Hydrogen peroxide is considered to be a dose- and time-dependent mediator in apoptotic and necrotic death. In this study, we examined the signaling of the E6 and E7 proteins with respect to apoptosis or necrosis after H2O2 injury using an in vitro model with overexpressed E6 or E7 genes. For this purpose, the E6 and E7 gene expressing astrocytes were exposed to 0.01 mM and 0.2 mM H2 O2 solutions. Twenty- four hours after treatment with the lower dosage(0.01 mM H2O2), control, E6-expressing cells suffered about 45% injury and LXSN-expressi ng cells decreased by 67% as assessed by LDH release. However, E7-expressing cells showed less injury, resulting in 20-30% of LDH release. Astrocytes expressing E6, E7, LXSN and mock-infected cells showed a typical apoptotic death patter n on the DNA gel after treatment with a low-dose of H2O2 (0.01 mM), however the y died from necrotic death after a high-dose (0.2 mM) H2O2. Overexpression of HPV-E7 genes protected the cells from apoptotic death after a low-dose of H2O2 and from necrotic death after a high-dose of H2O2, while the overexpression of E 6 genes from the necrotic death. E7 expressing astrocytes showed higher catalas e activity and the levels of E2F protein surged more than 100-folds compared with the control astrocytes. We believe that the activity of E7 protein to protect astrocytes from H2O2 injury was at least partly due to increased catalase, a scavenger protein.
Animal ; Apoptosis/*physiology ; Astrocytes/*drug effects/pathology/*physiology ; Cells, Cultured ; Hydrogen Peroxide/*pharmacology ; Mice ; Necrosis ; Oncogene Proteins, Viral/*genetics/*physiology ; Oxidants/*pharmacology ; Signal Transduction/physiology

BACKGROUNDSam68 plays an important role as a multiple functional RNA binding nuclear protein in cell cycle progress, RNA usage, signal transduction, and tyrosine phosphorylation by Src during mitosis. However, its precise impact on these essential cellular functions remains unclear. The purpose of this study is to further elucidate Sam68 functions in RNA metabolism, signal transduction regulation of cell growth and cell proliferation in DT40 cell line.

METHODSBy using gene targeting method, we isolated a mutation form of Sam68 in DT40 cells and described its effect on cell growth process and signal transduction. Southern, Northern, and Western blot, phosphorylation and flow-cytometric analyses were performed to investigate the Sam68 functions.

RESULTSA slower growth rate (2.1 hours growth elongation) and longer S phase (1.7 hours elongation) was observed in the Sam68 mutant cells. Serum depletion resulted in increased amounts of dead cells, and expansion of S phase in mutant cells. Upon B cell cross-linking, the maximal level of tyrosine phosphorylation on BLNK was observed to be significantly lower in mutant cells.

CONCLUSIONSThe proline rich domain of Sam68 is involved in cell growth control by modulating the function of mRNAs in S phase or earlier and the functions as an adaptor molecule in B cell signal transduction pathways.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; physiology ; Animals ; B-Lymphocytes ; cytology ; immunology ; physiology ; Binding Sites ; genetics ; Blotting, Western ; Cell Cycle ; physiology ; Cell Death ; physiology ; Cell Growth Processes ; drug effects ; physiology ; Cell Line, Tumor ; Culture Media, Serum-Free ; pharmacology ; Mutation ; genetics ; Phosphorylation ; Proline ; genetics ; RNA-Binding Proteins ; genetics ; metabolism ; physiology ; Receptors, Antigen, B-Cell ; immunology ; physiology ; Signal Transduction ; drug effects ; physiology ; Tyrosine ; metabolism

OBJECTIVETo investigate the role of extracellular signal-regulated kinase (ERK) in apoptosis of human colon cancer (HCT116) cells.

METHODSAfter the HCT116 cells were pretreated with specific ERK inhibitor (U0126) or specific siRNA and exposed to 10 mmol/L sodium butyrate (NaBT) for 24 h, their apoptosis was detected by flow cytometry, levels of SphK2 and ERK protein were measured by Western blot, and translocation of SphK2 was assayed by immunofluorescence microscopy.

RESULTSThe U0126 and siRNAs specific for SphK2 blocked the export of SphK2 from nuclei to cytoplasm and increased the apoptosis of HCT116 cells following NaBT exposure. Over-expression of PKD decreased NaBT-induced apoptosis of HCT116 cells, which was reversed by U0126. Furthermore, transfection of HCT116 cells with constitutively activated PKD plasmids recovered the U0126-blocked export of SphK2.

CONCLUSIONERK regulates the export of SphK2 and apoptosis of HCT116 cells by modulating PKD. Modulation of these molecules may help increase the sensitivity of colon cancer cells to the physiologic anti-colon cancer agent, NaBT.

Apoptosis ; drug effects ; physiology ; Butyric Acid ; pharmacology ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; HCT116 Cells ; drug effects ; Humans ; Phosphotransferases (Alcohol Group Acceptor) ; genetics ; metabolism ; Protein Kinase C ; genetics ; metabolism ; RNA, Small Interfering ; Signal Transduction ; drug effects

OBJECTIVETo explore the effects and molecular mechanisms of rhein on reducing starvation-induced autophagic protein expression in renal tubular epithelial ( NRK-52E) cells.

METHODHank's balanced salt solution (HBSS) was used to induce NRK-52E cells to be in the state of starvation. After the intervention of HBSS for 0, 0.5,1, 2 and 6 hours, firstly, the protein expression of microtubule-associated protein 1 light chain 3(LC3 I/II), which is a key protein in autophagy, was detected. Secondly, the protein expressions of mammalian target of rapamycin (mTOR) and phosphorylated-mTOR Ser2448 (p-mTOR S2448) were examined. And then, after the co-treatment of rhein (5 mg x L(-1)) and HBSS (1 mL) without or with mTOR inhibitor, rapamycin (100 nmol x L(-1)), the protein expressions of LC3 I/II, mTOR and p-mTOR S2448 were tested, respectively.

RESULTHBSS could induce the up-regulation of LC3 II and the down-regulation of p-mTOR S2448 at protein expression level in NRK-52E cells. The co-treatment of rhein and HBSS could reversely regulate the protein expressions of LC3 II and p-mTOR S2448 in NRK-52E cells significantly. The co-treatment of rapamycin, rhein and HBSS could recover the level of LC3 II protein expression in HBSS-intervened NRK-52E cells.

CONCLUSIONHBSS induces autophagy in renal tubular epithelial cells by inhibiting mTOR signaling pathway activation. Rhein reduces the autophagic protein expression in renal tubular epithelial cells through regulating mTOR signaling pathway activation, which is the possible effects and molecular mechanisms.

Animals ; Anthraquinones ; pharmacology ; Autophagy ; drug effects ; Cells, Cultured ; Epithelial Cells ; drug effects ; metabolism ; Isotonic Solutions ; pharmacology ; Kidney Tubules ; drug effects ; metabolism ; Microtubule-Associated Proteins ; genetics ; Rats ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; genetics ; physiology

OBJECTIVECardiotonic steroids (CTS) can bind to Na+, K+ -ATPase to activate complex intracellular signaling cascades regulating the proliferation and apoptosis of cells. The aim of this study was to investigate the effects of ouabain at different concentrations on growth regulation in various kinds of leukemia cell lines and explore the pathogenesis of leukemia, the functions of Na+, K+ -ATPase as a signal transduction conductor and its effects on cell growth.

METHODSUsing the MTT assay, the survival rates of leukemia cell lines were observed 6, 12 and 24 hrs after treatment with 1 or 10 nmol/L ouabain. The expression of Na+, K+ -ATPase alpha1 subunit of leukemia cells was detected by Western blot.

RESULTSThe MTT results showed that ouabain at 1 nmol/L or 10 nmol/L induced proliferation of lymphocytic leukemia B95 and Jhhan cell lines, as well as megakaryocytic leukemia M07e and Meg01 cell lines. Ouabain at 1 nmol/L or 10 nmol/L increased the expression of Na+, K+ -ATPase alpha1 subunit. There were significant differences in the proliferation and the expression of Na+, K+ -ATPase alpha1 subunit of the leukemia cell lines between the ouabain treatment and the blank control groups 24 hrs after ouabain treatment (P<0.05). The proliferation effect of leukemia cell lines was in a direct proportion with the ouabain concentration and incubation time.

CONCLUSIONSNa+, K+ -ATPase plays an important role in signal transductions. Through binding to ouabain, Na+, K+ -ATPase may regulate proliferation of leukemia cell lines of different origins. Ouabain at 1 nmol/L or 10 nmol/L may induce proliferation of lymphocytic leukemia cell lines (B95, Jhhan) and megakaryocytic leukemia cell lines (M07e, Meg01), and the proliferation effect was in a direct proportion with the concentration and incubation time of ouabain.

Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Leukemia ; pathology ; Ouabain ; pharmacology ; Signal Transduction ; Sodium-Potassium-Exchanging ATPase ; analysis ; genetics ; physiology

PURPOSE: The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), encoded by ATP2A2, is an essential component for G-protein coupled receptor (GPCR)-dependent Ca(2+) signaling. However, whether the changes in Ca(2+) signaling and Ca(2+) signaling proteins in parotid acinar cells are affected by a partial loss of SERCA2 are not known. MATERIALS AND METHODS: In SERCA2(+/-) mouse parotid gland acinar cells, Ca(2+) signaling, expression levels of Ca(2+) signaling proteins, and amylase secretion were investigated. RESULTS: SERCA2(+/-) mice showed decreased SERCA2 expression and an upregulation of the plasma membrane Ca(2+) ATPase. A partial loss of SERCA2 changed the expression level of 1, 4, 5-tris-inositolphosphate receptors (IP(3)Rs), but the localization and activities of IP3Rs were not altered. In SERCA2(+/-) mice, muscarinic stimulation resulted in greater amylase release, and the expression of synaptotagmin was increased compared to wild type mice. CONCLUSION: These results suggest that a partial loss of SERCA2 affects the expression and activity of Ca(2+) signaling proteins in the parotid gland acini, however, overall Ca(2+) signaling is unchanged.
Amylases/metabolism ; Animals ; Blotting, Western ; Calcium/metabolism ; Calcium Signaling/drug effects/genetics/*physiology ; Carbachol/pharmacology ; Immunohistochemistry ; Inositol 1,4,5-Trisphosphate Receptors/metabolism ; Mice ; Mice, Knockout ; Parotid Gland/*metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/genetics/*metabolism ; Signal Transduction/drug effects/genetics/physiology

Catalpol is the main active ingredient of an extract from Radix rehmanniae, which in a previous study showed a protective effect against various types of tissue injury. However, a protective effect of catalpol on uterine inflammation has not been reported. In this study, to investigate the protective mechanism of catalpol on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (bEECs) and mouse endometritis, in vitro and in vivo inflammation models were established. The Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway and its downstream inflammatory factors were detected by enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), western blot (WB), and immunofluorescence techniques. The results from ELISA and qRT-PCR showed that catalpol dose-dependently reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6, and chemokines such as C-X-C motif chemokine ligand 8 (CXCL8) and CXCL5, both in bEECs and in uterine tissue. From the experimental results of WB, qRT-PCR, and immunofluorescence, the expression of TLR4 and the phosphorylation of NF-κB p65 were markedly inhibited by catalpol compared with the LPS group. The inflammatory damage to the mouse uterus caused by LPS was greatly reduced and was accompanied by a decline in myeloperoxidase (MPO) activity. The results of this study suggest that catalpol can exert an anti-inflammatory impact on LPS-induced bEECs and mouse endometritis by inhibiting inflammation and activation of the TLR4/NF-κB signaling pathway.
Animals ; Cattle ; Chemokines/genetics* ; Cytokines/genetics* ; Endometritis/drug therapy* ; Epithelial Cells/drug effects* ; Female ; Inflammation/prevention & control* ; Iridoid Glucosides/therapeutic use* ; Lipopolysaccharides/pharmacology* ; Mice ; NF-kappa B/physiology* ; Signal Transduction/drug effects* ; Toll-Like Receptor 4/physiology*

Animals ; Calcineurin ; genetics ; physiology ; Cardiomegaly ; genetics ; physiopathology ; prevention & control ; Cyclosporine ; pharmacology ; therapeutic use ; Gene Expression ; Humans ; Immunosuppressive Agents ; pharmacology ; therapeutic use ; NFATC Transcription Factors ; genetics ; physiology ; Signal Transduction ; drug effects ; genetics ; physiology ; Tacrolimus ; pharmacology ; therapeutic use

Genetic Therapy ; Hepatitis B ; therapy ; virology ; Hepatocytes ; cytology ; Humans ; Interleukin-6 ; genetics ; physiology ; Liver Regeneration ; drug effects ; Receptors, Interleukin-6 ; genetics ; physiology ; Recombinant Fusion Proteins ; genetics ; physiology ; Signal Transduction

Hair follicle stem cells (FSCs), which have characteristics of slow cycling and multipotential differentiations, play an important role in maintaining hair follicle cycling and repairing the epidermis during wound healing. The researches in this subject may be helpful to provide new ideals in treating the diseases of hair and skin. As we know, the Wnt signal transduction pathway has a significant meaning in controlling the proliferation and differentiation of FSCs, and different Wnt proteins have different functions in this process. In this paper, we review how the Wnt signal transduction pathway controls the FSCs and what the functions of different Wnt proteins are.
Cell Differentiation ; drug effects ; Cell Proliferation ; Cells, Cultured ; Hair Follicle ; cytology ; Humans ; Signal Transduction ; Stem Cells ; cytology ; Wnt Proteins ; genetics ; physiology