alpha-chymotrypsin and Lymphoma

During recent years major advances have been made in our understanding of glucocorticoid mechanism of action. This progress has been made possible by access to purified glucocorticoid receptor in significant amounts as well as by application of hybrid DNA technology within the field of glucocorticoid control of gene expression. Especially the mammary tumour virus genome has turned out to be a convenient experimental system suitable for such investigations. This paper summarizes some of the work carried out in our own laboratory, partially in collaboration with Dr Keith Yamamoto and his associates at the Department of Biochemistry and Biophysics, University of California, San Francisco, U.S.A.

Topics: Animals; Antibodies, Monoclonal; Binding Sites; Chymotrypsin; DNA; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glucocorticoids; Liver; Lymphoma; Mammary Tumor Virus, Mouse; Mice; Rabbits; Rats; Receptors, Glucocorticoid; Receptors, Steroid; Trypsin

Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells.. We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed.. We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo.. These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bone Marrow Cells; Boronic Acids; Bortezomib; Caspases; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Chemokine CXCL12; Chymotrypsin; Coculture Techniques; DNA Fragmentation; Drug Synergism; Enzyme Activation; Female; Humans; JNK Mitogen-Activated Protein Kinases; Lymphoma; Mice; Mice, SCID; Oligopeptides; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Proteasome Inhibitors; Pyrazines; Serine Proteinase Inhibitors; Unfolded Protein Response; Waldenstrom Macroglobulinemia

Proteasome inhibition represents a valid antitumor approach and its use has been validated in Waldenström macroglobulinemia (WM), where bortezomib has been successfully tested in clinical trials. Nevertheless, a significant fraction of patients relapses, and many present toxicity due to its off-target effects. Selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive proteasome 20S (c20S) and immunoproteasome 20S (i20S) represents a sufficient and successful strategy to induce antineoplastic effect in hematologic tumors. We therefore studied ONX0912, a novel selective, irreversible inhibitor of the CT-L activity of i20S and c20S. Primary WM cells express higher level of i20S compared with c20S, and that ONX0912 inhibited the CT-L activity of both i20S and c20S, leading to induction of toxicity in primary WM cells, as well as of apoptosis through c-Jun N-terminal kinase activation, nuclear factor kappaB (NF-kappaB) inhibition, caspase cleavage, and initiation of the unfolded protein response. Importantly, ONX0912 exerted toxicity in WM cells, by reducing bone marrow (BM)-derived interleukin-6 (IL-6) and insulin-like growth factor 1 (IGF-1) secretion, thus inhibiting BM-induced p-Akt and phosphorylated extracellular signal-related kinase (p-ERK) activation in WM cells. These findings suggest that targeting i20S and c20S CT-L activity by ONX0912 represents a valid antitumor therapy in WM.

Topics: Apoptosis; Chymotrypsin; Dipeptides; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Humans; Immunoblotting; Insulin-Like Growth Factor I; Interleukin-6; JNK Mitogen-Activated Protein Kinases; Lymphoma; NF-kappa B; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Waldenstrom Macroglobulinemia

Systemic enzymes (wobenzime and wobe-mugos) were approved, that had been prescribed as part of polychemotherapy to 24 patients with malignant lymphomas, who presented with large masses of lymphatic nodes poorly resorbable against the background of polychemotherapy and who were assigned for a high-total dose irradiation, which fact would undoubtedly entail sclerosing of adjacent intact tissues. The use of the enzymes made for the optimum realization of the polychemotherapy effect and permitted avoiding a good many of undesirable side events and complication thereof. The above systemic enzymes are well tolerated by patients; they induce no significant changes in the cellular composition or in blood biochemical indices.

Topics: Adjuvants, Immunologic; Adolescent; Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Blood; Chymotrypsin; Combined Modality Therapy; Drug Combinations; Drug Evaluation; Female; Hodgkin Disease; Humans; Hydrolases; Lymphoma; Male; Middle Aged; Pancreatic Extracts; Papain; Recurrence; Rutin; Thymus Extracts; Trypsin

To investigate the mechanism of glucocorticoid-induced lymphocytolysis, we used two-dimensional gel electrophoresis to analyze the effect of dexamethasone on the synthesis of individual proteins in S49 mouse lymphoma cells. We found that synthesis of a 78-Kd protein is preferentially maintained following dexamethasone treatment, at a time when the synthesis of most other cellular proteins is decreased. Synthesis of this protein could also be induced by tunicamycin, suggesting that it might be the 78-Kd glucose-regulated protein (GRP78). The identity of the 78-Kd protein with GRP78 was confirmed by limited chymotryptic mapping and immunoprecipitation analysis. Preferential synthesis of GRP78 in dexamethasone-treated cells was not secondary to alterations in the glycosylation of cellular proteins. Significantly, steady-state levels of GRP78 mRNA were unchanged following dexamethasone treatment. Preferential synthesis of GRP78 in glucocorticoid-treated S49 cells may reflect the unique property of GRP78 mRNA to be translated under conditions that interfere with the translation of most other cellular mRNAs. GRP78 is a highly conserved protein that is essential for cell viability. Preferential synthesis of GRP78 may be a protective response to metabolic events that interfere with normal mRNA translation in glucocorticoid-treated mouse lymphoma cells.

Topics: Animals; Autoradiography; Carrier Proteins; Chymotrypsin; Dexamethasone; Electrophoresis, Gel, Two-Dimensional; Endoplasmic Reticulum Chaperone BiP; Glycosylation; Heat-Shock Proteins; Immunoglobulin Heavy Chains; Lymphoma; Mice; Mice, Inbred BALB C; Molecular Chaperones; Peptide Mapping; RNA, Messenger; Tumor Cells, Cultured; Tunicamycin

We characterized the glucocorticoid receptor fragments produced by neutrophil elastase and compared these receptor fragments to nuclear transfer increased (nti) mutant receptors. Neutrophil elastase and chymotrypsin digested [3H]dexamethasone 21-mesylate labeled receptors at different sites to produce 52 kDa and 42 kDa fragments respectively. Both the 52 kDa elastolytic receptor fragments and 42 kDa chymotryptic receptor fragments bound to DNA-cellulose and were immunoadsorbed by anti-glucocorticoid receptor monoclonal antibodies (BUGR2). More extensive digestion of labeled receptors by neutrophil elastase produced 29 kDa receptor fragments that did not bind to DNA-cellulose and did not react with BUGR2 antibodies. The size of nti mutant receptors from S49 mouse lymphoma cell variants is intermediate between that of the 52 kDa elastolytic receptor fragments and 42 kDa chymotryptic receptor fragments. The nti receptors bound to DNA-cellulose with the same affinity as the 52 kDa elastolytic receptor fragments. However, the nti receptors were not immunoadsorbed by BUGR2 antibodies and did not react with these antibodies on Western blot analysis of denatured cellular proteins. The results indicate that 52 kDa elastolytic receptor fragments, 42 kDa chymotryptic receptor fragments and nti mutant receptors correspond to the same region of the receptor molecule. The failure of nti receptors to react with BUGR2 antibodies suggests that the nti receptors may have an altered sequence compared to the corresponding region of normal receptors.

Topics: Animals; Antibodies, Monoclonal; Chromatography, Affinity; Chymotrypsin; Lymphoma; Mice; Neoplasm Proteins; Neutrophils; Pancreatic Elastase; Peptide Fragments; Receptors, Glucocorticoid

Glucocorticoid receptors in wild type and mutant S49 mouse lymphoma cells were affinity labeled with [3H]dexamethasone 21-mesylate and analyzed directly by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of receptors in cytosol from wild type cells and nuclear transfer decreased (nt-) mutants was 97,000 (97 kDa). The molecular weight of receptors in cytosol from nuclear transfer increased (nti) mutants was 48 kDa. The 97 kDa receptor in cytosol from wild type cells was digested by chymotrypsin to a 40 kDa steroid-binding receptor fragment but the 48 kDa receptor in cytosol from nti mutants was resistant to digestion by chymotrypsin. In addition to the 48 kDa receptor, cytosol from nti mutants contained 40 and 18 kDa receptor fragments. Cytosol from the nt- mutants also contained 18 kDa receptor fragments. The 40 and 18 kDa receptor fragments were present in multiple subclones of a nti mutant cell line. Formation of these receptor fragments was not prevented by protease inhibitors and was not increased by extended incubation of cytosol samples. Both 48 and 40 kDa forms of the receptor, but not the 18 kDa form, could be activated and bound by DNA-cellulose.

Topics: Affinity Labels; Animals; Cell Line; Chymotrypsin; Dexamethasone; DNA; DNA-Binding Proteins; Lymphoma; Mice; Molecular Weight; Mutation; Peptide Fragments; Receptors, Glucocorticoid

A retrospective study of 76 primary gastrointestinal lymphomas utilizing an avidin: biotinylated horseradish peroxidase complex (ABC) technique demonstrated 22 B-cell lymphomas, including two associated with alpha-heavy chain disease. Seven cases were classified as true histiocytic lymphomas based on a positive reaction for one or more of three histiocytic enzyme markers utilized, predominantly alpha-1-antitrypsin and alpha-1-antichymotrypsin. However, in 20 cases, an intense admixture of reactive histiocytes was noted and these cells stained preferentially for the enzyme, lysozyme. Twenty cases, which stained for both kappa and lambda light chains and positively or negatively for albumin, could not be classified and 27 cases failed to stain with any of the antisera utilized.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Antibodies; B-Lymphocytes; Chymotrypsin; Gastrointestinal Neoplasms; Heavy Chain Disease; Histocytochemistry; Humans; Immunoenzyme Techniques; Immunoglobulin alpha-Chains; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Lymphoma; Lymphoma, Large B-Cell, Diffuse; Muramidase

Histological material was studied in five unselected cases of intestinal large-cell non-Hodgkin's lymphoma, occurring in patients either with previously diagnosed coeliac disease, or with atrophic mucosa at the time of diagnosis. The morphological diagnosis in each case was centroblastic lymphoma: these tumours were composed of large cells with pale nuclei and prominent nucleoli. No phagocytosis was evident, but some cells showed considerable pleomorphism. Polykaryotic giant cells were infrequent. Immunohistochemical staining for lysozyme, alpha-1-anti-trypsin and alpha-1-anti-chymotrypsin failed to demonstrate any of these proteins in the tumour cells, although they were identified in accompanying reactive macrophages. There is thus no evidence for a histiocytic nature in these five cases. The tumours were immunoglobulin-negative. Again, polyclonal immunoglobulin could be demonstrated in reactive (plasma) cells in and near the tumour. The relevance of these immunological markers is discussed. We suggest that these tumours, and possibly some of those reported in a similar situation by other investigators, are in fact lymphocytic in origin. They are probably examples of centroblastic lymphoma, although T-cell lymphoma, rare in the gastrointestinal tract, cannot be ruled out by our immunohistological studies.

Topics: Adult; Aged; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Celiac Disease; Cell Nucleolus; Cell Nucleus; Chymotrypsin; Histocytochemistry; Humans; Intestinal Neoplasms; Lymphocytes; Lymphoma; Macrophages; Male; Middle Aged; Muramidase; Plasma Cells

The major cAMP-binding proteins isolated from [35S]methionine-labeled S49 mouse lymphoma cells or MDBK bovine kidney cells correspond in isoelectric point and apparent molecular weight to the regulatory subunit (R) of type I cAMP-dependent protein kinase. These proteins were compared directly by two-dimensional gel electrophoresis and by two-dimensional gel electrophoresis of peptides generated either from native R with thermolysin and chymotrypsin or from denatured R with papain. Both the undigested proteins and all their major peptides were identical in charge and apparent molecular weights, indicating a very high degree of structural homology.

Topics: Animals; Cattle; Cell Line; Chymotrypsin; Cyclic AMP; Electrophoresis, Polyacrylamide Gel; Isoelectric Focusing; Isoelectric Point; Kidney; Lymphoma; Mice; Molecular Weight; Papain; Peptide Fragments; Protein Kinases; Species Specificity; Thermolysin

8-Carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H )-one- mitozolomide (CCRG 81010, M & B 39565, NSC 353451) is a potent inhibitor of the growth of a number of experimental tumours and can potentially decompose to give either an isocyanate or the monochloroethyltriazene (MCTIC). In vitro CCRG 81010 is not cross-resistant with the bifunctional alkylating agents against the Walker carcinoma. To investigate the mechanism of the antitumour activity of CCRG 81010 a comparison has been made with BCNU and MCTIC on precursor incorporation into macromolecules in TLX5 mouse lymphoma cells. Whereas BCNU produces a rapid and extensive inhibition of both (methyl 3H) thymidine and [5-3H]uridine incorporation into acid-insoluble material, neither CCRG 81010 or MCTIC have an early effect on precursor incorporation. Inhibition of precursor uptake is also not produced by concentrations of 2-chloroethylisocyanate that inhibit intracellular glutathione reductase activity. The potential carbamoylating activity of CCRG 81010 has also been assessed by comparing its effect with that of BCNU and 2-chloroethyl isocyanate on enzymes known to be inhibited by carbamoylation. Such enzymes, glutathione reductase, chymotrypsin and gamma-glutamyltranspepidase are not inhibited by CCRG 81010 under conditions where BCNU and 2-chloroethyl isocyanate show complete inhibition of enzyme activity, suggesting an absence of carbamoylating species. The results suggest that the most likely antitumour metabonate produced from CCRG 81010 is the triazene MCTIC.

Topics: Adenosine; Aminoimidazole Carboxamide; Animals; Antineoplastic Agents; Carcinoma 256, Walker; Carmustine; Cells, Cultured; Chymotrypsin; DNA, Neoplasm; gamma-Glutamyltransferase; Glutathione Reductase; Imidazoles; Lymphoma; Mice; Nitrogen Mustard Compounds; Thymidine; Uridine

Glucocorticoid-resistant (CR), in contrast to glucocorticoid-sensitive (CS), mouse lymphoma P1798 was shown to lack antiglucocorticoid receptor immunoactivity. Antibodies raised against the purified rat liver glucocorticoid receptor (GR) cross-reacted with the GR from CS, but not with the GR from CR, P1798 lymphoma. Using highly specific antisera against the GR in an indirect competitive enzyme-linked immunosorbent assay, it was demonstrated that alpha-chymotrypsin digestion of the GR from CS P1798 lymphoma caused a separation of a "resistant-like" nonimmunogenic steroid and DNA-binding domain (Stokes' radius, 3.3 nm) from an immunoactive domain (Stokes' radius, 2.6 nm). In contrast to CS P1798 lymphoma, neither before nor after alpha-chymotrypsin digestion, immunoactivity could be found in the cytosol from CR P1798 lymphoma. This was assayed after chromatography on DNA-cellulose or gel filtration on Agarose A (0.5 m). These results suggest that the domain of the CS GR containing the immunoactive determinant(s), normally removed by limited proteolysis by alpha-chymotrypsin, appears to be missing in CR P1798 lymphoma cytosol. It seems that this domain plays an important role in the mechanism of action of glucocorticoids. This might suggest that a mutation has occurred affecting the genome resulting in defective transcription of the receptor gene(s) in CR P1798 lymphoma.

Topics: Animals; Chromatography, Affinity; Chymotrypsin; Cross Reactions; Cytosol; DNA; Enzyme-Linked Immunosorbent Assay; Epitopes; Lymphoma; Mice; Mutation; Neoplasms, Experimental; Receptors, Glucocorticoid; Receptors, Steroid

Glucocorticoid receptors of wild-type mouse lymphoma cells and two glucocorticoid resistant variants of nt- and nti phenotypes, respectively, were investigated. Photoaffinity labelling of receptor complexes with a radiolabelled glucocorticoid of high affinity enabled us to analyse crude receptor preparations by SDS gel electrophoresis. Wild-type and nt- receptors yielded radiolabelled polypeptide bands of 98,000 mol. wt while nti receptors had a mol. wt of 42,000. Monoclonal antibodies raised against purified rat liver glucocorticoid receptors reacted with wild-type and nt- receptors but not with nti receptors. Partial proteolysis of wild-type receptors with alpha-chymotrypsin resulted in a fragment of 39,000 mol. wt which contained the steroid binding site but had increased affinity for DNA indistinguishable from nti receptors. Mild proteolysis with trypsin yielded smaller fragments which contained the steroid binding site but did not bind to DNA. A model of the wild-type receptor is discussed.

Topics: Animals; Chromatography, Affinity; Chymotrypsin; Epitopes; Lymphoma; Mice; Molecular Weight; Mutation; Neoplasms, Experimental; Peptide Fragments; Receptors, Glucocorticoid; Receptors, Steroid

Topics: Adenylyl Cyclases; Animals; Brain; Cattle; Cell Membrane; Chymotrypsin; Cytosol; GTP-Binding Proteins; Kidney; Kinetics; Liver; Lymphoma; Male; Mice; Neoplasms, Experimental; Organ Specificity; Rats; Receptors, Cell Surface; Sheep; Species Specificity; Spermatozoa; Testis

In cultured lines of mouse lymphoma cells, resistant to glucocorticoids is frequently associated with the occurrence of glucocorticoid receptors with an abnormally low affinity (nt-) or an abnormally high affinity (nti) for nuclei and DNA. We have investigated whether the abnormal affinities for DNA are correlated with alterations in charge and surface properties of the receptors, that would be revealed through the partition coefficient in aqueous dextran/poly(ethylene glycol) two-phase systems. We have found that none of the receptor variants is defective in the activation step per se, and that only the nti receptors are abnormal in partition properties. Partial proteolysis of wild-type and nt- receptors with alpha-chymotrypsin produces forms which are indistinguishable from the nti receptors with respect to partition coefficients. Upon alpha-chymotrypsin treatment the wild-type receptors attain DNA-binding properties identical to those of the nti receptors, while the nt- receptors, in spite of some increase in DNA affinity, still bind less firmly to DNA than the alpha-chymotrypsin-treated wild-type receptors. alpha-Chymotrypsin treatment of the various receptor types also produces an increase in the binding to dextran sulphate, but the dextran sulphate affinity is higher and varies less between different receptor types than the DNA affinity. Trypsin-treated receptors were found to be devoid of affinity for DNA and dextran sulphate.

Topics: Animals; Cell Line; Chymotrypsin; Cytosol; Dextran Sulfate; Dextrans; DNA; Hot Temperature; Lymphoma; Mice; Neoplasms, Experimental; Receptors, Glucocorticoid; Receptors, Steroid; Solubility; Trypsin

Topics: Animals; Cell Nucleus; Chromatography, Affinity; Chymotrypsin; DNA; Lymphoma; Mice; Neoplasms, Experimental; Peptide Fragments; Receptors, Glucocorticoid; Receptors, Steroid; Trypsin

Topics: Adenylyl Cyclases; Animals; Binding Sites; Cell Line; Cell Membrane; Cholera Toxin; Chymotrypsin; Cytidine Triphosphate; Cytosine Nucleotides; Enzyme Activation; Female; Fluorides; Guanylyl Imidodiphosphate; Kinetics; Liver; Lymphoma; Neoplasms, Experimental; Papain; Protein Binding; Rats

Vertical studies indicate that, in general, acute phase reactant proteins (APRP) reflect disease activity in both Hodgkin's disease and non-Hodgkin's lymphoma. Longitudinal studies of the selected APRP profile demonstrate the following: 1. The stable profile is characteristic of remission. 2. Considerable elevation of APRPs coincides with relapsed disease. 3. An unstable profile is a feature of relapsing disease and may give early warning of relapse. 4. Patients responding inadequately to treatment frequently have unstable APRP profiles.

Topics: Adult; alpha 1-Antitrypsin; C-Reactive Protein; Chymotrypsin; Female; Glycoproteins; Hodgkin Disease; Humans; Lymphoma; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Non-Hodgkin; Male; Middle Aged; Orosomucoid