ABSTRACT
Granzyme B (GrB) is an immune protease implicated in the pathogenesis of several human diseases. In the current model of GrB activity, perforin determines whether the downstream actions of GrB occur intracellularly or extracellularly, producing apoptotic cytotoxicity or nonapoptotic effects, respectively. In the current study, we demonstrate the existence of a broad range of GrB-dependent signaling activities that 1) do not require perforin, 2) occur intracellularly, and 3) for which cell death is not the dominant outcome. In the absence of perforin, we show that GrB enzymatic activity still induces substoichiometric activation of caspases, which through nonlethal DNA damage response signals then leads to activity-associated phosphorylation of IFN regulatory factor-3. These findings illustrate an unexpected potential interface between GrB and innate immunity separate from the traditional role of GrB in perforin-dependent GrB-mediated apoptosis that could have mechanistic implications for human disease.
Subject(s)
Fibroblasts/physiology , Granzymes/metabolism , Interferon Regulatory Factor-3/metabolism , Apoptosis , Cell Survival , Cells, Cultured , Granzymes/genetics , HEK293 Cells , Humans , Interferon Regulatory Factor-3/genetics , Mutation/genetics , Perforin/metabolism , Phosphorylation , Proteolysis , Signal TransductionABSTRACT
Since the first description of infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in China in December 2019, it has evolved into a pandemic and emerged as an unprecedented worldwide crisis overwhelming healthcare systems globally. Analysis of the available literature to date suggests that, in addition to older age, patients with underlying co-morbidities including hypertension, diabetes, heart disease are at higher risk for severe disease with increased mortality. Practitioners around the world also have become increasingly concerned that immunosuppressed patients including those with autoimmune diseases may be at increased risk for developing Coronavirus Disease 2019 (COVID-19) with serious complications. Very little is known about how anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis modifies the susceptibility, clinical presentation and disease course of COVID-19. In this review, we discuss the mechanism of action and challenges of the current therapeutic armamentarium of ANCA-associated vasculitis and outline approaches to management of ANCA-associated vasculitis during the COVID-19 pandemic.
ABSTRACT
Background: The incidence of venous thromboembolism (VTE) is increased in ANCA-associated vasculitis (AAV). We assessed the frequency of VTE observed among patients with AAV evaluated at our center and identified risk factors. Methods: Patients from the Johns Hopkins Vasculitis Center cohort who were evaluated between 1998 and 2018 and had a diagnosis of granulomatosis with polyangiitis (GPA) or microscopic polyangiitis (MPA) were eligible for analysis. Baseline demographics and clinical and serologic data were extracted. Univariate and multivariate analyses were performed to identify factors associated with VTE in AAV. Results: A total of 162 patients with AAV were identified, 105 (65%) with GPA; 22 (14%) of these patients had a recorded VTE with a median time to VTE of 1 month. The mean (SD) age in the VTE versus non-VTE groups was 54±20 versus 55±17 years (P=0.99), 64% versus 60% female (P=0.93), 82% versus 49% PR3-ANCA positive (P=0.01), with a total mean BMI of 33.3±5.7 versus 28.3±6.1 kg/m2, (P<0.001) respectively. The median Birmingham Vasculitis Activity Score (BVAS version 3) was 19 versus 14 (P=0.02). Univariate analyses identified PR3-ANCA, rapidly progressive GN (RPGN), and hypoalbuminemia. In multivariate analysis, the significant associations with VTE included PR3-ANCA (OR, 4.77; P=0.02), hypoalbuminemia (OR, 4.84; P=0.004), and BMI (OR, 1.18; P<0.001). Conclusions: VTE is a surprisingly common complication of AAV. PR3-ANCA and hypoalbuminemia are risk factors for developing VTEs. Further studies are needed to confirm these findings. Podcast: This article contains a podcast at https://www.asn-online.org/media/podcast/K360/2020_04_30_KID0000572019.mp3.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis , Granulomatosis with Polyangiitis , Adult , Aged , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/complications , Antibodies, Antineutrophil Cytoplasmic , Female , Granulomatosis with Polyangiitis/complications , Humans , Incidence , Male , Middle Aged , Myeloblastin , Peroxidase , Risk FactorsABSTRACT
BACKGROUND: The introduction of combination therapy with glucocorticoids (GC) and cyclophosphamide (CYC) or rituximab (RTX) has resulted in remission rates exceeding 90% in patients with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). However, early treatment-related mortality remains a major concern and has driven the search for safer induction regimens exploring minimization or avoidance of GC and CYC. Most trials have excluded patients with severe renal disease. We report the outcomes of AAV patients with severe renal disease treated with sequential therapy (ST) starting with (GC) and oral (CYC) followed by transition to (RTX). METHODS: Patients with new or relapsing severe AAV who presented with severe renal disease and/or rapidly progressive glomerulonephritis (RPGN) were identified. RPGN was defined as at least a 20% decrease in estimated glomerular filtration rate (eGFR) over a 2-week period along with hematuria and proteinuria. Induction treatment included pulse (GC) for 3 days followed by oral prednisone tapered to 5 mg by month 6, oral (CYC) adjusted for GFR until improvement in Birmingham Vasculitis Activity Score (BVAS), and serum creatinine at which point (CYC) was stopped and induction dose of (RTX) was given. Use of plasmapheresis (PLEX) was allowed. The primary outcome was complete remission defined as BVAS of zero by 6 months. Descriptive data are presented as median with range and mean with SD. RESULTS: Nine patients met the inclusion criteria. Median age at diagnosis was 63 years. The majority were females, myeloperoxidase ANCA positive, and had a new diagnosis. The mean nadir (SD) eGFR was 12 (5) with 3 requiring dialysis. The median BVAS at the time of diagnosis was 15. All patients received ST and 3 received PLEX. The median exposure to oral CYC was 35 days. The mean (SD) eGFR and median BVAS were 26 (12) and 3, respectively, at the time of switching to RTX. The median prednisone dose at 6M was 5 mg. The median follow-up was 44 months. All patients achieved remission. One patient with relapsing disease reached ESRD. The mean (SD) eGFR in the remaining 8 patients at last FU was 37 (27), and the mean (SD) eGFR rise at 1 year was 26 (25). Adverse events included 2 patients with pneumonia and 3 with bone marrow suppression. There were no deaths. CONCLUSION: ST with GC and CYC followed by RTX is effective for in AAV patients with severe renal disease. Therapy-related adverse events are comparable to other studies, and further modification in ST with decrease in GC dosage should be explored.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/drug therapy , Glomerulonephritis/drug therapy , Immunosuppressive Agents/administration & dosage , Remission Induction/methods , Adult , Aged , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Cyclophosphamide/administration & dosage , Cyclophosphamide/adverse effects , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Therapy, Combination/adverse effects , Drug Therapy, Combination/methods , Female , Glomerular Filtration Rate/drug effects , Glomerular Filtration Rate/immunology , Glomerulonephritis/diagnosis , Glomerulonephritis/immunology , Glucocorticoids/administration & dosage , Glucocorticoids/adverse effects , Humans , Immunosuppressive Agents/adverse effects , Male , Middle Aged , Rituximab/administration & dosage , Rituximab/adverse effects , Severity of Illness Index , Treatment OutcomeABSTRACT
ANCA-associated vasculitis (AAV) can present in an atypical manner and obscure the clinical picture. We sought to characterize clinical characteristics and outcomes in these uncommon presentations. We conducted a retrospective study of 171 AAV patients in our vasculitis database to identify patients with atypical presentation of AAV. Patient demographics, serologies, renal indices, and treatment regimens were assessed. Of the 171 patients, eight were identified to have uncommon presentations. These patients were usually extremes of age with three being less than 30 years and four being more than 70 years. Six patients were positive for PR3 antibodies. The mean delay in diagnosis from time of symptom development was 12 months. All patients developed acute kidney injury during their clinical course. Pancreatitis was the most frequent atypical presentation (n = 3), with pulmonary pathologies (cystic lung disease and usual interstitial pneumonia) and splenic infarcts being present in two patients each. The diagnosis of AAV was established by positive ANCA serology and renal or lung biopsy evidence of vasculitis. Six patients received induction therapy with steroids and rituximab, while two received steroids and cyclophosphamide. One patient died of respiratory failure in the first month following diagnosis while the remaining patients achieved disease remission. One patient developed end-stage renal disease. Uncommon presentations of AAV afflict extremes of age with a PR3 ANCA predominance and are associated with subsequent development of AKI. This case series demonstrates that a significant delay in diagnosis can be associated with these presentations. KEY POINTS: ⢠Uncommon manifestations of AAV are seen more often with PR3 ANCA disease and respond to standard induction therapy of AAV. ⢠High index of suspicion is required to avoid delays in diagnosis.
Subject(s)
Acute Kidney Injury/complications , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/therapy , Adult , Age Factors , Aged , Aged, 80 and over , Antibodies, Antineutrophil Cytoplasmic/blood , Cyclophosphamide/administration & dosage , Databases, Factual , Delayed Diagnosis , Female , Granulomatosis with Polyangiitis/immunology , Humans , Lung Diseases/complications , Lung Diseases, Interstitial/complications , Male , Middle Aged , Pancreatitis/complications , Remission Induction , Respiratory Insufficiency/therapy , Retrospective Studies , Rituximab/administration & dosage , Steroids/administration & dosage , Treatment Outcome , Young AdultABSTRACT
The family name of the co-author of the article mentioned above was incorrectly spelled. The correct name should have been "Veena S. Katikineni"instead of "Veena Katikeneni". The original article has been corrected.
ABSTRACT
Objectives Intravenous immunoglobulin G (IVIG) is used to treat antineutrophil cytoplasmic antibody (ANCA) patients with recurrent infections as a result of hypogammaglobulinemia (HG) induced by treatment regimens. We sought to characterize clinical features, treatment, and outcomes for patients treated with the novel subcutaneous IgG (SCIG) for the aforementioned purpose. Methods We conducted a retrospective study of 136 patients in our ANCA database to identify patients with recurrent infections and HG subsequently treated with SCIG. Patient demographics, serologies, treatment, and immunological parameters were assessed. Results Of 136 patients, four were treated with SCIG. All were Caucasian, proteinase-3 (PR3)-positive, and the majority (n = 3) were females. All patients had pulmonary involvement, and regimens of cyclophosphamide (CYC) and/or rituximab (RTX) were employed for induction and remission. Three patients each experienced recurrent bouts of respiratory tract infections and shingles. Ig levels (G, M, and A) were reduced in all patients, except for one patient who had normal IgA levels. CD19/20 cells were depleted and CD3/4/8/NK cells were preserved in all patients. Three patients had no discernible antibody response to the pneumococcal vaccine (specific pneumococcal serotypes measured pre- and post-vaccine). The mean duration elapsed between the first rituximab administration and commencement of SCIG was 7.2 years. The IgG level normalized and none of the patients had a recurrence of infection since the initiation of SCIG. Conclusion This data, albeit preliminary, is the first series that demonstrates SCIG can be a reliable alternative to IVIG in ANCA patients with recurrent infections secondary to HG. Early identification of this subset of patients is likely to mitigate infectious risks, associated morbidity, and hospitalization.
ABSTRACT
Objectives The optimal duration of maintenance immunosuppressive therapy in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is still controversial. The aim of our study is to describe the characteristics and outcomes of patients with AAV who were able to stop maintenance agents completely while remaining on daily prednisone (< 5 mg) for at least 36 months. Materials and methods AAV patients treated at our center from 2000 to 2016 and who were not on maintenance agents while remaining on prednisone < 5 mg daily for at least 36 months were identified by the providers, and their records were retrospectively reviewed. Relapse was defined by the reinitiation of immunosuppressive therapy for biopsy-proven glomerulonephritis or any extra-renal organ involvement. Results Of the 18 patients who fulfilled the study inclusion criteria, 12 were male and 14 were Caucasian. The mean age at AAV diagnosis was 54 years. Seventeen patients had renal involvement and seven had lung involvement. Eleven patients received cyclophosphamide and eight patients received rituximab along with glucocorticoids for remission induction. Twelve patients were weaned completely off prednisone. The median duration of prednisone use was 20 months. Nine patients received maintenance therapy with azathioprine or mycophenolate mofetil. The median duration of maintenance therapy was 24 months. The mean follow-up time after stopping the maintenance agent was 64 months. During this period, three patients had disease relapse. Conclusions Stopping maintenance agents for > 36 months can be achieved in some patients with AAV. Prospective, randomized controlled trials are needed to confirm this finding.
ABSTRACT
PURPOSE OF REVIEW: The ANCA-associated vasculitides are a group of small vessel vasculitides characterized by autoantibodies recognizing the neutrophil cytoplasmic antigens PR3 and MPO. We examine the current clinical and molecular immunology understanding of ANCA-associated vasculitides and discuss the current needs in our understanding of the pathogenic mechanisms of these rare diseases. RECENT FINDINGS: The majority of efforts to understand the pathogenesis of these diseases have focused on dissecting neutrophil biology because the neutrophil is the primary expressor of ANCA autoantigens. However, a number of important genetic, clinical, and cellular biology observations suggest that attempts to understand the pathogenesis of ANCA vasculitides should move away from emphasis on the role of the neutrophil and instead re-focus on the potential role of other immune cell mediators. Whether or not neutrophils are the key determinant of ANCA-associated vasculitis pathogenesis should be revisited in detail. A neutrophil-centric view of the pathogenesis of these diseases cannot fully account for important genetic, clinical, and cellular biology observations that implicate important and under-appreciated roles for monocytes and T cells. Refocusing on these findings will likely lead to new discovery of novel therapeutic targets and the identification of clinically useful biomarkers for disease activity.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/immunology , Antibodies, Antineutrophil Cytoplasmic/biosynthesis , Antibodies, Antineutrophil Cytoplasmic/immunology , Evidence-Based Medicine/methods , Humans , Immune Tolerance/immunology , Neutrophils/immunologyABSTRACT
V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs(3A)) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.
Subject(s)
DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , V(D)J Recombination , Animals , Ataxia Telangiectasia Mutated Proteins/chemistry , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Catalytic Domain , Cells, Cultured , DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA-Activated Protein Kinase/chemistry , DNA-Activated Protein Kinase/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Endonucleases/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Mice , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphorylation , Precursor Cells, B-Lymphoid/metabolism , Protein Interaction Domains and MotifsABSTRACT
Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase catalytic subunits (DNA-PKcs) are members of the phosphatidylinositol 3-like family of serine/threonine kinases that phosphorylate serines or threonines when positioned adjacent to a glutamine residue (SQ/TQ). Both kinases are activated rapidly by DNA double-strand breaks (DSBs) and regulate the function of proteins involved in DNA damage responses. In developing lymphocytes, DSBs are generated during V(D)J recombination, which is required to assemble the second exon of all Ag receptor genes. This reaction is initiated through a DNA cleavage step by the RAG1 and RAG2 proteins, which together comprise an endonuclease that generates DSBs at the border of two recombining gene segments and their flanking recombination signals. This DNA cleavage step is followed by a joining step, during which pairs of DNA coding and signal ends are ligated to form a coding joint and a signal joint, respectively. ATM and DNA-PKcs are integrally involved in the repair of both signal and coding ends, but the targets of these kinases involved in the repair process have not been fully elucidated. In this regard, the RAG1 and RAG2 proteins, which each have several SQ/TQ motifs, have been implicated in the repair of RAG-mediated DSBs. In this study, we use a previously developed approach for studying chromosomal V(D)J recombination that has been modified to allow for the analysis of RAG1 and RAG2 function. We show that phosphorylation of RAG1 or RAG2 by ATM or DNA-PKcs at SQ/TQ consensus sites is dispensable for the joining step of V(D)J recombination.
Subject(s)
Chromosome Breakage , Chromosomes, Mammalian/metabolism , DNA Breaks, Double-Stranded , DNA Repair/physiology , DNA-Binding Proteins/metabolism , Homeodomain Proteins/metabolism , Recombination, Genetic/physiology , Amino Acid Motifs , Amino Acid Sequence , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Cell Cycle Proteins/immunology , Cell Cycle Proteins/metabolism , Cell Line, Transformed , Chromosomes, Mammalian/genetics , Chromosomes, Mammalian/immunology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Immunogenetic Phenomena/physiology , Lymphocytes/immunology , Lymphocytes/metabolism , Mice , Phosphatidylinositol 3-Kinases/genetics , Phosphatidylinositol 3-Kinases/immunology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation/physiology , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Sequence Deletion , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/immunology , Tumor Suppressor Proteins/metabolismABSTRACT
Lymphocyte antigen receptor genes are assembled through the process of V(D)J recombination, during which pairwise DNA cleavage of gene segments results in the formation of four DNA ends that are resolved into a coding joint and a signal joint. The joining of these DNA ends occurs in G1-phase lymphocytes and is mediated by the non-homologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. The ataxia telangiectasia mutated (ATM) and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), two related kinases, both function in the repair of DNA breaks generated during antigen receptor gene assembly. Although these proteins have unique functions during coding joint formation, their activities in signal joint formation, if any, have been less clear. However, two recent studies demonstrated that ATM and DNA-PKcs have overlapping activities important for signal joint formation. Here, we discuss the unique and shared activities of the ATM and DNA-PKcs kinases during V(D)J recombination, a process that is essential for lymphocyte development and the diversification of antigen receptors.
Subject(s)
Cell Cycle Proteins/physiology , DNA-Activated Protein Kinase/physiology , DNA-Binding Proteins/physiology , Protein Serine-Threonine Kinases/physiology , Recombination, Genetic , Tumor Suppressor Proteins/physiology , Animals , Ataxia Telangiectasia Mutated Proteins , Catalytic Domain , DNA Repair , HumansABSTRACT
Lymphocyte antigen receptor gene assembly occurs through the process of V(D)J recombination, which is initiated when the RAG endonuclease introduces DNA DSBs at two recombining gene segments to form broken DNA coding end pairs and signal end pairs. These paired DNA ends are joined by proteins of the nonhomologous end-joining (NHEJ) pathway of DSB repair to form a coding joint and signal joint, respectively. RAG DSBs are generated in G1-phase developing lymphocytes, where they activate the ataxia telangiectasia mutated (Atm) and DNA-PKcs kinases to orchestrate diverse cellular DNA damage responses including DSB repair. Paradoxically, although Atm and DNA-PKcs both function during coding joint formation, Atm appears to be dispensible for signal joint formation; and although some studies have revealed an activity for DNA-PKcs during signal joint formation, others have not. Here we show that Atm and DNA-PKcs have overlapping catalytic activities that are required for chromosomal signal joint formation and for preventing the aberrant resolution of signal ends as potentially oncogenic chromosomal translocations.
Subject(s)
Cell Cycle Proteins/metabolism , Chromosomes , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Mice , Mice, SCIDABSTRACT
A-type lamins are intermediate filament proteins that provide a scaffold for protein complexes regulating nuclear structure and function. Mutations in the LMNA gene are linked to a variety of degenerative disorders termed laminopathies, whereas changes in the expression of lamins are associated with tumourigenesis. The molecular pathways affected by alterations of A-type lamins and how they contribute to disease are poorly understood. Here, we show that A-type lamins have a key role in the maintenance of telomere structure, length and function, and in the stabilization of 53BP1, a component of the DNA damage response (DDR) pathway. Loss of A-type lamins alters the nuclear distribution of telomeres and results in telomere shortening, defects in telomeric heterochromatin, and increased genomic instability. In addition, A-type lamins are necessary for the processing of dysfunctional telomeres by non-homologous end joining, putatively through stabilization of 53BP1. This study shows new functions for A-type lamins in the maintenance of genomic integrity, and suggests that alterations of telomere biology and defects in DDR contribute to the pathogenesis of lamin-related diseases.
Subject(s)
DNA Repair , Lamin Type A/genetics , Lamin Type A/metabolism , Telomere/metabolism , Animals , Cell Line , Cell Nucleus/chemistry , Cell Nucleus/metabolism , Chromosomal Proteins, Non-Histone , DNA-Binding Proteins , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Deletion , Genomic Instability , Intracellular Signaling Peptides and Proteins/analysis , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Telomere/chemistry , Tumor Suppressor p53-Binding Protein 1ABSTRACT
DNA double-strand breaks are generated by genotoxic agents and by cellular endonucleases as intermediates of several important physiological processes. The cellular response to genotoxic DNA breaks includes the activation of transcriptional programs known primarily to regulate cell-cycle checkpoints and cell survival. DNA double-strand breaks are generated in all developing lymphocytes during the assembly of antigen receptor genes, a process that is essential for normal lymphocyte development. Here we show that in murine lymphocytes these physiological DNA breaks activate a broad transcriptional program. This program transcends the canonical DNA double-strand break response and includes many genes that regulate diverse cellular processes important for lymphocyte development. Moreover, the expression of several of these genes is regulated similarly in response to genotoxic DNA damage. Thus, physiological DNA double-strand breaks provide cues that can regulate cell-type-specific processes not directly involved in maintaining the integrity of the genome, and genotoxic DNA breaks could disrupt normal cellular functions by corrupting these processes.
Subject(s)
B-Lymphocytes/metabolism , DNA Breaks, Double-Stranded , Gene Expression Regulation, Developmental/genetics , Animals , Ataxia Telangiectasia Mutated Proteins , B-Lymphocytes/drug effects , Cell Cycle Proteins/drug effects , Cell Line , DNA-Binding Proteins/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Profiling , Gene Expression Regulation, Developmental/drug effects , Homeodomain Proteins/metabolism , Mice , Mice, Knockout , Mice, SCID , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/drug effects , Tumor Suppressor Proteins/drug effectsABSTRACT
Further structure-activity relationship (SAR) exploration of 3,4-methylenedioxy-5,4'-dimethoxy-3'-amino-Z-stilbene (1a) derivatives resulted in the efficient synthesis of tyrosine amide hydrochloride 9, two tyrosine amide phosphate prodrugs (3a and 6), and sodium aspartate amide 11. Two additional cancer cell growth inhibitors (14 and 16) were synthesized by employing peptide coupling between amine 1a and the Dap unit of dolastatin 10 (4a) to yield amide 14 followed by Dov-Val-Dil (15) to yield peptide 16. The latter represents a combination of stilbene 1a with the des-Doe tetrapeptide unit of the powerful tubulin assembly inhibitor dolastatin 10. Peptide 16 was examined for potential binding to tubulin in the vinca and/or colchicine regions and found to perform primarily as a relative of dolastatin 10. Amide 14 had anticryptococcal and antibacterial activities.
Subject(s)
Antineoplastic Agents/chemical synthesis , Dioxoles/chemical synthesis , Stilbenes/chemical synthesis , Tyrosine/chemical synthesis , Amides/chemical synthesis , Amides/chemistry , Amides/pharmacology , Anti-Infective Agents/chemical synthesis , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Colchicine , Depsipeptides , Dioxoles/chemistry , Drug Screening Assays, Antitumor , Growth Inhibitors , Humans , Mitosis/drug effects , Molecular Structure , Oligopeptides/pharmacology , Stereoisomerism , Stilbenes/chemistry , Structure-Activity Relationship , Tubulin/chemistry , Tumor Cells, Cultured , Tyrosine/analogs & derivatives , Tyrosine/chemistry , Tyrosine/pharmacologyABSTRACT
Previous work has shown that laulimalide, a sponge-derived natural product, resembles paclitaxel in enhancing tubulin assembly and in its effects on cellular microtubules. The two compounds, however, seem to have distinct binding sites on tubulin polymer. Nearly equimolar amounts of tubulin, laulimalide, and paclitaxel are recovered from microtubules formed with both drugs. In the present study, we searched for differences between laulimalide and paclitaxel in their interactions with tubulin polymer. Laulimalide was compared with paclitaxel and epothilone A, a natural product that competes with paclitaxel in binding to microtubules, for assembly properties at different temperatures and for effects of GTP and microtubule-associated proteins on assembly. Although minor differences were observed among the three drugs, their overall effects were highly similar, except that aberrant assembly products were observed more frequently with paclitaxel and that the polymers formed with laulimalide and epothilone A were more stable at 0 degrees C. The most dramatic difference observed between laulimalide and epothilone A was that only laulimalide was able to enhance assembly synergistically with paclitaxel, as would be predicted if the two drugs bound at different sites in polymer. Because stoichiometric amounts of laulimalide and paclitaxel can cause extensive tubulin assembly, maximum synergy was observed at lower temperatures under reaction conditions in which each drug alone is relatively inactive. Laulimalide-induced assembly, like paclitaxel-induced assembly, was inhibited by drugs that inhibit tubulin assembly by binding at either the colchicine- or vinblastine-binding site. When radiolabeled GTP is present in a reaction mixture with either laulimalide or paclitaxel, nucleotide hydrolysis occurs with incorporation of radiolabeled GDP into polymer.