OBJECTIVETo verify the existence and significance of calcium/calmodulin dependent serine protein kinase/inhibitors of differentiation 1 (CASK/Id1) pathway in fibroblasts of human keloid.

METHODSImmunofluorescence laser was used to confirm CASK and Id1 protein expression and localization in fibroblasts of the keloid and normal skin. RT-PCR and Western-blot were adopted to analysis the CASK and Id1 expression and differences between keloid and normal skin fibroblasts. The natural combination of CASK and Id1 protein of keloid fibroblasts was tested by immunoprecipitation.

RESULTSCASK and Id1 protein expression were both found in fibroblast cells of keloid and normal skin under normal circumstances. Most of CASK and Id1 were distributed in the cytoplasm and nucleus of fibroblasts. The results of RT-PCR showed that the expression of CASK mRNA in the keloid group was 0.658 +/- 0.024, which was lower than that in the normal control group (1.076 +/- 0.008, t = 11.159, P < 0.05). The expression of Id1 mRNA was 0.497 +/- 0.014, which was higher than that in the normal control group (0.307 +/- 0.017, t = 15.148, P < 0.05). The results of Western-blot showed that the expression level for CASK protein in the keloid group was 0.057 +/- 0.006, which was lower than that in the normal control group (0.168 +/- 0.012, t = 13.524, P < 0.05); the expression of Id1 protein was 0.812 +/- 0.035, which was higher than that in the normal control group (0.368 +/- 0.031, t = 16.356, P < 0.05). The results of immunoprecipitation showed that Id1 could be detected in the CASK precipitate, while CASK also could be detected in the Id1 precipitate. There was a natural binding of CASK and Id1 in keloid fibroblasts.

CONCLUSIONCASK/Id1 signal pathway may be existed and involved in the proliferation of keloid fibroblasts, which is related with the occurrence of keloid.

Cell Proliferation ; genetics ; Cyclin-Dependent Kinase Inhibitor Proteins ; genetics ; metabolism ; Fibroblasts ; metabolism ; Humans ; Inhibitor of Differentiation Protein 1 ; genetics ; metabolism ; Keloid ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Signal Transduction

The study was purposed to investigate the significance of id4 gene promoter methylation in monitoring AML patients with complete remission (CR). Methylation specific-PCR (MS-PCR) were used to detect the status of promoter methylation of id4 gene in bone marrow samples from AML patients with CR who had accepted induction with DA or IA and 4 to 5 consolidation chemotherapy with Ara-C. The results showed that in the all 32 patients, 15 were found to show id4 promoter methylation and 7 out of the 15 were found relapsed or tendency to relapse in the following-up period. While all the 17 patients with id4 unmethylation were still in their CR status in the same period. In conclusion, id4 promoter methylation might be a predictor for relapse of AML patients with CR in certain degree.
DNA Methylation ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Neoplasm, Residual ; diagnosis ; genetics ; Promoter Regions, Genetic ; genetics

OBJECTIVETo examine the relationship between effect of vascular endothelial growth factor (VEGF) on epithelial-myofibroblast transition (EMT) of HK-2 cells and changes in expressions of bone morphogenetic protein-7 (BMP-7) and inhibitor of DNA binding/differentiation (Id) 2, Id3.

METHODSThe cultured HK-2 cells were co-treated with transforming growth factor-beta1 (TGF-beta1) (5 ng/ml) and VEGF165 (0.1, 1, 10, 100 ng/ml), or with TGF-beta1 (5 ng/ml) and VEGF receptor-1 neutralized antibody (10 microg/ ml), and were also co-treated with TGF-beta1 (5 ng/ml) and VEGF165 (100 ng/ml) with or without activin receptor-like kinase 6 (Alk6)/Fc Chimera (2 microg/ml, to neutralize endogenous BMP-7) for 48 hours. mRNA and protein expressions of alpha-smooth muscle actin (alpha-SMA), E-cadherin, BMP-7, Id2 and Id3 of HK-2 cells were assessed with double-stain immunocytochemistry, real-time PCR and Western blot respectively.

RESULTSCompared with normal controls, alpha-SMA expression significantly increased, while E-cadherin, BMP-7, Id2, and Id3 mRNA and protein expressions markedly decreased in HK-2 cells treated with TGF-beta1 (5 ng/ml) (P < 0.05). VEGF165 interrupted TGF-beta1 induced alpha-SMA expression in a dose-dependent manner and upregulated BMP-7, Id2 mRNA and protein expressions of the cells (P < 0.05). alpha-SMA expression increased, while E-cadherin, BMP-7, and Id2 expressions decreased further in HK-2 cells co-treated with TGF-beta1 and VEGFR1 antibody compared with normal controls (P < 0.05). When endogenous BMP-7 was neutralized with Alk6/Fc Chimera in the cells co-treated with TGF-beta1 and VEGF165, alpha-SMA expression upregulated (P < 0.05), while Id2 was not changed.

CONCLUSIONSVEGF165 may partially inhibit TGF-beta1-induced EMT of HK-2 cells in vitro. This effect is related to the upregulated expressions of BMP-7 and Id2. Id2 may be upregulated directly by VEGF165, but not related to BMP-7.

Bone Morphogenetic Protein 7 ; genetics ; metabolism ; Cell Differentiation ; Cell Line ; Epithelial Cells ; cytology ; metabolism ; Gene Expression Regulation ; Humans ; Inhibitor of Differentiation Protein 2 ; genetics ; metabolism ; Inhibitor of Differentiation Proteins ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Transforming Growth Factor beta1 ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Multiple myeloma (MM) is an incurable heterogeneous disease derived from malignant clonal expansion of plasma cells. The evaluation of prognosis, detection of minimal residual disease (MRD) and treatment of MM are unclear for decades. Recently, Velcade and autotransplantation have been broadly applied to MM patients and achieved better outcomes, but there is yet no effective and universal marker for MRD detection in MM. Both genetic and epigene-tic aberrations play important roles in the pathogenesis and development of cancer. Our preliminary data showed that aberrant promoter methylation of zo-1 and id4 genes was correlated with their gene silencing in several types of hematological malignancies. Therefore, this study was aimed to identify the promoter methylation status of zo-1 and id4 genes in MM and their relationship with the prognosis, MRD and treatment of MM. The methylation status of zo-1 and id4 genes of MM cell lines U266, H929 and IM9 was tested by using MS-PCR; the methylation status of zo-1, id4 gene promoters in bone marrow samples of 20 MM patients and 6 healthy persons was detected by MS-PCR. The results showed that the zo-1, id4 gene in MM cell lines all were methylation positive (complete or partial methylation), the zo-1, id4 gene in samples of 5 healthy persons all were completely unmethylated. The methylation positive rate of zo-1 and id4 genes were 50% and 85% respectively, which were significantly higher than that in normal bone marrow (0%). The coverage rate of zo-1 and id4 gene methylation was 95%. There were no significant differences in the methylation status of both genes among the patients with different heavy chains, different light chains and symptoms. It is concluded that the change of zo-1, id4 genes methylation status occurs in MM patients and has specificity, which may be a new gene marker of MM, methylation analysis of zo-1 and id4 genes may be important for MRD monitoring in patients with MM.
Adult ; Aged ; Bone Marrow ; metabolism ; Cell Line, Tumor ; DNA Methylation ; Female ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Middle Aged ; Multiple Myeloma ; genetics ; metabolism ; Neoplasm, Residual ; genetics ; Phosphoproteins ; genetics ; metabolism ; Zonula Occludens-1 Protein

OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

BACKGROUNDNeural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS), which are active during development and maintain homeostasis and tissue integrity throughout life. Microglias are an immune cell population resident in the CNS, which have crucial physiological functions in the developing and adult CNS. This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.

METHODSMicroglia and NSCs were co-cultured in 24-well insert plates. NSCs were plated in the bottom of the well and microglia in the insert. Fluorescent staining, Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation.

RESULTSCo-culture of microglia and NSCs promoted astrogliogenesis from NSCs. Several key genes, such as Notch 1, Notch 2, Notch 3, Hes 5, and NRSF were downregulated, while the critical genes Id1 and Id2 were upregulated. BMP2 and FGF2 were upregulated.

CONCLUSIONMicroglias act as a regulator of NSCs astrogliogenesis.

Animals ; Astrocytes ; cytology ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Coculture Techniques ; methods ; Fibroblast Growth Factor 2 ; genetics ; Inhibitor of Differentiation Protein 1 ; genetics ; Inhibitor of Differentiation Protein 2 ; genetics ; Microglia ; cytology ; metabolism ; Microscopy, Fluorescence ; Neural Stem Cells ; cytology ; metabolism ; Rats ; Repressor Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

This study was purpose to investigate the difference of Id4 gene promoter methylation between healthy individuals and acute leukemia patients. MS-PCR methods were used to detect the status of Id4 gene methylation in healthy individuals and acute leukemia patients. The results showed that Id4 gene was unmethylated in bone marrow samples from healthy individuals. In new diagnosed AML and ALL patients, the rate of Id4 gene methylation was 84% and 86% respectively. Id4 gene methylation was found in all 8 cases of relapsed acute leukemias. In 14 ALL-CR patients with Id4 gene methylation, 8 patients relapsed within 12 months, while in 9 ALL-CR patients with Id4 gene unmethylation only 1 patient relapsed within 12 months. In AML-CR patients, the 12-months relapse rate in patients with Id4 gene methylation was 62.5%, while it was 10% in Id4 gene unmethylation patients, there was significant difference between them. The rate of Id4 gene methylation in ALL-CR patients was 64.3%, while it was 28.6% in AML-CR patients, there was significant difference between them. In the all 39 new diagnosed AL patients, Id4 gene methylation could be detected in 33 patients. In all 8 relapsed AL patients Id4 gene methylation was found, out of 58 AL-CR cases, 24 patients was found with Id4 gene methylation. It is concluded that as compared with healthy individuals, Id4 gene in acute leukemia patients was methylated in different degrees. The percentage of Id4 gene methylation in AL-CR group is lower than that in AL-non remission group. The change of Id4 gene methylation is thought to be associated with occurrence of acute leukemia.
Acute Disease ; Case-Control Studies ; DNA Methylation ; Female ; Humans ; Inhibitor of Differentiation Proteins ; metabolism ; Leukemia ; genetics ; metabolism ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; Promoter Regions, Genetic ; genetics

Small heterodimer partner (SHP) is an atypical member of nuclear receptor superfamily that lacks a DNA-binding domain. In previous study, we showed that SHP, c-jun, p65 of NF-kappaB subunits, and p21WAF1 expression was increased during monocytic differentiaton with the exposure of human leukemia cells to a differentiation agent, PMA. In this study, c-Jun and p65 were shown to mediate the transcriptional activation of the SHP promoter. In addition, SHP induced the cell cycle regulatory protein levels and cooperatively increased an induction of p21WAF1 expression with p65. Furthermore, SHP protected differentiated cells from etoposide-induced cellular apoptosis through the induction and cytoplasmic sequestration of p21WAF1. Complex formation between SHP and p21WAF1 was demonstrated by means of coimmunoprecipitation. These results suggest that SHP prolongs a cellular survival of differentiating monocytes through the transcriptional regulation of target genes of cell survival and differentiation.
*Apoptosis ; Cell Differentiation ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/genetics/*metabolism ; Gene Expression Regulation ; Humans ; Monocytes/cytology ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-jun/genetics/metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Transcription Factor RelA/genetics/metabolism

BACKGROUNDId3 plays a key role in the progression of breast cancer. Previously, four and a half LIM protein (FHL2) was identified as a repressor of Id family proteins by interacting with them. This study aimed to investigate the effects of FHL2 on the transcriptional regulation and oncogenic activities of Id3 in human breast cancer cells.

METHODSCell transfection was performed with SuperFect reagent. Stable transfectants that overexpressed Id3 were obtained by selection on G418. The level of Id3 protein was determined by Western blotting analysis. Dual luciferase assays were used to measure the effect of Id3 and FHL2 on E47-mediated transcriptional activity in MCF-7 human breast cancer cells. The MTT assay was used to measure cell proliferation. The transwell assay was used to measure the invasive capacity of MCF-7 cancer cells.

RESULTSId3 markedly repressed transcription mediated by the basic helix-loop-helix (bHLH) factor E47 in MCF-7 cells. This Id3-mediated repression was effectively antagonized by FHL2. Overexpression of Id3 markedly promoted the proliferation and invasive capacity of MCF-7 cells; however, these effects were significantly suppressed by the overexpression of FHL2.

CONCLUSIONSFHL2 can inhibit the proliferation and invasive growth of human breast cancer cells by repressing the functional activity of Id3. The functional roles of FHL2-Id3 signaling in the development of human breast cancer need further research.

Blotting, Western ; Breast Neoplasms ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Inhibitor of Differentiation Proteins ; genetics ; metabolism ; LIM-Homeodomain Proteins ; genetics ; metabolism ; MCF-7 Cells ; Muscle Proteins ; genetics ; metabolism ; Neoplasm Proteins ; genetics ; metabolism ; Transcription Factor 3 ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism

The study was purposed to explore the effect and mechanisms of decitabine and/or Trichostatin A (TSA) on SKM-1 cells in vitro. The effect of decitabine and/or TSA on proliferation of SKM-1cells was analyzed with trypan blue exclusion; the differentiation of SKM-1 cells was detected by nitro-blue tetrazolium (NBT) reduction and flow cytometry; the apoptosis of cells was measured by Annexin V-FITC; the mRNA expression of Fas, survivin and P15(INK4B) in cells treated with decitabine and/or TSA was evaluated by RT-PCR. The results showed that decitabine and/or TSA were capable of inhibiting SKM-1 cell growth and promoting cell differentiation; they stimulated the expression of CD14 and CD11b and inhibited HLA-DR expression; meanwhile and decitabine or/and TSA could induce cell apoptosis, up-regulate mRNA expression of Fas and P15(INK4B), and down-regulate survivin mRNA expression. It is concluded that decitabine can induce apoptosis/differentiation of SKM-1 cells, whose mechanisms may related to the expression of Fas, survivin and P15(INK4B). Decitabine has the synergistic effect with TSA.
Antimetabolites, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Differentiation ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Drug Synergism ; Humans ; Hydroxamic Acids ; pharmacology ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; Myelodysplastic Syndromes ; pathology ; fas Receptor ; genetics ; metabolism