OBJECTIVE: To study the significance of dishevelled (DVL) proteins in the Wnt signaling pathway in the pathogenesis and prognosis of childhood acute lymphoblastic leukemia (ALL). METHODS: A total of 33 children with new-onset ALL were enrolled as the case group. According to the degree of risk, they were divided into 3 groups: low-risk (n=14), intermediate-risk (n=5) and high-risk (n=14). A total of 29 children with immune thrombocytopenia were enrolled as the control group. At diagnosis and on day 33 of induction therapy, 2 mL bone marrow samples were collected from the case and control groups, and qRT-PCR was used to measure the mRNA expression of DVL1, DVL2 and DVL3 in blood cells of bone marrow. RESULTS: The mRNA expression of DVL1, DVL2 and DVL3 in the case group in the incipient stage was significantly higher than that in the remission stage and the control group (P<0.05). Compared with the control group, the case group had a significant increase in the mRNA expression of DVL2 in the remission stage (P<0.05). The mRNA expression of DVL2 was significantly higher than that of DVL1 and DVL3 in both remission and incipient stages (P<0.05). The high- and intermediate-risk groups had significantly higher mRNA expression of DVL1 and DVL2 than the low-risk group (P<0.05). The mRNA expression of DVL2 was significantly higher than that of DVL1 and DVL3 in the low-, intermediate- and high-risk groups (P<0.05). CONCLUSIONS The change in the expression of DVL, especially DVL2, in the Wnt signal pathway has certain significance in the pathogenesis and prognosis of childhood ALL.
Child ; Dishevelled Proteins ; Humans ; Phosphoproteins ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; Wnt Signaling Pathway

OBJECTIVETo construct the recombinant vectors that express mouse Dishevelled 2 (Dvl2), and to evaluate its expression level in transfected RAW264.7 cells.

METHODSA pair of specific primers were designed according to the mouse Dvl2 cDNA sequence published in GenBank. Total RNA of RAW264.7 cells was extracted, and open reading frame of Dvl2 was obtained by RT-PCR, which was then cloned into pEZ-M29 plasmid. Electrophoresis after macrorestriction and DNA sequence analysis were used to identify the reconstructed plasmids. After transient transfection via liposome, the transfection of RAW264.7 cells was confirmed under a fluorescence microscope, and the expression level of Dvl2 was evaluated by real-time RT-PCR.

RESULTSThe recombinant plasmid containing mouse Dvl2, namely pEZ-M29/ Dvl2, was successfully constructed, and confirmed by DNA sequence analysis. After 48 h of tranfection, the expression of enhanced green fluorescene protein was observed under a fluorescence microscope, and real-time RT-PCR analysis revealed that the Dvl2 mRNA level was prominantly elevated in the transient transfected RAW264.7 cells.

CONCLUSIONThe recombinant plasmids pEZ-M29/Dvl2 are successfully constructed and can elevate the Dvl2 mRNA level in the transient transfected RAW264.7 cells, which can be used in further studies aiming at revealing the functional significance of Dvl2 in the osteoclastogenesis.

Adaptor Proteins, Signal Transducing ; biosynthesis ; Animals ; Cell Line, Tumor ; Dishevelled Proteins ; Genetic Vectors ; Mice ; Phosphoproteins ; biosynthesis ; Plasmids ; RNA, Messenger ; Transfection

OBJECTIVE: This study aims to investigate the expression of disheveled 2 (Dvl2) around the implant of hyperlipidemic rats at an early stage after the implantation. METHODS: A total of 24 Wistar rats were divided equally into the experimental group fed with high-fat diet group and control group fed with a normal diet. After 8 weeks, the serum lipid levels were detected, and rats received implants in the femur metaphysis. Rats were sacrificed at 1, 3, and 5 days after implantation, and the bones around implants were obtained. Methylene blue-acid fuchsin staining was performed to observe the implant-bone interface. Real-time polymerase chain reaction was performed on runt-related transcription factor 2 (Runx2), cathepsin K (CatK), and Dvl2. Dvl2 Western blot or immunoprecipitation, phosphorylation, and ubiquitination were also conducted. RESULTS: In the experimental group, less osteoblasts, lower expression of Runx2 and Dvl2, and lower Dvl2 phosphorylation (P<0.05) than those of the control group were observed; furthermore, the CatK expression and Dvl2 ubiquitination were higher than those in the control group (P<0.05). CONCLUSIONS Hyperlipidemia may suppress bone remodeling around the implant at an early stage by Dvl2 down-regulation, phosphorylation, and up-regulated ubiquitination.
Animals ; Bone Remodeling ; Cathepsin K ; metabolism ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Dishevelled Proteins ; metabolism ; Osteoblasts ; Rats ; Rats, Wistar

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.
Adaptor Proteins, Signal Transducing ; chemistry ; genetics ; physiology ; Animals ; Dishevelled Proteins ; Gene Library ; Kruppel-Like Transcription Factors ; physiology ; Mice ; Nerve Tissue Proteins ; physiology ; Phosphoproteins ; chemistry ; genetics ; physiology ; Plasmids ; Signal Transduction ; Two-Hybrid System Techniques ; Wnt Proteins ; physiology ; Zinc Finger Protein Gli3

The NF-kappaB transcription factor plays important regulatory roles in inflammation, apoptosis, immune and stress responses. IkappaB kinase (IKK) composed of two catalytic subunits and a regulator subunit, acts as a key component of NF-kappaB activation pathway through phosphorylation of IkappaB, the inhibitor of NF-kappaB. CIKS (connection to IKK and SAPK), a newly identified cellular protein, is involved in NF-kappaB and JNK activation. Although it has been known that CIKS interacts with IKK complex, and activates both IKK and SAPK when overexpressed; the underling mechanisms are poorly understood. To better understand the physiological roles of CIKS, we have screened human HeLa MATCHMAKER cDNA library for new binding partners of CIKS by using the yeast two-hybrid system with truncated CIKS (151-574) as the bait. The yeast strain AH109 was sequentially transformed with the bait plasmid and the library. The transformants were screened on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal)selective plates. Positive clones were restreaked on SD(-Leu/-Trp / + X-alpha-gal)plates three times to allow loss of some of the AD/library plasmids while maintaining selective pressure on both the DNA-BD and AD vectors. After 3 screenings on SD(-Leu/-Trp / + X-alpha-gal), the positive clones were further verified on SD(-Leu/-Trp/-His/-Ade/ + X-alpha-gal) plates. The inserts in AD/library plasmids were amplified by PCR and PCR products were characterized by Hae III digestion to eliminate the duplicates. After screening in selective plates, the positive AD/library plasmids were rescued via transformation of E. coli HB101 and the interactions of CIKS (151-574) with positive AD/library plasmids were further assessed by yeast two-hybrid analysis. Finally, the DNA sequences of the positive AD/library plasmids were determined and BLAST analysis against the databases was performed. The BLAST results indicate that the inserts in the positive plasmids encode RIKEN cDNA 473340F03, PLAC8, CD27BP (Siva-1), CDC5L, SnRNP smB, and DVL2. CDC5L is a key component of the multi-protein complex essential for the formation of pre-mRNA splicing product and is not required for spliceosome assembly. A role for CDC5L in the cell division cycle has been precious suggested as its overexpression of this protein in mammalian cells leads to a shortening G2 phase of the cell cycle, and a negative-dominant mutant of CDC5L lacking the N-terminal activation domain delays the G2 phase cell's entry into the mitosis. It has been reported that SnRNP smB participates in pre-mRNA splicing and CD27BP (Siva-1) binds to and inhibits BCL-XL-mediated protection against UV radiation-induced apoptosis and regulates T cell homeostasis. The functions of RIKEN cDNA 473340F03, PLAC8 and DVL2 are unknown. It has been suggested that CIKS functions as a critical component for cross-talk between NF-kappaB and JNK signaling pathways. IKK subunits, which have been demonstrated to interact with CIKS, were not shown up in this experiment. We speculate that the truncated CIKS (151-574) bait may not contain the binding domain that mediates the interaction of IKK subunits with CIKS. Taken together, the above results suggest that CIKS may play a role in cell regulation through interacting with various cellular proteins. Further investigations are required to characterize these interactions.
Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Dishevelled Proteins ; Gene Library ; HeLa Cells ; Humans ; Oncogene Proteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Plasmids ; genetics ; Polymerase Chain Reaction ; Protein Binding ; genetics ; physiology ; Proteins ; Tumor Necrosis Factor Receptor-Associated Peptides and Proteins ; genetics ; metabolism ; Two-Hybrid System Techniques

OBJECTIVETo study the diagnostic usefulness of immunohistochemical markers in distinguishing between invasive ductal carcinoma and invasive lobular carcinoma of breast.

METHODSTwenty-four cases of grade I invasive ductal carcinoma, 12 cases of classic invasive lobular carcinoma and 14 cases of invasive carcinoma with mixed ductal and lobular features were retrieved from the archival files of Peking University First Hospital during the period from January, 1998 to December, 2001. Immunohistochemical study for E-cadherin, p120 catenin, epithelia membrane protein 1 (EMP1) and DVL1 was performed.

RESULTSThe positivity rates for E-cadherin in grade I invasive ductal carcinoma and classic invasive lobular carcinoma were 83.3% (20/24) and 0, respectively (P < 0.01). The positivity rates for p120 catenin were 100% in both grade 1 invasive ductal carcinoma (membranous staining) and classic invasive lobular carcinoma (cytoplasmic staining). The positivity rates for EMP1 and DVL1 in gradeI invasive ductal carcinoma were 95.8% (23/24) and 54.2% (13/24), respectively; while those in classic invasive lobular carcinoma were 12 and 5 cases, respectively.

CONCLUSIONSE-cadherin and p120 catenin are useful immunomarkers for distinguishing between invasive ductal carcinoma and invasive lobular carcinoma. On the other hand, EMP1 and DVL1 are of limited value in this respect.

Adaptor Proteins, Signal Transducing ; metabolism ; Breast Neoplasms ; metabolism ; pathology ; surgery ; Cadherins ; metabolism ; Carcinoma, Ductal, Breast ; metabolism ; pathology ; surgery ; Carcinoma, Lobular ; metabolism ; pathology ; surgery ; Catenins ; metabolism ; Diagnosis, Differential ; Disease-Free Survival ; Dishevelled Proteins ; Female ; Humans ; Mastectomy ; methods ; Middle Aged ; Neoplasm Proteins ; metabolism ; Phosphoproteins ; metabolism ; Receptors, Cell Surface ; metabolism ; Survival Rate

OBJECTIVETo investigate the role of Wnt signaling pathway on paraquat (PQ)induced PC12 cells damage.

METHODSUsing PC12 cells, in this study CCK8 assay was used to detect the effect of cell viability. The cell apoptosis and cell cycle was detected by flow cytometry. The real-time polymerase chain reaction (RT-PCR) was used to measure the mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and β-catenin and downstream genes including Bax, Bcl2, Survivin, Cyclin D1 and C-myc.

RESULTCompared with the control, PC12 cells viability in 50.00 and 100.00 µmol/L PQ treatment groups were obviously decreased, the cell cycle S phase arrest, and cell apoptosis increased (P<0.05). The 25.00, 50.00 and 100.00 µmol/L PQ treatment groups mRNA expression of Wnt pathway key genes including Fzd1, Dvl2 and β-catenin and downstream genes including apoptosis suppressor genes (Bcl-2 and survivin)and cyclin gene (Cyclin D1) were downregulated (P<0.05). The mRNA expression of pro-apoptosis gene (Bax) and cyclin gene (C-myc) were upregulated (P<0.05).

CONCLUSIONIt suggested that PQ can activate Wnt pathway to regulate downsteam genes expression, resulting in PC12 cell cycle arrest and apoptosis.

Adaptor Proteins, Signal Transducing ; metabolism ; Animals ; Apoptosis ; Apoptosis Regulatory Proteins ; metabolism ; Cell Cycle ; Cell Survival ; Dishevelled Proteins ; Down-Regulation ; Flow Cytometry ; Frizzled Receptors ; metabolism ; Gene Expression ; drug effects ; PC12 Cells ; Paraquat ; toxicity ; Phosphoproteins ; metabolism ; Rats ; Receptors, Neurotransmitter ; metabolism ; Wnt Signaling Pathway