Proteinase 3 Gene Research Articles

Identification of Proteinase 3 (PR3) Gene Overexpresison and Protein Delocalization in Core Binding Factor Leukemias as a Mechanism Leading to Increased Chemosensitivity.

Proteinase 3 (PR3) gene codes for a serine protease with a broad spectrum of proteolytic activity. PR3 is involved in the control of proliferation of myeloid leukemia cells. When abnormally expressed it confers factor-independent growth to hematopoietic cells. The aim of this study was to investigate the role of PR3 gene in leukemic haematopoiesis. We analyzed the expression levels of PR3 by RQ-PCR in 113 BM samples collected from AML patients at diagnosis. The FAB distribution was as follows: M0=5, M1=12, M2=38, M3=12, M4=37, M5=5, M6=4. 19 patients were characterized by t(8;21) and 16 by inv(16). PR3 expression level was also analyzed in 57 BM and 42 PB samples from 88 MDS patients (44 RA, 32 RAEB and 12 secondary-AML) and in 15 BM and 40 PB samples from healthy volunteers. PR3 protein was analyzed by western blot (WB) and its localization determined by immunofluorescence assay using specific antibodies. The transcription factor C/EBPα, which negatively regulates PR3 expression was studied in parallel at the RNA and protein level by RQ-PCR and WB. The DNA binding activity of C/EBPα was investigated by EMSA assay. Gain and loss of function experiments were performed by transfecting COS and 293T cell lines with a plasmid containing the full length PR3 sequence and HL60, Me-1, and Kasumi cell lines with specific shRNA. We found that PR3 is significantly overexpressed in AML samples. The median value of 2−Δ ΔCt is 740, (range 15-5043). Interestingly, patients affected by Core Binding Factor leukemias showed significantly higher PR3 values compared to patients with normal karyotypes (NK) (p<0,0002 for t(8;21), p<0,001 for inv16) and lower C/EPBα levels. EMSA assay demonstrated the absence of C/EBPα DNA binding activity in CBF AML cells but not in NK AML. In addition, PR3 overexpression was detected in 60% of RA patients (mean value: 10, range 3–268), and in all the cases of RAEB (mean value 201: range:128–803) and secondary AML (mean value 589, range 207–7131). WB demonstrated the correlation between the mRNA and protein amount. Interestingly, immunofluorescence demonstrated the de-localization of the protein within the nucleous in CBF AML but it is completely cytoplasmatic in leukemic cells with normal karyotype and in MDS. Transfection experiments with PR3 plasmid demonstrated that PR3 overexpression results into a significantly increased proliferation and reduced apoptosis. By contrast transfection with shRNA triggers apoptosis and cell growth inhibition. In addition, WB demonstrated that nuclear PR3 is able to cleavage the p65subunit of NF-kB into a p56 isoform which lacks any transcriptional activity as confirmed by EMSA. In conclusion, PR3 gene expression and protein are significantly increased in AML and MDS, particularly in CBF leukemias in which the protein is not only increased but also completely delocalized within the nucleous. PR3 overepression My be due to a significant downmodulation of C/EBPα. Ectopic expression of PR3 induces increased proliferation and apoptosis arrest. The abnormal nuclear localization of PR3 in CBF leukemias results into the loss of function of NF-kB thus representing one mechanism of chemo sensitivity in this group of patients.

High PR3 or ELA2 expression by CD34+ cells in advanced-phase chronic myeloid leukemia is associated with improved outcome following allogeneic stem cell transplantation and may improve PR1 peptide–driven graft-versus-leukemia effects

AbstractThe primary granule proteins elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1, which can induce cytotoxic T lymphocyte (CTL) responses against chronic myeloid leukemia (CML) cells. To investigate whether eradication of CML after allogeneic stem cell transplantation (SCT) was influenced by PR3 and ELA2 gene expression or PR1-specific CTL responses, we studied cells from 87 CML patients and 27 HLA-A*0201+ donors collected prior to T-cell–depleted HLA-identical sibling SCT. For patients in advanced phase (AdP), a higher expression of both PR3 and ELA2 in CD34+ progenitors before SCT was associated with a lower incidence of relapse-related death, improved leukemia-free survival (LFS), and overall survival (OS); in chronic phase patients, no differences were observed. PR1-CTL responses were detected in 7 of 27 HLA-identical sibling donors, and associated with improved LFS and OS after SCT on follow-up. PR1-CTL responses detected in 7 of 28 CML patients before transplantation were not predictive of outcome and correlated inversely with PR3 and ELA2 expression. These findings suggest that assessment of PR3 and ELA2 expression in leukemic progenitors is useful for predicting posttransplantation outcome in AdP patients undergoing SCT. The presence of a donor immune response against PR1 may be advantageous and could be exploited therapeutically.

Cytotoxic T Lymphocyte Responses to PR1 Peptide in Chronic Myeloid Leukemia Patients Inversely Correlate with Proteinase 3 and Elastase Expression but Donor PR1 Responses Determine Survival after Stem Cell Transplantation.

The primary granule proteins (PGP) neutrophil elastase (ELA2) and proteinase 3 (PR3) both contain the nonapeptide PR1 which can induce cytotoxic T lymphocyte (CTL) responses in chronic myeloid leukemia (CML). The relative contribution of PR3 and ELA2 to PR1 expression is not known. We previously found that higher levels of PR3 and ELA2 gene expression in CD34+ progenitor cells were associated with longer survival in CML patients. Eradication of leukemia depends on the elimination of leukemic stem cells which are thought to reside within the CD34+ progenitor cell pool. We therefore studied PGP expression and T cell response to PR1 in 23 CML patients and their HLA-identical family donors prior to T-cell depleted allogeneic stem cell transplantation (SCT) with T-cell add-back on day 45–100 post-SCT. CD8+ T cells and CD34+ progenitor cells were purified from mononuclear cells (MNC) of cryopreserved leukapheresis products from HLA-A*0201+ CML patients. Following reverse transcription of RNA from MNC and CD34+ cells, ELA2 and PR3 gene expression was measured using real-time quantitative polymerase chain reaction (RQ-PCR). To assess PR1-CTL responses, T2 cells were loaded with PR1 peptide (VLQELNVTV) at 0.1, 1, and 10μM for 2h, irradiated and subsequently co-cultured with CD8+ T cells for 3h. PR1-CTL response was measured as interferon-γ mRNA expression (relative to CD8) obtained by RQ-PCR. PR1-specific CTL responses were detected in 5/23 CML patients and 6/16 HLA-identical donors. In CML patients, pre-SCT expression of both PR3 and ELA2 in MNC was strongly correlated with the expression in CD34+ cells (p=0.009 and p=0.0006 respectively). There was an inverse relationship between PR1-CTL response in CML patients pre-SCT and PR3 or ELA2 expression (p=0.02 and p=0.01 respectively). This data suggest that both PR3 and ELA2 expression in CD34+ cells and their progeny are a potential source of PR1. However, high expression of these proteins may result in selective deletion of PR1 T cell clones. The presence of PR1-responses in HLA-identical donors was associated with an improved overall survival post-SCT. However, there was no impact of PR1-response in either CML patients pre-SCT or donors, on time to achieve molecular remission post-SCT but a greater proportion of patients whose donors did not have a PR1-response succumbed to fatal graft-versus-host disease. These findings support post-transplant vaccination strategies with PR1 peptide to eradicate minimal residual disease but in patients who are not eligible for SCT, the lower PR1 response in the presence of high PGP expression suggests that there is a risk that vaccines given to patients with minimal residual disease could induce tolerance. [Display omitted]

PR1-Specific T Cell Responses in the First Months Following T-Cell Depleted Allogeneic Stem Cell Transplantation Occur in Both Myeloid and Non-Myeloid Malignancies but Are Only Associated with a GVL Effect in Myeloid Leukemias.

The immune milieu in the first few months post-stem cell transplantation (SCT) is favorable for graft-versus-leukemia (GVL) responses. A homeostatic drive caused by lymphopenia stimulates expansions of transplanted donor T-cells responding to diverse antigenic stimuli in the recipient. Human leukemia antigens such as proteinase 3 (PR3) and elastase (ELA2) are self-antigens which induce low but detectable frequencies of autoreactive T cells in normal individuals. PR1, an HLA-A*0201 restricted peptide shared by PR3 and ELA2, is expressed in normal neutrophils and overexpressed in myeloid (but not lymphoid) leukemias. T-cell responses against PR1 have been linked to GVL. We studied patients early post SCT to look for early induction of T cell responses to PR1, and correlate them with ELA2 and PR3 expression and GVL effects. Using PR1/HLA-A*0201 tetramers and flow cytometry for intracellular IFN-gamma, we analyzed PBMC for CD8+ T-cell responses against PR1 on days 30, 60, 90 and 120 following a T-depleted SCT in 28 patients (13 CML, 10 ALL, 5 solid tumor). Ten patients with CML, 6 with ALL and 3 with solid tumors had detectable PR1 responses post-SCT. PR1 specific CD8+ T cells had a predominantly effector-memory phenotype (CD45RO+CD27−CD57+). PR3 and ELA2 gene expression in these samples was assessed by RQ-PCR and found to be significantly correlated (P < 0.001). There was a strong association between the expression of PR3 and ELA2 and the emergence of PR1 specific CD8+ T-cell responses. Conversely, reduction or disappearance of PR3 and ELA-2 expression from blood coincided with reduction or disappearance of PR1 specific CD8+ T-cell responses (P <0.001) (as depicted below). The in-vivo anti-leukemia effect of the PR1 response was assessed in CML patients by BCR-ABL transcript numbers at day 90 post-SCT. Eight of ten patients with significant PR1 responses post-SCT were BCR-ABL negative at day 90 compared to 1 of 3 without PR1 responses (P <0.001). This GVL association was restricted to CML patients: in ALL using WT1 gene expression as a measure of minimal residual disease (MRD) 2 of 5 patients with PR1 responses and 3 of 5 patients without were MRD positive on day 90 post-SCT (P =0.36). Since PR1 responses were not restricted to CML and because these transplant approaches usually induce 100% donor myeloid chimerism by day 30 post-SCT, the recovering donor marrow is the likely antigenic source of PR3 and ELA2 driving the PR1 response. Our findings suggest that the post-SCT milieu is favorable for exaggerating weak autoimmune responses to self antigens such as PR1 causing antigen-specific T-cell proliferation. GVL effects may follow if the self antigen is expressed on the leukemia as occurs in CML. These results suggest that vaccination in conjunction with induction of T cell homeostatic proliferation is likely to enhance the anti-leukemia response and effectiveness of a transplant procedure. [Display omitted]

Increased neutrophil membrane expression and plasma level of proteinase 3 in systemic vasculitis are not a consequence of the − 564 A/G promotor polymorphism

Several findings link proteinase 3 (PR3) to small vessel vasculitis. Besides being a major target of anti-neutrophil cytoplasm antibodies (ANCA), previous findings have shown increased circulating levels of PR3 in vasculitis patients, increased levels of neutrophil membrane-PR3 (mPR3) expression and a skewed distribution of the - 564 A/G polymorphism in the promotor region of the PR3 gene. In this study we elucidate how these three findings relate to each other. The plasma concentration of PR3 was measured by enzyme-linked immunosorbent assay (ELISA), mPR3 expression by fluorescence activated cell sorter (FACS) and the gene polymorphism by real-time polymerase chain reaction (PCR). We compared results from 63 patients with ANCA-associated systemic vasculitis (AASV) with 107 healthy blood donors. In accordance with previous reports, AASV patients had increased plasma concentrations of PR3 compared to healthy controls (mean 224 microg/l versus 155 microg/l, P < 0.0001). They also showed an increased number of mPR3-positive neutrophils (60%versus 42%, P < 0.001). However, contrary to a previous report, we found no skewed distribution of the polymorphism in PR3 gene. There was a weak correlation between mPR3 mean fluorescence intensity (MFI) and plasma PR3 among healthy controls and myeloperoxidase-ANCA (MPO-ANCA)-positive patients (r = 0.24, P = 0.015 and r = 0.52, P = 0.011, respectively). In conclusion, increased plasma PR3 and high expression of mPR3 are associated with small vessel vasculitis, but neither of them is a consequence of the - 564 A/G polymorphism of the PR3 gene promotor.

New insights that link microbes with the generation of antineutrophil cytoplasmic autoantibodies: the theory of autoantigen complementarity

Reviewed are recent discoveries that provide insights into novel mechanisms involved in the aetiology and pathology of anti-neutrophil cytoplasmic autoantibodies (ANCA) disease. Gene expression profiles of circulating leukocytes from anti-neutrophil cytoplasmic autoantibody immunogenesis patients revealed high levels of proteinase 3 (PR3) and myeloperoxidase (MPO) mRNA. Combined with reports of increased expression of these proteins, it appears that increased antigen availability is a pathologic component of anti-neutrophil cytoplasmic autoantibody immunogenesis disease, which might be equally as important as the presence of anti-MPO or anti-PR3 autoantibodies. Genetic predisposition to develop anti-neutrophil cytoplasmic autoantibody immunogenesis disease may include a polymorphism in the promoter region of the PR3 gene. Signalling pathways affected by anti-neutrophil cytoplasmic autoantibody immunogenesis binding to neutrophils involve the p21 pathway. Lastly, a topic discussed at length in this review is the seminal observation that PR3-ANCA patients harbour antibodies reactive with a protein produced from PR3-antisense RNA, whose amino acid sequence has homologies with proteins from many microbes and viruses. Delineated in the Theory of Autoantigen Complementarity, it is proposed that the initiator of an autoimmune response is not the autoantigen, but instead is a protein that is 'antisense' or complementary to the autoantigen (e.g. from bacteria or PR3). The progress in research efforts in the past year, including the identification of complementary proteins as a potential cause of anti-neutrophil cytoplasmic autoantibody immunogenesis, should highly impact future approaches therapeutic intervention.

Proteinase 3 gene polymorphisms and Wegener's granulomatosis

Wegener's granulomatosis (WG) is a rare systemic autoimmune disease characterized by small-vessel vasculitis leading to organ damage and the presence of antineutrophil cytoplasmic autoantibodies (ANCAs). ANCAs were shown to be involved in the pathogenesis of the disease by increasing adhesion of polymorphonuclear cells (PMNs) to endothelial cells and through activation of primed PMN. The main autoantigen of ANCA in WG is proteinase 3 (PR3), a neutrophil- and monocyte-derived neutral serine protease. The association of WG with individuals continuously expressing a high level of PR3 on the surface of PMNs suggests that PR3 variants or altered regulation of PR3 expression might be directly involved in the pathogenesis of the disease. We screened the entire coding and promoter sequences of the PR3 gene for polymorphisms by means of polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Allelic, genotypic, and haplotype frequencies were compared between 79 WG patients and a cohort of 129 healthy controls. Seven single-nucleotide polymorphisms (SNPs), one amino acid change (Val119Ile), one 84 bp insertion/deletion, and a microsatellite were identified. An association with WG could be demonstrated for the A-564G polymorphism in the PR3 promoter affecting a putative transcription factor-binding site. This study excludes certain PR3 epitope variants as autoantigenic stimuli in WG, since the Val119Ile polymorphism showed no differences between patients and controls. Overexpression of PR3, however, might predispose the patient to the development of autoimmune ANCA-associated vasculitis.

Structure, chromosomal assignment, and expression of the gene for proteinase-3. The Wegener's granulomatosis autoantigen.

Proteinase-3 (PR-3) is a neutral serine proteinase present in the azurophil granules of human polymorphonuclear leukocytes. It degrades a variety of extracellular matrix proteins including elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters. It is identical to the target autoantigen (c-ANCA) associated with Wegener's granulomatosis and to myeloblastin, a serine proteinase first identified in HL-60 leukemia cells. In this study, the gene encoding PR-3 was cloned and sequenced. The gene spans approximately 6.5 kilobase pairs and consists of five exons and four introns. The genomic organization of PR-3 is similar to that of the other serine proteinases expressed in hemopoietic cells. Each residue of the catalytic triad of PR-3 is located on a separate exon, and the positions of the residues within the exons are similar to those in human leukocyte elastase and cathepsin G. The phase and placement of the introns in the PR-3 gene are also similar to those in human leukocyte elastase and cathepsin G. The 400-base pair (bp) 5'-flanking sequence of the PR-3 gene contains a TATA box at position 379. There is no CAAT box promoter element. The 3'-untranslated region is 200 bp, extending from a TGA stop codon to the site of polyadenylation 10 bp after the canonical AATAAA signal. Amplification of PR-3 from a human/hamster hybrid cell line localizes the gene to human chromosome 19. Evidence from Northern analysis suggests that PR-3 expression is primarily confined to the promyelocytic/myelocytic stage of bone marrow development.

Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter.

The human neutrophil and monocyte-derived serine protease homologues neutrophil elastase (NE), proteinase 3 (PR3), and azurocidin (AZU) are involved in a variety of immune defense reactions. NE and PR3 assist in the destruction of phagocytosed microorganisms, cleave the important connective-tissue protein elastin, and generate chemotactic activities by forming alpha 1-proteinase inhibitor complexes and elastin peptides. AZU is cytotoxic to certain microorganisms and chemotactic for monocytes. All three proteins are produced and packaged into azurophil granules in large quantities during neutrophil differentiation. We have isolated several cosmid clones each of which contains the functional genes for AZU, PR3, and NE in this order. The PR3 gene is separated by 8 kilobases from the 3' end of the AZU gene and by 3 kilobases from the 5' end of the NE gene. We report a physical map of the gene cluster, its location on chromosome 19pter, and the exon-intron organization of the AZU and PR3 genes. Our fluorescence in situ hybridization studies disprove the previous chromosomal assignment of the human NE gene to 11q14. The five exons of AZU and PR3 are organized like those of NE and other granule-associated serine proteases of hematopoietic cells. NE, PR3, and AZU are coordinately downregulated in the premonocytic cell line U937 during induced terminal differentiation. The cluster-like physical organization of these genes and concerted regulation during hematopoietic differentiation suggests that they are located in a developmentally activated chromatin domain promoting high-level, cell-specific expression in the monocyte-myelocyte lineage.