BACKGROUNDThere is a higher rate of cytomegalovirus (CMV) reactivation in patients with multiple myeloma after an autologous stem cell transplantation, but no attention has been given thus far to a possible pathogenetic interplay between CMV and multiple myeloma. CMV can infect many kinds of cells, and CMV infection has been shown to inhibit apoptotic responses in several cell systems. In this study the authors investigated the alterations in apoptosis in the multiple myeloma cell line KM3 after infection with CMV and proposed a possible mechanism.

METHODSKM3 cells were infected with 100, 10, or 1 TCID50 of CMV and then cultured in serum-free RPMI 1640. An RT-PCR-based assay was used to detect mRNA expression of CMV-IE, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and IL-6 in CMV-infected and mock-infected cells. Flow cytometry was used to detect apoptotic cells. CMV particles and apoptotic cells were also examined with an electron microscope.

RESULTSCMV-infected KM3 cells clearly expressed immediate early (IE) antigen mRNA when compared to uninfected cells, and there were fewer apoptotic cells among cells treated with 100 or 10 TCID50 of CMV after culturing in serum-free RPMI 1640. CMV particles were observable in infected cells under an electron microscope. Expression of IL-6 mRNA increased after infection.

CONCLUSIONCMV can infect the multiple myeloma cell line KM3, inhibit the apoptotic response in these cells after apoptosis induction in serum-free culture, and increase the expression of IL-6 mRNA.

Apoptosis ; physiology ; Cell Line, Tumor ; Cytomegalovirus ; physiology ; Flow Cytometry ; Humans ; Immediate-Early Proteins ; analysis ; Interleukin-6 ; genetics ; physiology ; Multiple Myeloma ; pathology ; RNA, Messenger ; analysis ; Viral Proteins ; analysis

OBJECTIVETo study the expression and location of early growth response gene-1 (Egr-1), transforming growth factor-beta(1) (TGF-beta(1)), fibronectin (FN) in silicotic rat and to discuss the role of Egr-1 in the development of silicosis.

METHODSSilicotic animal model of rat was established, and the expressions of Egr-1, TGF-beta(1), FN in various lung cells of silicotic rat were analysed by using immunohistochemical technique (SP) and the image analysis.

RESULTSThe expressions of Egr-1 in bronchial epithelial cell, pulmonary macrophage, alveolar epithelium cell and interstitial cell in lung silicotic tissue (gray values: 118.58 +/- 5.65 - 168.52 +/- 5.67) were higher than those of controls (gray values: 166.23 +/- 5.23 - 188.12 +/- 8.35) during 1 - 28 days, and the expression was mainly in nucleus; the expressions of TGF-beta(1) in these cells (gray values: 123.49 +/- 5.65 - 170.24 +/- 3.56) were also higher than those of controls (166.53 +/- 6.25 - 198.56 +/- 4.53), and the expression was mainly in cytoplasm. The expressions of FN in bronchial epithelial cell, pulmonary macrophage and alveolar epithelial cell (gray values: 150.32 +/- 6.54 - 201.54 +/- 7.38) were lower, while those in interstitial cell (gray values: 121.43 +/- 5.65 - 167.55 +/- 6.35) were higher than those of controls. The changes of TGF-beta(1) and Egr-1 expression level in bronchial epithelial cell, pulmonary macrophage, alveolar epithelium cell and interstitial cell were synchronous during the experiment (1 - 28 days). Both of them were correlated with each other (r = 0.61, P < 0.01), while the expression of FN was not correlated with Egr-1, but correlated to TGF-beta(1) in interstitial cell (r = 0.46, P < 0.01).

CONCLUSIONSilicon dioxide could up-regulate the expression of nuclear transcription factor Egr-1 in several kinds of cell in lung. The activated Egr-1 may coordinate the expression of TGF-beta(1) and FN to regulate the development of silicosis.

Animals ; DNA-Binding Proteins ; analysis ; physiology ; Disease Models, Animal ; Early Growth Response Protein 1 ; Fibronectins ; analysis ; physiology ; Immediate-Early Proteins ; analysis ; physiology ; Immunohistochemistry ; Lung ; chemistry ; physiopathology ; Rats ; Silicosis ; etiology ; metabolism ; Transcription Factors ; analysis ; physiology ; Transforming Growth Factor beta ; analysis ; physiology

OBJECTIVELung fibrosis is the ultimate outcome of hyperoxia-induced chronic lung diseases (CLD) and connective tissue growth factor (CTGF) is correlated with fibrosis. This study investigated the role of CTGF in hyperoxia-induced CLD.

METHODSFifty premature rats were randomly exposed to hyperoxia (Model group) and to room air (Control group) (n = 25 each). CLD was induced by hyperoxia exposure. The expression of CTGF was detected by immunohistochemical method at 1, 3, 7, 14 and 21 days after exposure. The severity of pulmonary fibrosis was evaluated.

RESULTSIn the Control group there was a slight expression of CTGF in the bronchial epithelial cells and vascular endothelial cells. The intensity and range of CTGF expression in the Model group were similar to the Control group on days 1, 3 and 7 of exposure. On the 14th day, CTGF was expressed in some alveolar epithelial cells, fibroblasts and interstitial cells, and the intensity of CTGF expression increased significantly compared with the Control group, with the IODT of CTGF of 10.53 +/- 4.24 vs 5.58 +/- 1.18 (P < 0.01). On day 21, the expression intensity and range of CTGF in the Model group (IODT: 16.61 +/- 5.39) increased compared with that of Control group (P < 0.01). The expression of CTGF was correlated with the degree of fibrosis in the Model group on days 14 and 21 (r = 0.903, r = 0.926 respectively, P < 0.01).

CONCLUSIONSThe CTGF expression increased with the time of hyperoxia exposure and the development of fibrosis. CTGF is closely related to the development of hyperoxia-induced pulmonary fibrosis.

Animals ; Chronic Disease ; Connective Tissue Growth Factor ; Female ; Hyperoxia ; complications ; Immediate-Early Proteins ; analysis ; physiology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; physiology ; Lung ; chemistry ; pathology ; Pulmonary Fibrosis ; etiology ; pathology ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; physiology

The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.
Cells, Cultured ; Connective Tissue Growth Factor/genetics ; Connective Tissue Growth Factor/*metabolism ; Glucose/*pharmacology ; Immediate-Early Proteins/metabolism ; Immediate-Early Proteins/*physiology ; Mesangial Cells/cytology ; Mesangial Cells/*metabolism ; Protein-Serine-Threonine Kinases/metabolism ; Protein-Serine-Threonine Kinases/*physiology ; Signal Transduction/drug effects

BACKGROUNDRenal hypertrophy has been regarded as the early feature of diabetic nephropathy (DN), which may eventually lead to proteinuria and renal fibrosis. However, the exact mechanism of renal hypertrophy is still unclear. The aim of this study was to investigate the possible association of connective tissue growth factor (CTGF) with renal hypertrophy in uninephrectomized diabetic rats.

METHODSSeventy-two Sprague-Dawley (SD) rats were randomly divided into two groups: control group (group C, n = 32) and diabetic nephropathy (group DN, n = 40). Each group was re-divided into 4 subgroups according to the experimental period. The rats were sacrificed at 1, 2, 4, and 8 weeks respectively after induction of diabetes. Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) after rats had received uninephrectomy. Blood glucose (BG), body weight (BW), 24-h urinary albumin excretion (24hUalb), kidney weight (KW), KW/BW, glomerular tuft area (AG), glomerular tuft volume (VG), proximal tubular area (AT) at each time point, the width of glomerular basement membrane (GBM) and tubular basement membrane (TBM) at week 8 were measured when the rats were sacrificed. Renal expression of CTGF and p27kip1 were detected by immunohistochemical staining. The relationship between CTGF expression and increasing of VG and AT was analyzed.

RESULTSThere was a significant increase of 24hUalb, KW, and KW/BW from week 1 onward in diabetic rats compared to those in group C (P < 0.05, respectively), diabetic rats also had a significant increase of AG, VG, and AT from week 1 onward. It was also shown that diabetic rats had a thickening of GBM [(245.7 +/- 103.0) nm vs (121.8 +/- 19.1) nm, P < 0.01] and TBM [(767.7 +/- 331.1) nm vs (293.0 +/- 110.5) nm, P < 0.01] at week 8. There was a weak expression for CTGF and p27kip1 in normal glomeruli and tubuli, while a significant increasing expression of CTGF and p27kip1 was found in glomeruli and tubuli in diabetic kidney from week 1 onward (P < 0.05, respectively), and the extent of CTGF expression was positively correlated with AG (r = 0.92, P < 0.05), VG (r = 0.86, P < 0.05), AT (r = 0.94, P < 0.01) and positively correlated with the expression of p27kip1 (r = 0.96, P < 0.01).

CONCLUSIONThe expression of CTGF increases in diabetic rat kidney at the early stage, which might be an important mediator of renal hypertrophy through arresting cell cycling.

Albuminuria ; etiology ; Animals ; Connective Tissue Growth Factor ; Cyclin-Dependent Kinase Inhibitor p27 ; analysis ; Diabetes Mellitus, Experimental ; pathology ; Hypertrophy ; Immediate-Early Proteins ; analysis ; physiology ; Intercellular Signaling Peptides and Proteins ; analysis ; physiology ; Kidney ; pathology ; Male ; Nephrectomy ; Rats ; Rats, Sprague-Dawley ; Streptozocin

OBJECTIVETo study the role of connective tissue growth factor (CTGF) in the pathogenesis of human keloid.

METHODSHuman keloid fibroblasts (HKF) were isolated from human keloid and cultured in vitro. The cells were then divided into 3 groups according to different processing, i.e. ASODN treatment (AT), in which phosphorothioate CTGF antisense oligonucleotides (ASODN) labeled by fluorescent isothiocyananate were transfected into the HKFs by liposome; liposome control (LC, with liposome only); control groups (without liposome or ASODN). The distribution of CTGF ASODN in all groups of cells was observed under fluorescent microscope. The CTGF mRNA index (RI) of HKF was assessed by reverse transcription polymerase chain reaction method (RT-PCR). The collagen synthesis of HKF was assessed by (3)H-proline incorporation method.

RESULTSA large amount of fluorescence could be observed in the cytoplasm of HKFs in AT 12 hours after transfection, but not in LC and C groups. The CTGF mRNA index of HKF in AT group 48 hours after transfection was significantly lower than that in LC and C groups (0.12 +/- 0.62 vs 0.51 +/- 0.18 vs 0.54 +/- 0.35, P < 0.01). The (3)H-proline incorporation rate in AT group (108.96 +/- 79.05) was lower than that in LC and C groups (P < 0.01).

CONCLUSIONThe expression of CTGF gene and collagen synthesis of the cultured HKF could be inhibited by CTGF ASODN, implying that CTGF played a role in the development of excessive fibrosis of human keloid.

Collagen ; biosynthesis ; Connective Tissue Growth Factor ; Fibroblasts ; metabolism ; Humans ; Immediate-Early Proteins ; antagonists & inhibitors ; genetics ; physiology ; Intercellular Signaling Peptides and Proteins ; genetics ; physiology ; Keloid ; etiology ; metabolism ; Oligonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; Transfection

OBJECTIVE: To screen for aberrantly expressed genes in osteosarcoma cells and investigate the role of RHPN2 in regulating the proliferation, apoptosis, migration and tumorigenic abilities of osteosarcoma cells. METHODS: We used GEO2R to analyze the differential gene expression profile between osteosarcoma cells and normal cells in the GSE70414 dataset. RTqPCR and Western blotting were performed to detect RHPN2 expression in osteosarcoma cell lines MG-63, 143B and SAOS2. Two RHPN2-shRNA and a control NC-shRNA were designed to silence the expression of RHPN2 in 143B cells, and CCK8 assay, colony-forming assay, annexin V-FITC/PI staining and scratch assays were carried out to examine the changes in proliferation, apoptosis and migration of the cells. We also established nude mouse models bearing osteosarcoma xenografts derived 143B cells and RHPN2-shRNA-transfected 143B cells, and assessed the effect of RHPN2 silencing on osteosarcoma cell tumorigenesis using HE staining. Kaplan-Meier survival curves were used to analyze the correlation between RHPN2 expression and survival outcomes of patients with osteosarcoma. RESULTS: RHPN2 expression was significantly upregulated in osteosarcoma cell lines MG-63, 143B and SAOS2 (P < 0.01). Silencing of RHPN2 significantly inhibited the proliferation and migration of 143B cells in vitro, promoted cell apoptosis (P < 0.01), and suppressed tumorigenic capacity of the cells in nude mice. A high expression of RHPN2 was significantly correlated with a poor prognosis of patients with osteosarcoma (P < 0.05). CONCLUSION RHPN2 is highly expressed in osteosarcoma cells to promote cell proliferation and migration and inhibits cell apoptosis. A high expression of RHPN2 is associated with a poorer prognosis of the patients with osteosarcoma.
Adaptor Proteins, Signal Transducing/metabolism* ; Animals ; Apoptosis ; Bone Neoplasms/metabolism* ; Carcinogenesis ; Cell Line, Tumor ; Cell Movement/physiology* ; Cell Proliferation/physiology* ; Humans ; Immediate-Early Proteins ; Mice ; Mice, Nude ; Osteosarcoma/metabolism* ; RNA, Small Interfering/genetics*

Herpes simplex virus type 1 (HSV-1) is a common human pathogen causing cold sores and even more serious diseases. It can establish a latent stage in sensory ganglia after primary epithelial infections, and reactivate in response to stress or sunlight. Previous studies have demonstrated that viral immediate-early protein ICP0 plays a key role in regulating the balance between lytic and latent infection. Recently, It has been determined that promyelocytic leukemia (PML) nuclear bodies (NBs), small nuclear sub-structures, contribute to the repression of HSV-1 infection in the absence of functional ICP0. In this review, we discuss the fundamentals of the interaction between ICP0 and PML NBs, suggesting a potential link between PML NBs and ICP0 in regulating lytic and latent infection of HSV-1.
Herpes Simplex ; virology ; Herpesvirus 1, Human ; genetics ; physiology ; Humans ; Immediate-Early Proteins ; metabolism ; Intranuclear Inclusion Bodies ; metabolism ; virology ; Leukemia, Promyelocytic, Acute ; metabolism ; Ubiquitin-Protein Ligases ; metabolism ; Virus Latency ; physiology

Animals ; Collagen Type I ; genetics ; Connective Tissue Growth Factor ; Corneal Injuries ; Fibronectins ; analysis ; Immediate-Early Proteins ; analysis ; genetics ; physiology ; Immunohistochemistry ; Intercellular Signaling Peptides and Proteins ; analysis ; genetics ; physiology ; RNA, Messenger ; analysis ; Rabbits ; Signal Transduction ; physiology ; Smad Proteins ; physiology ; Transforming Growth Factor beta ; analysis ; genetics ; physiology ; Transforming Growth Factor beta1 ; Wound Healing ; physiology

OBJECTIVETo inhibit the proliferation and metastasis of colon cancer cells by increasing the expression level of B-cell translocation gene-2 (BTG2).

METHODSWestern blot assay was used to detect the expression level of BTG2 protein in the normal intestinal epithelial HIEC cells and three colon cancer cell lines SW620, HT-29 and LS174T. The expression of BTG2 protein in normal colonic epithelial tissue, colon adenoma and colon cancer tissue was detected by immunohistochemistry. The plasmid with BTG2 gene full-length sequence was transfected into colon cancer SW620 cells, and the expression of BTG2 protein was detected by Western blot. The cell growth curve was drawn by MTT test. The Ki-67-positive rate was calculated using immunofluorescence staining. The cell migration of colon cancer cells was detected by scratch test and Transwell double chamber culture system, and the pseudopodia growth of tumor cells was detected by Matrigel 3D culture system.

RESULTSWestern blot results showed that BTG2 relative expression levels were 0.83 ± 0.12, 0.18 ± 0.04, 0.20 ± 0.05 and 0.36 ± 0.07 in normal human intestinal epithelial cells HIEC, and human colon cancer cell line SW620, HT-29 and LS174T, respectively. The results of immunohistochemistry showed that the positive expression of BTG2 protein in normal colorectal tissue, colorectal adenoma and colorectal carcinoma tissues were 82.5% (33/40), 77.5%(31/40) and 17.5% (7/40), respectively, with a significant difference between two groups (P < 0.05). Immunofluorescence results showed that the positive rate of Ki-67 in the control group, empty vector group and BTG2 transfection group was (76.2 ± 8.0)%, (81.4 ± 9.7)% and (50.1 ± 7.1)%, respectively, showing a significant difference between two groups (P < 0.05). The scratch test results showed that in the control group, empty vector group and BTG2 transfection group, the distance of SW620 cells between two sides was (79.27 ± 11.24) µm, (80.65 ± 12.17) µm and (124.77 ± 19.63) µm, respectively, with a significant difference between two groups (P < 0.05). Transwell results showed that in the control group, empty plasmid group and BTG2 transfection group, the SW620 cell migration rate was (78.5 ± 13.1)%, (73.2 ± 12.9)% and (47.4 ± 9.1)%, respectively, showing a significant difference between two groups (P < 0.05). The number of neurospheres of BTG2 transfection group was decreased SW620, which had poor ductility.

CONCLUSIONSBTG2 gene is involved in colon cancer cell proliferation and metastasis, and effectively restores the function of BTG2 protein. Therefore, it may be expected to become a new option in gene therapy for colon cancer.

B-Lymphocytes ; physiology ; Cell Cycle ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; genetics ; Colonic Neoplasms ; Genetic Vectors ; Humans ; Immediate-Early Proteins ; genetics ; Immunohistochemistry ; Plasmids ; Transfection ; Tumor Suppressor Proteins ; genetics