tosylphenylalanyl-chloromethyl-ketone and Cell-Transformation--Neoplastic

N(alpha)-tosyl-L-phenylalanine chloromethylketone (TPCK) has been widely used to investigate signal transduction pathways that are involved in gene expression and cell survival/cell death. However, contradictory effects of TPCK on apoptosis have been reported, and the underlying signaling events leading to TPCK-induced promotion or prevention of apoptosis are not fully understood. Here, we show that TPCK induces caspase-dependent apoptosis in Epstein-Barr virus (EBV)-transformed human B cell lines with release of pro-apoptotic proteins from mitochondria. TPCK treatment also results in down-regulation of the anti-apoptotic proteins, cIAP1, cIAP2, and HAX-1, and caspase-dependent cleavage of the anti-apoptotic proteins, Bcl-2 and XIAP. Quantitative PCR analysis confirmed that the TPCK-induced down-regulation of HAX-1 occurred at the transcriptional level, and experiments using the specific pharmacological inhibitor, Bay 11-7082, suggested that HAX-1 expression is subject to regulation by the transcription factor, NF-kappaB. B cell lines derived from patients with homozygous HAX1 mutations were more sensitive to TPCK-induced apoptosis when compared with normal donor cell lines. Furthermore, N-acetylcysteine effectively blocked TPCK-induced apoptosis in EBV-transformed B cell lines and prevented the down-regulation or cleavage of anti-apoptotic proteins. Taken together, our studies demonstrate that TPCK induces apoptosis in human B cell lines and exerts multiple effects on pro- and anti-apoptotic factors.

Topics: Acetylcysteine; Adaptor Proteins, Signal Transducing; Antioxidants; Apoptosis; B-Lymphocytes; Baculoviral IAP Repeat-Containing 3 Protein; Caspases; Cell Line, Transformed; Cell Transformation, Neoplastic; Coumarins; Cysteine; Dose-Response Relationship, Drug; Down-Regulation; Fluorescent Dyes; Herpesvirus 4, Human; Humans; Inhibitor of Apoptosis Proteins; Jurkat Cells; Mitochondria; NF-kappa B; Oligopeptides; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Tosylphenylalanyl Chloromethyl Ketone; Transcription, Genetic; Ubiquitin-Protein Ligases; X-Linked Inhibitor of Apoptosis Protein

Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356-4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Complementary; Female; Fibroblasts; Genetic Vectors; Hepacivirus; Mice; Mice, Nude; Mutation; Phenylmethylsulfonyl Fluoride; Rats; Serine Proteinase Inhibitors; Tosylphenylalanyl Chloromethyl Ketone; Transfection; Viral Nonstructural Proteins

The human papillomaviruses (HPV) which are commonly found in anogenital malignancies express a viral E7 oncoprotein which possesses both immortalizing and transforming activities. The E7 protein forms a complex with the cellular tumor suppressor protein Rb and alters its interaction with transcription factor E2F, presumably interfering with cell cycle control and contributing to cellular transformation/immortalization. We demonstrated earlier that the serine protease inhibitors tosyl-L-phenylalanine chloromethyl ketone (TPCK) and tosyl-L-lysine chloromethyl ketone (TLCK) modified the HPV type 18 E7 protein in cell extracts as well as in living cells and abrogated its ability to form a complex with Rb. In the present study we evaluated the effect of TLCK or TPCK treatment on the immortalization of primary keratinocytes by transfected HPV-18 DNA. Supplementing the medium of primary foreskin keratinocytes with TLCK or TPCK during their immortalization with HPV 18 DNA demonstrated that either TLCK (5--10 microM) or TPCK (0.25 microM) could inhibit cellular immortalization by 50-100% without altering keratinocyte proliferation, suggesting that the topical application of these reagents in vivo could significantly interfere with HPV-mediated cellular transformation.

Topics: Cell Division; Cell Line; Cell Transformation, Neoplastic; Humans; Keratinocytes; Papillomaviridae; Serine Proteinase Inhibitors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone

In these studies, we performed experiments designed to elucidate the role that arachidonic acid metabolism plays in oncogenic transformation in vitro. The levels of TxB2 and 6-keto-PGF1 alpha were elevated in cells treated with X-rays. A significant increase in the levels of these eicosanoids was observed following irradiation. Treatment of cells with the anticarcinogenic protease inhibitors, Bowman-Birk Inhibitor (BBI) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), significantly reduced the levels of TxB2 and 6-keto-PGF1 alpha present. Indomethacin treatment significantly reduced the levels of TxB2 and 6-keto-PGF1 alpha to < 10% of those present in untreated or irradiated cells. We also report that addition of lipoxygenase or minoxidil [a selective inhibitor of prostacyclin (PGl2) synthetase] led to a highly significant decrease in transformation. In addition, minoxidil treatment resulted in a significant reduction in the levels of 6-keto-PGF1 alpha in irradiated cells. Our results suggest the hypothesis that the relative levels of 6-keto-PGF1 alpha are important in radiation induced transformation.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acids; Cell Line; Cell Transformation, Neoplastic; Indomethacin; Mice; Mice, Inbred C3H; Minoxidil; Models, Biological; Protease Inhibitors; Thromboxane B2; Tosylphenylalanyl Chloromethyl Ketone; Trypsin Inhibitor, Bowman-Birk Soybean; X-Rays

X-irradiation and the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act in a synergistic manner to increase the yield of transformed C3H10T1/2 cells in vitro. TPA modulated both translocation from the cytosol to the plasma membrane, and down regulation of protein kinase C (PKC) after prolonged (48 h) TPA exposure. N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), antipain, and soybean-derived Bowman-Birk inhibitor, protease inhibitors that suppress transformation of C3H10T1/2 cells, had no effect on these TPA-mediated alterations of PKC activity, suggesting that protease inhibitors suppress TPA-stimulated promotion in vitro via a PKC-independent pathway. Several experiments were performed to determine whether non-toxic concentrations of the PKC inhibitors, N-p-tosyl-L-lysine chloromethyl ketone (TLCK), TPCK, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), or 1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine (H-7), modulated the movement of cells from a quiescent state into the cell cycle. TPCK and the combination of H-7 and W-7 lowered DNA synthesis when cells were stimulated to divide by TPA. Because other protease inhibitors that slow transformation in vitro did not have the same suppressive effect on DNA synthesis, the inhibitory pathway that suppresses carcinogenic activity is likely to be different from the suppression of DNA synthesis.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Cytosol; DNA Replication; Isoquinolines; Kinetics; Mice; Mice, Inbred C3H; Piperazines; Protease Inhibitors; Protein Kinase C; Tetradecanoylphorbol Acetate; Thymidine; Time Factors; Tosylphenylalanyl Chloromethyl Ketone; X-Rays

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Carcinoma, Squamous Cell; Cell Transformation, Neoplastic; Cocarcinogenesis; Croton Oil; Fluocinolone Acetonide; Mice; Mice, Inbred Strains; Papilloma; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tosylphenylalanyl Chloromethyl Ketone; Tretinoin

Topics: alpha-Macroglobulins; Aminocaproates; Aprotinin; Cell Line; Cell Transformation, Neoplastic; Fibrinolysis; Fluorescent Antibody Technique; gamma-Glutamyltransferase; Membrane Proteins; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Trypsin Inhibitors

Virally transformed fibroblasts have on their surfaces zero or reduced amounts of a large external transformation-sensitive (LETS) glycoprotein. This protein is extremely sensitive to proteolysis. When prelabeled normal fibroblasts are cocultivated with transformed cells, the LETS glycoprotein of the normal cells shows an increased rate of turnover. Experiments are described which investigate the possibility that this phenomenon and the absence of LETS glycoprotein are due to proteolysis by the transformed cells. In particular, the role of plasminogen activation is examined by the use of protease inhibitors and plasminogen-depleted serum. It is concluded that activation of plasminogen is not required for the disappearance of the LETS glycoprotein although the involvement of other proteases cannot be ruled out. The role of proteases in affecting cell growth and behavior is discussed.

Topics: Aminocaproates; Blood; Cell Division; Cell Line; Cell Transformation, Neoplastic; Fibrinolysis; Glycoproteins; Ovomucin; Peptide Hydrolases; Plasminogen; Tosylarginine Methyl Ester; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Trypsin Inhibitors

Topics: Cell Adhesion; Cell Line; Cell Membrane; Cell Transformation, Neoplastic; Kinetics; Peptide Hydrolases; Phenylmethylsulfonyl Fluoride; Polyomavirus; Time Factors; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Trypsin

Topics: Binding Sites; Cell Adhesion; Cell Aggregation; Cell Division; Cell Membrane; Cell Transformation, Neoplastic; Cyclic AMP; Cyclic GMP; Enzyme Inhibitors; Glucose; Glycosaminoglycans; Lectins; Models, Biological; Peptide Hydrolases; Phenotype; Plasminogen; Protease Inhibitors; Proteins; Thrombin; Tosyllysine Chloromethyl Ketone; Tosylphenylalanyl Chloromethyl Ketone; Trypsin Inhibitors; Viruses