated and used to detect the PCAF-mediated Fascin acetylation in esophageal squamous cell carcinoma (ESCC) cells using Western blotting by overexpressing and knocking down PCAF expression. An in vitro cell migration assay was performed, and a xenograft model was established to study in vivo tumor metastasis. Live-cell imaging and fluorescence recovery after photobleaching were used to evaluate the function and dynamics of acetylated Fascin in filopodium formation. The clinical significance of acetylated Fascin and PCAF in ESCC was evaluated using immunohistochemistry. RESULTS: Fascin directly interacted and colocalized with PCAF in the cytoplasm and was acetylated at lysine 471 (K471) by PCAF. Using the specific anti-AcK471-Fascin antibody, Fascin was found to be acetylated in ESCC cells, and the acetylation level was consequently increased after PCAF overexpression and decreased after PCAF knockdown. Functionally, Fascin-K471 acetylation markedly suppressed in vitro ESCC cell migration and in vivo tumor metastasis, whereas Fascin-K471 deacetylation exhibited a potent oncogenic function. Moreover, Fascin-K471 acetylation reduced filopodial length and density, and lifespan of ESCC cells, while its deacetylation produced the opposite effect. In the filipodium shaft, K471-acetylated Fascin displayed rapid dynamic exchange, suggesting that it remained in its monomeric form owing to its weakened actin-bundling activity. Clinically, high levels of AcK471-Fascin in ESCC tissues were strongly associated with prolonged overall survival and disease-free survival of ESCC patients. CONCLUSIONS: Fascin interacts directly with PCAF and is acetylated at lysine 471 in ESCC cells. Fascin-K471 acetylation suppressed ESCC cell migration and tumor metastasis by reducing filopodium formation through the impairment of its actin-bundling activity.

Transmembrane helix of novel oncogene with kinase-domain (NOK) influences its oligomerization and limits the activation of RAS/MAPK signaling.

Ligand-dependent or independent oligomerization of receptor protein tyrosine kinase (RPTK) is often an essential step for receptor activation and intracellular signaling. The novel oncogene with kinase-domain (NOK) is a unique RPTK that almost completely lacks an ectodomain, expresses intracellularly and activates constitutively. However, it is unknown whether NOK can form oligomer or what function oligomerization would have. In this study, two NOK deletion mutants were generated by either removing the ectodomain (NOKDeltaECD) or including the endodomain (NOK-ICD). Co-immunoprecipitation demonstrated that the transmembrane (TM) domain of NOK was essential for its intermolecular interaction. The results further showed that NOK aggregated more closely as lower order oligomers (the dimer- and trimer-sized) than either deletion mutant did since NOK could be cross-linked by both Sulfo-EGS and formaldehyde, whereas either deletion mutant was only sensitive to Sulfo-EGS. Removing the NOK TM domain (NOK-ICD) not only markedly promoted higher order oligomerization, but also altered the subcellular localization of NOK and dramatically elevated the NOK-mediated constitutive activation of extracellular signal-regulated kinase (ERK). Moreover, NOK-ICD but not NOK or NOKDeltaECD was co-localized with the upstream signaling molecule RAS on cell membrane. Thus, TM-mediated intermolecular contacting may be mainly responsible for the constitutive activation of NOK and contribute to the autoinhibitory effect on RAS/MAPK signaling.

[Suppression of cell proliferation by inhibitors to redox signaling in human lens epithelial cells].

OBJECTIVE: Physiological level of reactive oxygen species (ROS) has been shown to play an important role in mitogen-stimulated cell signaling in many cell types. Both EGF and bFGF can induce ROS generation in human lens epithelial cells. But the role of ROS and Redox signaling on EGF and bFGF-stimulated cell proliferation is not clear. This study was to investigate the control of EGF and bFGF-induced cell proliferation by Redox signaling in human lens epithelial cells (SRA 01/04), using specific inhibitors to Redox signaling. METHODS: EGF and bFGF-induced cell proliferation was measured by [methyl-3H] thymidine incorporation assay. In some experiments, cell proliferation was also measured by trypan blue negative cell counting parallel with 3H-thymidine incorporation assay. The inhibitors used in this study include: catalase (specific enzyme to detoxify hydrogen peroxide), N-acetyl-L-cysteine (free radical scavenger), DPI (inhibitor for NADPH oxidase) and AACOCF3 (specific inhibitor for cytosolic phospholipase A2, which had been shown to play important role in ROS generation in our previous study). Serum starved SRA 01/04 cells were pretreated with these inhibitors for 30 minutes before exposure to EGF or bFGF (20 microg/L). In short term study, all these inhibitors were removed before adding growth factor, while in long term study, inhibitors were maintained in the medium along with growth factor. Cells were kept growing in the medium with 20 microg/L EGF or 20 microg/L bFGF for 48 hours. Then cell proliferation was quantified by [methyl-3H] thymidine incorporation assay or by cell counting. RESULTS: We found that catalase, NAC, DPI and AACOCF3 were able to suppress EGF and bFGF-induced cell proliferation in both short term and long term study. In EGF study, 20 microg/L EGF produced about 26% (t = 7.093, P <0.01) increase in DNA synthesis after 48 hours. Pretreatment of the cells for 30 minutes with 1 x 10(5) U/L catalase, 0.5 mmol/L NAC, 0.1 micromol/L DPI or 0.5 micromol/L AACOCF3 inhibited EGF-stimulated DNA synthesis by 18.0% (t=6.132, P<0.01), 24.6% (t=6.188, P<0.01), 28.5% (t=6.386, P<0.01) and 16.4% (t =3.705, P =0.001) respectively. The inhibition was dose-dependent and was proved by trypan-blue negative cell counting. If the cells were treated with inhibitors for 48.5 hours (long term study), the lowest concentrations to inhibit cell proliferation were much lower than those used in short term study. Treatment of the cells with 0.5 x 10(5) U/L catalase, 0.2 mmol/L NAC, 0.01 micromol/L DPI and 0.1 micromol/L AACOCF3 led to suppression on DNA synthesis significantly. Similar results were detected in bFGF study. 48 hours treatment with 20 microg/L bFGF induced about 28.8% (t =9.523, P <0.01) increase in cell proliferation. If the cells were pretreated with 1 x 10(5) U/L catalase, 0.5 mmol/L NAC, 0.1 micromol/L DPI or 0.5 micromol/L AACOCF3 for 30 minutes, bFGF-stimulated cell proliferation was suppressed by 24.5% (t = 6.697, P < 0.01), 22.2% (t = 6.693, P<0.01), 23.9% (t =6.661, P<0.01) and 30.5% (t =8.959, P <0.01) respectively. If cells were treated with inhibitors for 48.5 hours, the lowest concentration of catalase, NAC, DPI and AACOCF3 to inhibit cell proliferation significantly was 0.5 x 10(5) U/L( t =21.641, P <0.01), 0.2 mmol/L (t =11.218, P < 0.01), 0.01 micromol/L (t = 4.570, P <0.01) and 0.1 micromol/L (t = 5.426, P < 0.01) respectively, lower than those used in short term study. CONCLUSIONS: We conclude that mitogenic stimulus function of EGF and bFGF in human lens epithelial cells appears to be mediated via ROS to activate cell proliferation. Inhibition of Redox signaling, either by removal of ROS (the role of catalase and NAC) or blocking ROS generation (the role of DPI and AACOCF3), eradicate EGF and bFGF-stimulated cell proliferation. It is proposed that Redox signaling may play an important role in cell proliferation in human lens epithelial cells.

Overexpression of CREPT confers colorectal cancer sensitivity to fluorouracil.

AIM: To investigate expression of cell cycle-related and expression-elevated protein in tumor (CREPT) in colorectal cancer (CRC) and determine its prognostic value in response to 5-fluorouracil (5-FU). METHODS: The relative expression of CREPT in CRC tumor samples was determined using immunohistochemistry. The protein content in cell lines was analyzed by immunoblotting. Cell viability was measured with the CCK-8 assay. Cell cycle and apoptosis analyses were performed with flow cytometry. RESULTS: CREPT was overexpressed in CRC tissues and correlated with histological grade. Clinicopathological analysis indicated that CREPT was positively related to tumor progression. Exogenous expression of CREPT stimulated cell proliferation and accelerated the cell cycle. More importantly, high expression of CREPT sensitized CRC cells to 5-FU treatment. Furthermore, we demonstrated that 5-FU elicited significant apoptosis in CREPT-positive cells. CONCLUSION: Aberrant overexpression of CREPT contributes to tumorigenesis of CRC by promoting cell proliferation and accelerating the cell cycle, and confers sensitivity to 5-FU. CREPT is a potential prognostic biomarker for 5-FU in CRC.

Identification and functional characterization of a novel interleukin 17 receptor: a possible mitogenic activation through ras/mitogen-activated protein kinase signaling pathway.

Interleukin-17 receptor (IL-17R) is increasingly emerged as a distinct receptor family functioning in diverse cellular processes including inflammation and cancer. In this study, we uncovered a novel member of IL-17R from mouse tissue that was named mouse IL-17RE (mIL-17R). Mouse IL-17RE cDNA is composed of at least 14 exons and presents at least 6 spliced isoforms (mIL-17RE1-6) with a molecular weight ranging from 34.2 to 70.1 kD. Mouse IL-17RE is expressed in limited tissues such as lung, kidney, stomach, intestine and testis, etc., and is mainly localized in the cytoplasm and on cell membrane. IL-17RE can also be detected in numerous tumor cell lines. Importantly, a mitogenic effect was detected in BaF3 cells stably transfected with the chimeric receptor fused by the ectodomain of erythropoietin receptor (EPOR) with the transmembrane and endomain of IL-17RE in a serum-dependent but EPO-independent manner. Moreover, ERK1/2 phosphorylation was significantly up-regulated as the dose of mIL-17RE increased. Specific RNAi targeting at mIL-17RE dramatically inhibited the activation of ERK1/2, indicating that mIL-17RE could functionally activate RAS/MAPK signaling pathway. Using dominant negative MEK (Dn-MEK) or RAS (Dn-RAS) as a signaling blocker, we were able to show that mIL-17RE probably activated RAS/MAPK signaling at or upstream of RAS. Overall, our results strongly indicate that mIL-17RE may belong to a novel growth-receptor like molecule that has the capability to support cellular mitogenesis through RAS/MAPK pathway.

Point mutation at single tyrosine residue of novel oncogene NOK abrogates tumorigenesis in nude mice.

Receptor protein-tyrosine kinases (RPTKs) are tightly regulated during normal cellular processes including cell growth, differentiation, and metabolism. Recently, a RPTK-like molecule named novel oncogene with kinase-domain (NOK) has been cloned and characterized. Overexpression of NOK caused severe cellular transformation as well as tumorigenesis and metastasis in nude mice. In the current study, we generated two tyrosine-->phenylalanine (Y-->F) point mutations (Y327F and Y356F) within the endodomain of NOK that are well conserved in many RPTK subfamilies and are the potential tyrosine phosphorylation sites important for major intracellular signaling. Using BaF3 cells stably expressing the ectodomain of mouse erythropoietin receptor, and the transmembrane and endodomain of NOK (BaF3-E/N), we were able to show that point mutations at either Y327 or Y356 dramatically blocked cellular transformation by NOK as examined by colony formation and cellular DNA synthesis. In addition, tumorigenesis induced by BaF3-E/N was completely abrogated upon the introduction of either single mutation. Importantly, signaling studies revealed that the activation of extracellular signal-regulated kinase was inhibited by Y356F and was significantly reduced by Y327F. Both mutations significantly impaired Akt phosphorylation. Interestingly, both mutations did not affect the kinase activity of NOK. Moreover, apoptotic analysis revealed that both mutations accelerated cell death by activating caspase-3-mediated pathways. Thus, our study shows that these potential tyrosine phosphorylation sites may play critical roles in NOK-mediated tumorigenesis both in vitro and in vivo.

A novel protein tyrosine kinase NOK that shares homology with platelet- derived growth factor/fibroblast growth factor receptors induces tumorigenesis and metastasis in nude mice.

Receptor protein tyrosine kinases (RPTKs) play important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Here, we report the identification and characterization of a novel RPTK-like molecule that has a critical role in induction of tumorigenesis and metastasis and is termed Novel Oncogene with Kinase-domain (NOK). NOK contains a putative single transmembrane domain and a conserved intracellular tyrosine kinase domain that shares homology with members of the platelet-derived growth factor/fibroblast growth factor receptor superfamily. NOK was exclusively located in the cytoplasm. NOK mRNAs were detected in limited human organs and expressed with the highest abundance in the prostate. A variety of tumor cells also expressed the NOK mRNAs. We demonstrated that NIH3T3 and BaF3 cells could be strongly transformed by the expression of the NOK gene as examined by colony formation experiment. In addition, BaF3 cells with the stable expression of NOK induced rapid tumorigenesis in nude mice. Interestingly, these NOK-expressing tumor cells could promptly invade and spread into various distinct organs and form metastatic foci, eventually leading to the rapid death of these animals. Moreover, molecular mechanism studies indicated that NOK could concomitantly activate both MAP kinase and phosphatidylinositol 3'-kinases (PI3K) pathways in stable BaF3 cells. Thus, our results both in vitro and in vivo suggest that NOK is a novel oncogene with the capacity of promoting cell transformation, tumorigenesis, and metastasis.

[Cloning, eukaryotic expression and function assay of recombinant leukemia inhibitory factor gene LIF].

Leukemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, which was discovered to inhibit the proliferation of murine myeloid leukemic cell line M1, has multiple functions in various biological processes. This factor is highly glycosylated when it binds to receptor to activate the signal transduction. Therefore, expression of LIF through eukaryotic system is the best way to obtain the correct glycosylation. In this study, human LIF cDNA with the sequence of signal peptide was cloned from adult blood cells by RT-PCR, and then subcloned into pcDNA3 for expression in HEK-293T cells. After transfection of the recombinant plasmid pcDNA3/LIF into HEK-293T cells, the conditioned medium containing the secreted LIF was obtained. The activity of the secreted LIF in the conditioned medium was detected through the phosphorylation of STAT3, EMSA and luciferase reporter (pGL2-APRE-luc) assays in Hep3B and 293T cells. Furthermore, to investigate the biological functions of the overexpressed recombinant LIF, a [(3)H]-thymedine incorporation assay was performed, and the results showed that the recombinant LIF inhibited the growth of M1 cells strongly. Taken together, all the function assays suggest that the recombinant LIF has the normal functions, suggesting that the recombinant LIF could be used for further studies.

[Establishment of high-throughput drug screening cell models based on JAK-STAT signal pathway].

AIM: To discover new drugs which may be applied to diseases of the immune system, hemogenesis system diseases and tumors, several high-throughput drug screening cell models based on JAK-STAT signal pathway have been established. METHODS: Four repeats of STAT DNA binding conserved sequences were synthesized, subcloned into pGL-Luc reporter vector and stably transfected into cell lines in vitro. Cell clones with high copy numbers of STAT binding sites and reporter genes were chosen as high-throughput drug screening cell models. The cell models were tested with known anti-allergic drugs and anti-tumor drugs by determining luciferase activity. The reaction was performed in 96 well micro-plates with a final volume of 50 microL. RESULTS: The cell models by performing rapid fluorescence assay were shown to be highly sensitive and stable after testing with cytokine and drugs. The modification of the expression plasmid simplified this method and made it more practical. It also provided good linear correlation, wide range of assay, highly sensitive and good reproducibility. CONCLUSION: The method can be performed by high-throughput drug screening for effective extraction of Chinese traditional herbs.

[Wnt/Frizzled signaling pathway in renal carcinoma].

OBJECTIVE: To investigate the transduction of Wnt/Frizzled signaling pathway, especially the function of T cell factor 4 (TCF(4)), in renal carcinoma. METHODS: A renal carcinoma yeast two hybrid library and a human TCF(4) yeast two hybrid expression vector were constructed. Proteins interacting with the bait protein human TCF(4) were obtained from the renal carcinoma yeast two hybrid library by reverse yeast two hybrid system. RESULTS: 67 positive clones interacting with the bait protein TCF(4) were obtained by reverse yeast two hybrid system, including 18 beta-catenin clones, 24 TCF(4) clones and 25 unknown clones. CONCLUSION: Wnt/Frizzled signaling pathway exists in renal carcinoma. TCF(4), its important signal factor, interacts with beta-catenin and forms homodimer or homocopolymer by itself, thus displaying its constitutive transcriptional activity.

Autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells.

Late-onset hypogonadism (LOH) is closely related to secondary androgen deficiency in aged males, but the mechanism remains unclear. In this study, we found that reduced testosterone production in aged rat Leydig cells is associated with decreased autophagic activity. Primary rat Leydig cells and the TM3 mouse Leydig cell line were used to study the effect of autophagic deficiency on Leydig cell testosterone production. In Leydig cells from young and aged rats, treatment with wortmannin, an autophagy inhibitor, inhibited luteinising hormone (LH)-stimulated steroidogenic acute regulatory (StAR) protein expression and decreased testosterone production. In contrast, treatment with rapamycin, an autophagy activator, enhanced LH-stimulated steroidogenesis in Leydig cells from aged, but not young, rats. Intracellular reactive oxygen species (ROS) levels were increased in both young and aged Leydig cells treated with wortmannin but decreased only in aged Leydig cells treated with rapamycin. Furthermore, an increased level of ROS, induced by H(2)O(2), resulted in LH-stimulated steroidogenic inhibition. Finally, knockdown of Beclin 1 decreased LH-stimulated StAR expression and testosterone production in TM3 mouse Leydig cells, which were associated with increased intracellular ROS level. These results suggested that autophagic deficiency is related to steroidogenic decline in aged rat Leydig cells, which might be influenced by intracellular ROS levels.

[Effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea].

OBJECTIVE: To establish a mice model of cisplatin-induced ototoxicity, and to investigate the effect of cisplatin on apoptosis of spiral ganglion cell and expression of caspase-3 in mouse cochlea. METHODS: Terminal deoxynucleotidyl transferase-mediated nick end labeling method (TUNEL) was used to monitor the apoptosis of spiral ganglion cell. Envision method of immunohistochemistry was applied to detect the expression of caspase-3 in cochlea. Auditory brainstem response (ABR) was measured to observe the change of hearing. RESULTS: The weight and hearing of mice in different dose of cisplatin groups were declined significantly as compared with those of control group (P < 0.05, P < 0.01), and the TUNEL positive cell number and expression of caspase-3 were greater remarkably with the more cisplatin injected. CONCLUSION: A mouse model of cisplatin-induced ototoxicity can be established. Cisplatin can lead to the apoptosis of spiral ganglion cells, and caspase-3 has participated in this apoptosis process, which approves further that apoptosis might be one of the mechanisms of cisplatin ototoxicity.

The construction of siRNA plasmid targeting mouse HIF-1alpha and in vitro study of its inhibition effect.

OBJECTIVE: To construct effective RNA-interference plasmids targeting mouse HIF-1alpha gene and testify their effects and specificities in interfering HIF-1alpha expression. METHODS: Three RNA-interference plasmids targeting mouse HIF-1alpha gene, pBS/U6/HIF-1alpha-siRNAI~III, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1alpha in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1alphai-II in SH-SY5Y cell line was further investigated. RESULTS: All the three RNA-interference plasmids, especially pBS/U6/HIF1alphai-II, showed significant inhibition in HIF-1alpha expression in 293T cell line. pBS/U6/HIF1alphai-II could also inhibit HIF-1alpha expression in SH-SY5Y cell line, in a dose-dependent way. CONCLUSION: Plasmid pBS/U6/HIF1alphai-II constructed in our study can effectively and specifically inhibit HIF-1alpha expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/HIF1alphai-II plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and ischemia development.

The protein level of hypoxia-inducible factor-1alpha is increased in the plateau pika (Ochotona curzoniae) inhabiting high altitudes.

The plateau pika (Ochotona curzoniae) is a high hypoxia-tolerant species living only at 3,000-5,000 m above sea-level on the Qinghai-Tibetan plateau. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. To investigate how the plateau pika adapts to a high-altitude hypoxic environment at the molecular level, we examined the expression pattern of the HIF-1alpha protein in the pika by Western blot and immunohistochemical analysis. We found that HIF-1alpha protein is expressed at a significantly high level in the pika, which is higher in most tissues (particularly in the lung, liver, spleen and kidney) of the plateau pika than that of mice living at sea-level. Importantly, we found that the protein levels of HIF-1alpha in the lung, liver, spleen and kidney of the pika were increased with increased habitat altitudes. We observed that the plateau pika HIF-1alpha localized to the nucleus of cells by an immunostaining analysis, and enhanced HRE-driven gene expression by luciferase reporter assays. Our study suggests that the HIF-1alpha protein levels are related to the adaptation of the plateau pika to the high-altitude hypoxic environment.

[Screening of proteins interacting with Dishevelled2 in mouse 11.5dpc embryo library].

Dishevelled proteins are multifunctional and highly conserved. These proteins are also required for the specification of cell fate and polarity by secreted Wnt proteins. To investigate the molecular mechanism of Dishevelled in mediating Wnt signal transduction, a mouse 11.5dpc embryo library was screened by yeast-two-hybrid system to find mouse Dishevelled2 DEP domain and C-terminal interacting proteins. 15 possitive clones were identified from 4.1 x 10(6) transformants. The DNA sequences of the positive AD/library plasmids were determined. The BLAST results revealed that one of the positive clones contained N-terminus cDNA fragments (amino acids 6-122) of Gli3 protein. The interaction between Dv12 and Gli3 detected by yeast two-hybrid system suggests that Gli3 might play a role in some biological processes with Dishevelled.