leupeptins and Hodgkin-Disease

The proteasome, which plays a pivotal role in the control of many cell cycle-regulatory processes, has become the focus of new approaches to the treatment of cancer, including B-cell malignancies, and the first proteasome inhibitor, bortezomib (VELCADE; formerly PS-341), has entered clinical trials. The proteasome controls the stability of numerous proteins that regulate progression through the cell cycle and apoptosis, such as cyclins, cyclin-dependent kinases, tumor suppressors, and the nuclear factor-kB. By altering the stability or activity of these proteins, proteasome inhibitors sensitize malignant cells to apoptosis. Bortezomib is a dipeptidyl boronic acid proteasome inhibitor that effectively and specifically inhibits proteasome activity. In preclinical studies, bortezomib and other proteasome inhibitors have shown activity against a variety of B-cell malignancies, including multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, and Hodgkin's lymphoma. These agents can induce apoptosis and sensitize tumor cells to radiation or chemotherapy. Based on these findings, phase I clinical trials were conducted with bortezomib in various solid and hematologic malignancies. In these studies, bortezomib was generally well tolerated with manageable toxicities. Phase II trials have been initiated for relapsed and refractory multiple myeloma, refractory chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Preliminary data from the multiple myeloma phase II study indicate that a significant number of patients responded to therapy or exhibited stable disease and that the drug had manageable toxicities. These findings, along with extensive preclinical data, suggest that bortezomib and other proteasome inhibitors may have far-reaching potential in the treatment of various cancers, including B-cell malignancies.

Topics: Acetylcysteine; Animals; Boronic Acids; Bortezomib; Cell Cycle Proteins; Clinical Trials as Topic; Drug Screening Assays, Antitumor; Enzymes; Gene Expression Regulation; Hodgkin Disease; Humans; Leukemia, B-Cell; Leupeptins; Lymphoma, B-Cell; Lymphoma, Large B-Cell, Diffuse; Lymphoma, Mantle-Cell; Mice; Multiple Myeloma; Neoplasm Proteins; NF-kappa B; Oncogene Proteins; Peptide Hydrolases; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Substrate Specificity; Transcription Factors; Treatment Outcome

Topics: Chromosomes, Human, Pair 8; Genes, myc; Genetic Loci; Genetic Predisposition to Disease; Hodgkin Disease; Humans; Leupeptins; Polymorphism, Single Nucleotide; Prognosis; RNA, Long Noncoding; Survival Analysis

About 30-50% of Hodgkin lymphomas (HLs) harbor the Epstein-Barr virus (EBV), but the impact of EBV infection on clinical outcomes has been unclear. EBV-encoded small RNAs (EBERs) are presented in all EBV-infected cells, but their functions are still less understood.. EBER1 was transfected into two HL cell lines, KMH2 and L428, and microarrays were used to screen for EBER1-induced changes. We found that EBER1 suppressed p21cip1/waf1 transcription in HL cell lines. In addition, positive regulators of p21cip1/waf1 transcription, such as p53, EGR1, and STAT1, were decreased. Suppression of p21cip1/waf1 in the EBER1+ HL cell lines was associated with increased resistance to histone deacetylase inhibitors or proteasome inhibitors, drugs known to cause apoptosis by increasing p21cip1/waf1 levels. On biopsy specimens, EBV+ HLs had weaker expression of both p21cip1/waf1 and active caspase 3. Clinically, suppression of p21cip1/waf1 in EBV+ HLs was associated with a worse 2-year disease-free survival rate (45% for EBV+ HLs vs. 77% for EBV- HLs, p = 0.002).. Although the underlying mechanisms are still relatively unclear, EBER1 inhibits p21cip1/waf1 transcription and prevents apoptosis through down-regulation of p53, EGR1, and STAT1. The anti-apoptotic activity of EBER1 may be important in the rescue of Reed-Sternberg cells from drug-induced apoptosis and in the clinical behaviors of EBV+ HLs.

Topics: Apoptosis; Cell Cycle; Cell Line, Tumor; Cyclin D2; Cyclin-Dependent Kinase Inhibitor p21; Drug Resistance, Neoplasm; Early Growth Response Protein 1; Gene Expression Regulation, Neoplastic; Herpesvirus 4, Human; Hodgkin Disease; Humans; Hydroxamic Acids; Leupeptins; Models, Biological; Prognosis; RNA, Viral; STAT1 Transcription Factor; Transcription, Genetic; Tumor Suppressor Protein p53

Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-kappaB, the precise physiologic and clinical significance of NF-kappaB expression is currently undefined. Expression of active NF-kappaB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-kappaB inhibitors gliotoxin and MG132 and the effect of NF-kappaB inhibition utilizing an adenovirus vector carrying a dominant-negative IkappaBalpha mutant (Ad5IkappaB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin's disease. After chemical NF-kappaB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-kappaB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-kappaB inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD-FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IkappaB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-kappaB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-kappaB in HRS cell survival and resistance to apoptosis.

Topics: Adenoviridae; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Cysteine Endopeptidases; DNA-Binding Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flow Cytometry; Genetic Vectors; Gliotoxin; Hodgkin Disease; Humans; Immunohistochemistry; Jurkat Cells; Leupeptins; Multienzyme Complexes; NF-kappa B; Oligopeptides; Proteasome Endopeptidase Complex; Reed-Sternberg Cells; Transcription Factor RelA; Ubiquitins

Malignant cells from Hodgkin's disease have been reported to be defective in regulation of NF-kappaB activity. Ionizing radiation is known to activate NF-kappaB, and it has been suggested that this pathway may protect cells from apoptosis following exposure to radiation and other therapeutic agents. Defective NF-kappaB regulation in Hodgkin cells could therefore dictate the response of this disease to therapy, as well as be responsible for maintaining the malignant phenotype. The purpose of this study was to explore whether NF-kappaB activity could be modulated in Hodgkin cells and whether it determines the response of these cells to treatment with ionizing radiation and/or dexamethasone.. Activation of NF-kappaB in cells is accomplished in large part by degradation of its inhibitor IkappaB through the 26s proteasome. HD-My-Z Hodgkin cells were treated with the proteasome inhibitor MG-132 or transduced with a dominant negative super-repressor IkappaBalpha. Clonogenic survival, apoptosis, proteasome activity, and NF-kappaB binding activity were monitored in response to ionizing radiation and/or dexamethasone treatment.. HD-My-Z Hodgkin cells had modest NF-kappaB levels but, unlike other cell types, did not decrease their level of constitutively active NF-kappaB in response to proteasome inhibition with MG-132. In contrast, transduction with a non-phosphorable IkappaBalpha construct abolished expression. MG-132 did, however, induce apoptosis in HD-My-Z cells and sensitized them to ionizing radiation. Dexamethasone treatment had no effect on NF-kappaB activity or clonogenic survival of Hodgkin cells, but protected them from irradiation.. We conclude that inhibition of 26s proteasome activity can induce apoptosis in HD-My-Z Hodgkin cells and radiosensitize them, in spite of the fact that their constitutively active NF-kappaB levels are unaltered. The proteasome may be a promising new therapeutic target for intervention in this disease. In contrast, the use of glucocorticoids in conjunction with radiation treatment for this tumor may require re-evaluation.

Topics: Antineoplastic Agents, Hormonal; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dexamethasone; Dose-Response Relationship, Drug; Hodgkin Disease; Humans; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; NF-kappa B; Proteasome Endopeptidase Complex; Radiation Tolerance; Radiobiology; Tumor Cells, Cultured

The molecular mechanisms underlying Hodgkin's disease remain obscure, but it has been recognized that the neoplastic cells display high levels of constitutively active nuclear NF-kappaB. Here we demonstrate that although nuclear NF-kappaB is transcriptionally active, the Hodgkin cells fail to activate NF-kappaB dependent transcription in response to CD40 ligand. In three Hodgkin cell lines examined each had abnormalities in expression of IkappaBalpha which could account for the deregulated NF-kappaB. Although all three cell lines had greater than normal levels of IkappaBalpha mRNA no IkappaBalpha protein could be detected in the KM-H2 cells, while the L428 cell line contains a C-terminally truncated IkappaBalpha species that fails to associate with NF-kappaB. The HDLM-2 cell line contains a more slowly migrating form of IkappaBalpha that can associate with NF-kappaB, but increasing the level of this protein within the cell fails to inhibit nuclear NF-kappaB. Addition of recombinant IkappaBalpha to nuclear extracts from all three cell lines resulted in complete inhibition of NF-kappaB DNA binding activity and introduction of a plasmid expressing IkappaBalpha into the cells inhibited the transcriptional activity of an NF-kappaB dependent reporter plasmid. Thus the constitutive expression of NF-kappaB in Hodgkin cells is a direct consequence of the abnormal expression of IkappaBalpha rather than changes in NF-kappaB that render it refractory to inhibition by IkappaB proteins. These changes could, at least in part, account for the characteristic activated phenotype of Hodgkin cells and their pattern of cytokine secretion, which determine the pathological appearance and clinical manifestations of Hodgkin's disease.

Topics: CD40 Ligand; Cell Nucleus; Dexamethasone; DNA; DNA-Binding Proteins; Hodgkin Disease; Humans; I-kappa B Proteins; Leupeptins; Membrane Glycoproteins; NF-kappa B; NF-KappaB Inhibitor alpha; RNA, Messenger; Transcriptional Activation; Tumor Cells, Cultured