leupeptins and Cell-Transformation--Viral

The purpose of this study was to determine the mechanism by which 1α, 25-dihydroxyvitamin D (1,25(OH)(2)D) alters hypoxia-inducible factor-1α (HIF-1α) protein in untransformed and Harvey-ras (H-ras) oncogene transfected MCF10A breast epithelial cells. Treatment with 1,25(OH)(2)D (10nM) increased both mRNA (2.55±0.6-fold vs. vehicle, p=0.03) and protein levels (2.37±0.3-fold vs. vehicle, p<0.0001) of HIF-1α in MCF10A cells in 12h, which remained elevated at 24h. However, in H-ras transfected MCF10A cells, 1,25(OH)(2)D treatment increased HIF-1α protein level (2.08±0.38-fold vs. vehicle, p=0.05) at 12h, with no change in mRNA level and HIF-1α protein level returned to baseline after 24h. A transcription inhibitor prevented the 1,25(OH)(2)D induction of HIF-1α protein and mRNA levels in MCF10A cells, but failed to alter the induction of HIF-1α protein level in H-ras transfected MCF10A cells. On the other hand, inhibition of proteasomal degradation prevented the 1,25(OH)(2)D-induced HIF-1α protein level in H-ras transfected MCF10A but not in MCF10A cells. These results support that 1,25(OH)(2)D regulates HIF-1α protein level via transcriptional regulation in MCF10A cells in contrast to through proteosomal degradation with the presence of H-ras oncogene in MCF10A cells.

Topics: Blotting, Western; Calcitriol; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Proteinase Inhibitors; Dactinomycin; Gene Expression Regulation; Genes, ras; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Protein Synthesis Inhibitors; Receptors, Calcitriol; Reverse Transcriptase Polymerase Chain Reaction; Transfection; Vitamins

BPV-4 E5 inhibits transcription of the bovine MHC class I heavy chain (HC) gene, increases degradation of HC and downregulates surface expression of MHC class I by retaining the complex in the Golgi apparatus (GA). Here we report that transcription inhibition can be alleviated by interferon treatment and the degradation of HC can be reversed by treatment with inhibitors of proteasomes and lysosomes. However, the inhibition of transport of MHC class I to the cell surface is irreversible. We show that E5 is capable of physically interacting with HC. Together with the inhibition of the vacuolar ATPase (due to the interaction between E5 and 16k subunit c), the interaction between E5 and HC is likely to be responsible for retention of MHC class I in the GA. C-terminus deletion mutants of E5 are incapable of either downregulating surface MHC class I or interacting with HC, establishing that the C-terminus domain of E5 is important in the inhibition of MHC class I.

Topics: Animals; Antiviral Agents; Bovine papillomavirus 1; Bovine papillomavirus 4; Cattle; Cell Transformation, Viral; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Inhibitors; Fetus; Golgi Apparatus; Histocompatibility Antigens Class I; Immunoprecipitation; Interferon-beta; Interferon-gamma; Leupeptins; Macrolides; Oncogene Proteins, Viral; Protein Biosynthesis; Protein-Tyrosine Kinases; Sequence Deletion; Transcription, Genetic; Vacuolar Proton-Translocating ATPases

Down-regulation of interleukin (IL)-6-type cytokine signaling has been shown to occur, among other mechanisms, via induction of the feedback inhibitor SOCS3 (suppressor of cytokine signaling 3). Binding of SOCS3 to the phosphorylated Tyr(759) in the cytoplasmic region of gp130, the common signal transducing receptor chain of all IL-6-type cytokines, is necessary for inhibition of Janus kinase-mediated signaling. In the present study, we analyzed the effect of SOCS3 on signal transduction by the proinflammatory cytokine oncostatin M (OSM), which signals through a receptor complex of gp130 and the OSM receptor (OSMR). OSM leads to a much stronger and prolonged induction of SOCS3 in HepG2 hepatoma cells and murine embryonal fibroblasts (MEF) compared with IL-6. A negative effect of SOCS3 on OSM signaling was confirmed using MEF cells lacking SOCS3. We can show that the OSMR-mediated signaling is inhibited by SOCS3 to a similar extent as previously described for gp130. However, the inhibition occurs independent of tyrosine motifs within the OSMR. Instead, SOCS3 interacts directly with JAK1 in a stimulation-dependent manner, a mechanism so far only known for SOCS1.

Topics: Animals; Antineoplastic Agents; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cytokine Receptor gp130; Cytokines; Fibroblasts; Gene Expression Regulation; Genes, Reporter; Humans; Interleukin-6; Janus Kinase 1; Leupeptins; Liver Neoplasms; Luciferases; Mice; Oncostatin M; Precipitin Tests; Protein Binding; Protein-Tyrosine Kinases; Receptors, Amino Acid; Receptors, Cytokine; Receptors, Oncostatin M; Signal Transduction; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.

Topics: Arsenites; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cycloheximide; Enzyme Inhibitors; Human T-lymphotropic virus 1; Humans; I-kappa B Kinase; Leukemia-Lymphoma, Adult T-Cell; Leupeptins; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; T-Lymphocytes

The human herpes virus 8 (HHV8)-encoded viral FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme) inhibitory protein (vFLIP) is believed to protect cells against death receptor-mediated apoptosis. In the present study we demonstrate that expression of HHV8 vFLIP in a growth factor-dependent TF-1 leukemia cell line protects against growth factor withdrawal-induced apoptosis. Unlike vector-expressing cells, those expressing HHV8 vFLIP maintain their mitochondrial membrane potential upon withdrawal from growth factor and also exhibit a block in the activation of caspases. The protective effect of HHV8 vFLIP is associated with its ability to activate the nuclear factor-kappa B (NF-kappaB) pathway and is missing in the vFLIP encoded by equine herpes virus 2 that lacks this activity. Inhibition of the NF-kappaB pathway by IkappaB superrepressor, lactacystin, MG132, arsenic trioxide, and phenylarsine oxide reverse the protection against growth factor withdrawal-induced apoptosis conferred by HHV8 vFLIP. HHV8 vFLIP up-regulates the expression of Bcl-x(L), an antiapoptotic member of the Bcl2 family, which is a known target of the NF-kappaB pathway. Collectively, the above results suggest that HHV8 vFLIP-induced NF-kappaB activation may contribute to cellular transformation seen in association with HHV8 infection by preventing the apoptosis of cells destined to die because of growth factor deprivation.

Topics: Acetylcysteine; Acute Disease; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-X Protein; Cell Transformation, Viral; Enzyme Activation; Gene Expression Regulation, Viral; Granulocyte-Macrophage Colony-Stimulating Factor; Herpesvirus 8, Human; Humans; I-kappa B Proteins; Leukemia, Myeloid; Leupeptins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; NF-kappa B; Oxides; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Recombinant Proteins; Rhadinovirus; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Viral Proteins

Murine erythroblasts infected with the anemia-inducing strain of Friend virus (FVA cells) terminally differentiate to the reticulocyte stage after 48 hours of culture in vitro in response to erythropoietin (EPO). The objective of this study was to determine the possible role of proteasome-mediated proteolysis during the terminal differentiation of FVA cells.. The proteasome inhibitors MG132 and lactacystin were used to perturb the normal function of proteasomes during terminal differentiation. Effects of proteasome inhibitors on terminal differentiation were quantitated by evaluation of cellular morphology after benzidine staining and by Western blot analyses.. Treatment of EPO-stimulated FVA cells with lactacystin or MG132 at later periods of culture increased accumulations of nuclear and cytosolic ubiquitinated proteins and decreased nuclear extrusion to less than 40% of controls.. Our results suggest that the proteasomal degradation of ubiquitinated proteins plays an important role in the enucleation of mammalian erythroblasts.

Topics: Acetylcysteine; Animals; Blotting, Western; Cell Differentiation; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Endopeptidases; Cytoplasm; Erythroid Precursor Cells; Erythropoiesis; Friend murine leukemia virus; Leupeptins; Mice; Multienzyme Complexes; Nuclear Proteins; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Reticulocytes; Ubiquitin

High-risk human papillomaviruses (HPVs) are etiologically linked to human cervical and oral cancers. The E6 and E7 oncoproteins encoded by HPV target host cell tumor suppressor proteins. E6 induces proteolysis of p53 through the ubiquitin-proteasome pathway. Recent studies showed that overexpression of E7 caused proteolytic degradation of the tumor suppressor Rb. However, unlike p53, Rb is not regulated by proteolysis in normal cells. In addition, it was unclear whether in its natural context E7 regulates Rb through the ubiquitin-proteasome pathway. Therefore, we sought to determine whether Rb is regulated by the ubiquitin-proteasome pathway in HPV-containing tumor cells. We carried out a detailed analysis in Caski cells, that are derived from HPV-containing cervical cancer tissues. Studies with various protease inhibitors revealed that Rb is regulated specifically by the ubiquitin-proteasome pathway in HPV-containing cervical tumor cells. Several inhibitors of the 26S proteasome significantly increased the level of Rb in the Caski cells. Rb controls cell growth by forming complexes with the E2F-family transcription factors. Surprisingly, in spite of a significant accumulation of the hypophosphorylated form of Rb, no Rb/E2F complex was detectable in the proteasome inhibitor treated cells. Further analysis revealed that there was an increased accumulation of the E7 oncoprotein. We showed that the proteasome inhibitors simultaneously blocked the proteolysis of E7 and Rb, suggesting that E7 is also regulated by the ubiquitin-dependent proteolysis in cervical cancer cells. Taken together, this study suggests that targeted inhibition of Rb proteolysis will be required for restoring Rb function in HPV-containing cervical cancer cells.

Topics: Carcinoma; Cell Cycle Proteins; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; E2F Transcription Factors; Female; Humans; Leupeptins; Multienzyme Complexes; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus E7 Proteins; Phosphorylation; Proteasome Endopeptidase Complex; Retinoblastoma Protein; Transcription Factors; Ubiquitins; Uterine Cervical Neoplasms

Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is induced in response to IL-2 but as yet its function has not been determined. The results of this study show that DUB-2 is expressed in human T-cell lymphotropic virus-I (HTLV-1)-transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB-2 markedly prolonged IL-2-induced STAT5 phosphorylation. Although DUB-2 did not enhance IL-2-mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2-induced genes cis and c-myc. Moreover, DUB-2 expression markedly inhibited apoptosis induced by cytokine withdrawal allowing cells to survive. Taken together these data suggest that DUB-2 can enhance signaling through the JAK/STAT pathway, prolong lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells.

Topics: Apoptosis; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Endopeptidases; DNA-Binding Proteins; Endopeptidases; Human T-lymphotropic virus 1; Humans; Immediate-Early Proteins; Interleukin-2; Leukemia-Lymphoma, Adult T-Cell; Leupeptins; Milk Proteins; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Signal Transduction; STAT5 Transcription Factor; T-Lymphocytes; Trans-Activators; Transcriptional Activation; Transfection; Ubiquitins

Previous studies in this laboratory have quantified the fibrinolytic activity of herpesvirus-transformed cell lines and implicated the proteolytic capacity of cloned cell lines in the formation of primary and metastatic tumours. Because of the involvement of proteases in tumourigenesis in this system, we examined the effect of various protease inhibitors (alpha-1-antitrypsin, leupeptin and alpha-2-macroglobulin), as well as hamster serum containing acute phase proteins generated in response to physiological trauma, on the fibrinolytic capacity of a herpes simplex virus type 2-transformed hamster cell line. The effects of the various inhibitors on cell growth, fibrinolysis in vitro and tumourigenesis were examined. Leupeptin, alpha-1-antitrypsin, alpha-2-macroglobulin and hamster serum containing acute phase proteins were capable of inhibiting fibrinolysis in vitro, were not toxic to the cells and their action was reversible, while rejection of a mixture of protease inhibitor and transformed cells resulted in delayed tumour development.

Topics: alpha 1-Antitrypsin; alpha-Macroglobulins; Animals; Blood Proteins; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cricetinae; Fibrinolysis; Leupeptins; Neoplasms, Experimental; Protease Inhibitors; Simplexvirus

Topics: Animals; Antipain; Cell Transformation, Viral; Cells, Cultured; Idoxuridine; Leupeptins; Mice; Oligopeptides; Protease Inhibitors; Retroviridae; Virus Replication