Increased CXCL4 expression in hematopoietic cells links inflammation and progression of bone marrow fibrosis in MPN.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that leads to progressive bone marrow (BM) fibrosis. Although the cellular mutations involved in the pathogenesis of PMF have been extensively investigated, the sequential events that drive stromal activation and fibrosis by hematopoietic-stromal cross-talk remain elusive. Using an unbiased approach and validation in patients with MPN, we determined that the differential spatial expression of the chemokine CXCL4/platelet factor-4 marks the progression of fibrosis. We show that the absence of hematopoietic CXCL4 ameliorates the MPN phenotype, reduces stromal cell activation and BM fibrosis, and decreases the activation of profibrotic pathways in megakaryocytes, inflammation in fibrosis-driving cells, and JAK/STAT activation in both megakaryocytes and stromal cells in 3 murine PMF models. Our data indicate that higher CXCL4 expression in MPN has profibrotic effects and is a mediator of the characteristic inflammation. Therefore, targeting CXCL4 might be a promising strategy to reduce inflammation in PMF.

Comprehensive clinical-molecular transplant scoring system for myelofibrosis undergoing stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation is curative in myelofibrosis, and current prognostic scoring systems aim to select patients for transplantation. Here, we aimed to develop a prognostic score to determine prognosis after transplantation itself, using clinical, molecular, and transplant-specific information from a total of 361 patients with myelofibrosis. Of these, 205 patients were used as a training cohort to create a clinical-molecular myelofibrosis transplant scoring system (MTSS), which was then externally validated in a cohort of 156 patients. Multivariable analysis on survival identified age at least 57 years, Karnofsky performance status lower than 90%, platelet count lower than 150 × 109/L, leukocyte count higher than 25 × 109/L before transplantation, HLA-mismatched unrelated donor, ASXL1 mutation, and non-CALR/MPL driver mutation genotype being independent predictors of outcome. The uncorrected concordance index for the final survival model was 0.723, and bias-corrected indices were similar. Risk factors were incorporated into a 4-level MTSS: low (score, 0-2), intermediate (score, 3-4), high (score, 5), and very high (score, >5). The 5-year survival according to risk groups in the validation cohort was 83% (95% confidence interval [CI], 71%-95%), 64% (95% CI, 53%-75%), 37% (95% CI, 17%-57%), and 22% (95% CI, 4%-39%), respectively (P < .001). Increasing score was predictive of nonrelapse mortality (P < .001) and remained applicable to primary (0.718) and post-essential thrombocythemia (ET)/polycythemia vera (PV) myelofibrosis (0.701) improving prognostic ability in comparison with all currently available disease-specific systems. In conclusion, this MTSS predicts outcome of patients with primary and post-ET/PV myelofibrosis undergoing allogeneic stem cell transplantation.

Reduced intensity allogeneic stem cell transplantation for younger patients with myelofibrosis.

Allogeneic stem cell transplantation (alloSCT) is a curative procedure for myelofibrosis. Elderly people are mainly affected, limiting the feasibility of myeloablative regimens. The introduction of reduced-intensity conditioning (RIC) made alloSCT feasible for older patients. Nevertheless, the incidence of myelofibrosis is not negligible in young patients, who are theoretically able to tolerate high-intensity therapy. Very few data are available about the efficacy of RIC-alloSCT in younger myelofibrosis patients. This study included 56 transplanted patients aged <55 years. Only 30% had a human leucocyte antigen (HLA)-matched sibling donor, the others were transplanted from a fully-matched (36%) or partially-matched (34%) unrelated donor. All transplants were conditioned according the European Society for Blood and Marrow Transplantation protocol: busulfan-fludarabine + anti-thymocyte globulin, followed by ciclosporin and mycophenolate. One patient experienced primary graft failure. Incidence of graft-versus-host disease grade II-IV was 44% (grade III/IV 23%). One-year non-relapse mortality was 7% and the 5-year cumulative incidence of relapse was 19%. After a median follow-up of 8·6 years, the estimated 5-year progression-free survival and overall survival (OS) was 68% and 82%, respectively. Patients with fully-matched donor had a 5-year OS of 92%, in contrast to 68% for those with a mismatched donor (P = 0·03). The most important outcome-determining factor is donor HLA-matching. In conclusion, RIC-alloSCT ensures optimal engraftment and low relapse rate in younger myelofibrosis patients, enabling the possibility of cure in this group.

Runx1 downregulates stem cell and megakaryocytic transcription programs that support niche interactions.

Disrupting mutations of the RUNX1 gene are found in 10% of patients with myelodysplasia (MDS) and 30% of patients with acute myeloid leukemia (AML). Previous studies have revealed an increase in hematopoietic stem cells (HSCs) and multipotent progenitor (MPP) cells in conditional Runx1-knockout (KO) mice, but the molecular mechanism is unresolved. We investigated the myeloid progenitor (MP) compartment in KO mice, arguing that disruptions at the HSC/MPP level may be amplified in downstream cells. We demonstrate that the MP compartment is increased by more than fivefold in Runx1 KO mice, with a prominent skewing toward megakaryocyte (Meg) progenitors. Runx1-deficient granulocyte-macrophage progenitors are characterized by increased cloning capacity, impaired development into mature cells, and HSC and Meg transcription signatures. An HSC/MPP subpopulation expressing Meg markers was also increased in Runx1-deficient mice. Rescue experiments coupled with transcriptome analysis and Runx1 DNA-binding assays demonstrated that granulocytic/monocytic (G/M) commitment is marked by Runx1 suppression of genes encoding adherence and motility proteins (Tek, Jam3, Plxnc1, Pcdh7, and Selp) that support HSC-Meg interactions with the BM niche. In vitro assays confirmed that enforced Tek expression in HSCs/MPPs increases Meg output. Interestingly, besides this key repressor function of Runx1 to control lineage decisions and cell numbers in progenitors, our study also revealed a critical activating function in erythroblast differentiation, in addition to its known importance in Meg and G/M maturation. Thus both repressor and activator functions of Runx1 at multiple hematopoietic stages and lineages likely contribute to the tumor suppressor activity in MDS and AML.

Impact of ruxolitinib pretreatment on outcomes after allogeneic stem cell transplantation in patients with myelofibrosis.

INTRODUCTION: Ruxolitinib is the first approved drug for treatment of myelofibrosis, but its impact of outcome after allogeneic stem cell transplantation (ASCT) is unknown. PATIENTS AND METHODS: We reported on 159 myelofibrosis patients (pts) with a median age of 59 years (r: 28-74) who received reduced intensity ASCT between 2000 and 2015 in eight German centers from related (n = 23), matched (n = 86) or mismatched (n = 50) unrelated donors. Forty-six (29%) patients received ruxolitinib at any time point prior to ASCT. The median daily dose of ruxolitinib was 30 mg (range 10-40 mg) and the median duration of treatment was 4.9 months (range 0.4-39.1 months). RESULTS: Primary graft failure was seen in 2 pts (4%) in the ruxolitinib and 3 (2%) in the non-ruxolitinib group. Engraftment and incidence of acute GVHD grade II to IV and III/IV did not differ between groups (37% vs 39% and 19% vs 28%, respectively), nor did the non-relapse mortality at 2 years (23% vs 23%). A trend for lower risk of relapse was seen in the ruxolitinib group (9% vs 17%, P = .2), resulting in a similar 2 year DFS and OS (68% vs 60% and 73% vs 70%, respectively). No difference in any outcome variable could be seen between ruxolitinib responders and those who failed or lost response to ruxolitinib. CONCLUSIONS: These results suggest that ruxolitinib pretreatment in myelofibrosis patient does not negatively influence outcome after allogeneic stem cell transplantation.

Impact of Molecular Genetics on Outcome in Myelofibrosis Patients after Allogeneic Stem Cell Transplantation.

Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P = .05), improved PFS (HR, .393; P = .01), and OS (HR, .448; P = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [P = .008], and HR, 5.451 [P = .002], respectively), whereas no impact was observed for "triple negative" patients. Molecular genetics, especially CALR, IDH2, and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis.

Endogenous retrovirus induces leukemia in a xenograft mouse model for primary myelofibrosis.

The compound immunodeficiencies in nonobese diabetic (NOD) inbred mice homozygous for the Prkdc(scid) and Il2rg(null) alleles (NSG mice) permit engraftment of a wide-range of primary human cells, enabling sophisticated modeling of human disease. In studies designed to define neoplastic stem cells of primary myelofibrosis (PMF), a myeloproliferative neoplasm characterized by profound disruption of the hematopoietic microenvironment, we observed a high frequency of acute myeloid leukemia (AML) in NSG mice. AML was of mouse origin, confined to PMF-xenografted mice, and contained multiple clonal integrations of ecotropic murine leukemia virus (E-MuLV). Significantly, MuLV replication was not only observed in diseased mice, but also in nontreated NSG controls. Furthermore, in addition to the single ecotropic endogenous retrovirus (eERV) located on chromosome 11 (Emv30) in the NOD genome, multiple de novo germ-line eERV integrations were observed in mice from each of four independent NSG mouse colonies. Analysis confirmed that E-MuLV originated from the Emv30 provirus and that recombination events were not necessary for virus replication or AML induction. Pathogenicity is thus likely attributable to PMF-mediated paracrine stimulation of mouse myeloid cells, which serve as targets for retroviral infection and transformation, as evidenced by integration into the Evi1 locus, a hotspot for retroviral-induced myeloid leukemia. This study thus corroborates a role of paracrine stimulation in PMF disease progression, underlines the importance of target cell type and numbers in MuLV-induced disease, and mandates awareness of replicating MuLV in NOD immunodeficient mice, which can significantly influence experimental results and their interpretation.

Cytokine Expression Pattern in Bone Marrow Microenvironment after Allogeneic Stem Cell Transplantation in Primary Myelofibrosis.

The only curative therapy for primary myelofibrosis (PMF) is allogeneic stem cell transplantation (ASCT). However, although we know that patients can benefit from ASCT, we do not know the extent of the changes of the expression profile of cytokines and matrix modulation factors. In this first systematic analysis, we evaluated the expression profile of 103 factors before and after transplantation to identify potential biomarkers. The expression of fibrosis-, inflammation-, and angiogenesis-associated genes was analyzed in a total of 52 bone marrow biopsies: PMF patients (n = 14) before and after ASCT and, for control purposes, post-ASCT multiple myeloma patients (n = 14) and non-neoplastic hematopoiesis (n = 10). In post-ASCT PMF cases, decreased expression of tissue inhibitor of metalloproteinases (TIMP) and platelet-derived growth factor alpha (PDGFA) correlated with bone marrow remodeling and hematological remission. Expression of several other matrix factors remained at high levels and may contribute to post-ASCT remodeling. This is the first systematic analysis of cytokine expression in post-ASCT PMF bone marrow that shows that normalization of bone marrow microenvironment is paralleled by decreased expression of TIMP and PDGFA.

JAK1/2 inhibition impairs T cell function in vitro and in patients with myeloproliferative neoplasms.

Ruxolitinib (INCB018424) is the first JAK1/JAK2 inhibitor approved for treatment of myelofibrosis. JAK/STAT-signalling is known to be involved in the regulation of CD4(+) T cells, which critically orchestrate inflammatory responses. To better understand how ruxolitinib modulates CD4(+) T cell responses, we undertook an in-depth analysis of CD4(+) T cell function upon ruxolitinib exposure. We observed a decrease in total CD3(+) cells after 3 weeks of ruxolitinib treatment in patients with myeloproliferative neoplasms. Moreover, we found that the number of regulatory T cells (Tregs), pro-inflammatory T-helper cell types 1 (Th1) and Th17 were reduced, which were validated by in vitro studies. In line with our in vitro data, we found that inflammatory cytokines [tumour necrosis factor-α (TNF), interleukin (IL)5, IL6, IL1B] were also downregulated in T cells from patients (all P < 0·05). Finally, we showed that ruxolitinib does not interfere with the T cell receptor signalling pathway, but impacts IL2-dependent STAT5 activation. These data provide a rationale for testing JAK inhibitors in diseases triggered by hyperactive CD4(+) T cells, such as autoimmune diseases. In addition, they also provide a potential explanation for the increased infection rates (i.e. viral reactivation and urinary tract infection) seen in ruxolitinib-treated patients.

CD133 marks a stem cell population that drives human primary myelofibrosis.

Primary myelofibrosis is a myeloproliferative neoplasm characterized by bone marrow fibrosis, megakaryocyte atypia, extramedullary hematopoiesis, and transformation to acute myeloid leukemia. To date the stem cell that undergoes the spatial and temporal chain of events during the development of this disease has not been identified. Here we describe a CD133(+) stem cell population that drives the pathogenesis of primary myelofibrosis. Patient-derived circulating CD133(+) but not CD34(+)CD133(-) cells, with a variable burden for JAK2 (V617F) mutation, had multipotent cloning capacity in vitro. CD133(+) cells engrafted for up to 10 months in immunocompromised mice and differentiated into JAK2-V617F(+) myeloid but not lymphoid progenitors. We observed the persistence of human, atypical JAK2-V617F(+) megakaryocytes, the initiation of a prefibrotic state, bone marrow/splenic fibrosis and transition to acute myeloid leukemia. Leukemic cells arose from a subset of CD133(+) cells harboring EZH2 (D265H) but lacking a secondary JAK2 (V617F) mutation, consistent with the hypothesis that deregulation of EZH2 activity drives clonal growth and increases the risk of acute myeloid leukemia. This is the first characterization of a patient-derived stem cell population that drives disease resembling both chronic and acute phases of primary myelofibrosis in mice. These results reveal the importance of the CD133 antigen in deciphering the neoplastic clone in primary myelofibrosis and indicate a new therapeutic target for myeloproliferative neoplasms.

Assessment of bone marrow inflammation in patients with myelofibrosis: an 18F-fluorodeoxyglucose PET/CT study.

PURPOSE: Myelofibrosis is a haematopoietic stem cell neoplasm characterized by bone marrow inflammation, reactive marrow fibrosis and extramedullary haematopoiesis. The aim of this study was to determine if (18)F-FDG PET/CT can be used to noninvasively visualize and quantify the extent and activity of bone marrow involvement. METHODS: In 30 patients, the biodistribution of (18)F-FDG was analysed by measuring the standardized uptake value in the bone marrow compartment and spleen. Imaging findings were compared with laboratory, cytogenetic and histopathological data. RESULTS: Retention of (18)F-FDG was observed in bone marrow and spleen. Bone marrow involvement varied, ranging from mildly increased uptake in the central skeleton to extensive uptake in most parts of the skeleton. The extent of bone marrow involvement decreased over time from initial diagnosis (r s = -0.43, p = 0.019). Metabolic activity of the bone marrow decreased as the histopathological grade of fibrosis increased (r s = -0.37, p = 0.04). There was a significant positive correlation between the metabolic activity of the bone marrow and that of the spleen (p = 0.04). CONCLUSION: (18)F-FDG PET/CT is as a promising technique for the quantitation of bone marrow inflammation in myelofibrosis. Our data indicate that the intensity of bone marrow (18)F-FDG uptake decreases as bone marrow fibrosis increases. Further evaluation in prospective studies is required to determine the potential clinical impact and prognostic significance of PET.

Osteoblast-specific inactivation of p53 results in locally increased bone formation.

Inactivation of the tumor suppressor p53 (encoded by the Trp53 gene) is relevant for development and growth of different cancers, including osteosarcoma, a primary bone tumor mostly affecting children and young adolescents. We have previously shown that deficiency of the ribosomal S6 kinase 2 (Rsk2) limits osteosarcoma growth in a transgenic mouse model overexpressing the proto-oncogene c-Fos. Our initial aim for the present study was to address the question, if Rsk2 deficiency would also influence osteosarcoma growth in another mouse model. For that purpose, we took advantage of Trp53fl/fl mice, which were crossed with Runx2Cre transgenic mice in order to inactivate p53 specifically in osteoblast lineage cells. However, since we unexpectedly identified Runx2Cre-mediated recombination also in the thymus, the majority of 6-month-old Trp53fl/fl;Runx2-Cre (thereafter termed Trp53Cre) animals displayed thymic lymphomas, similar to what has been described for Trp53-deficient mice. Since we did not detect osteosarcoma formation at that age, we could not follow our initial aim, but we studied the skeletal phenotype of Trp53Cre mice, with or without additional Rsk2 deficiency. Here we unexpectedly observed that Trp53Cre mice display a unique accumulation of trabecular bone in the midshaft region of the femur and the humerus, consistent with its previously established role as a negative regulator of osteoblastogenesis. Since this local bone mass increase in Trp53Cre mice was significantly reduced by Rsk2 deficiency, we isolated bone marrow cells from the different groups of mice and analyzed their behavior ex vivo. Here we observed a remarkable increase of colony formation, osteogenic differentiation and proliferation in Trp53Cre cultures, which was unaffected by Rsk2 deficiency. Our data thereby confirm a critical and tumorigenesis-independent function of p53 as a key regulator of mesenchymal cell differentiation.

Vertically transferred maternal immune cells promote neonatal immunity against early life infections.

During mammalian pregnancy, immune cells are vertically transferred from mother to fetus. The functional role of these maternal microchimeric cells (MMc) in the offspring is mostly unknown. Here we show a mouse model in which MMc numbers are either normal or low, which enables functional assessment of MMc. We report a functional role of MMc in promoting fetal immune development. MMc induces preferential differentiation of hematopoietic stem cells in fetal bone marrow towards monocytes within the myeloid compartment. Neonatal mice with higher numbers of MMc and monocytes show enhanced resilience against cytomegalovirus infection. Similarly, higher numbers of MMc in human cord blood are linked to a lower number of respiratory infections during the first year of life. Our data highlight the importance of MMc in promoting fetal immune development, potentially averting the threats caused by early life exposure to pathogens.

ASXL1/EZH2 mutations promote clonal expansion of neoplastic HSC and impair erythropoiesis in PMF.

Primary myelofibrosis (PMF) is a hematopoietic stem cell (HSC) disease, characterized by aberrant differentiation of all myeloid lineages and profound disruption of the bone marrow niche. PMF samples carry several mutations, but their cell origin and hierarchy in regulating the different waves of clonal and aberrant myeloproliferation from the prime HSC compartment is poorly understood. Genotyping of >2000 colonies from CD133+HSC and progenitors from PMF patients confirmed the complex genetic heterogeneity within the neoplastic population. Notably, mutations in chromatin regulators ASXL1 and/or EZH2 were identified as the first genetic lesions, preceding both JAK2-V617F and CALR mutations, and are thus drivers of clonal myelopoiesis in a PMF subset. HSC from PMF patients with double ASXL1/EZH2 mutations exhibited significantly higher engraftment in immunodeficient mice than those from patients without histone modifier mutations. EZH2 mutations correlate with aberrant erythropoiesis in PMF patients, exemplified by impaired maturation and cell cycle arrest of erythroid progenitors. These data underscore the importance of post-transcriptional modifiers of histones in neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones in PMF.

High Bone Turnover in Mice Carrying a Pathogenic Notch2 Mutation Causing Hajdu-Cheney Syndrome.

Hajdu-Cheney syndrome (HCS) is a rare autosomal-dominant disorder primarily characterized by acro-osteolysis and early-onset osteoporosis. Genetically, HCS is caused by nonsense or deletion mutations within exon 34 of the NOTCH2 gene, resulting in premature translational termination and production of C-terminally truncated NOTCH2 proteins that are predicted to activate NOTCH2-dependent signaling. To understand the role of Notch2 in bone remodeling, we developed a mouse model of HCS by introducing a pathogenic mutation (6272delT) into the murine Notch2 gene. By µCT and undecalcified histology, we observed generalized osteopenia in two independent mouse lines derived by injection of different targeted embryonic stem (ES) cell clones, yet acro-osteolysis did not occur until the age of 52 weeks. Cellular and dynamic histomorphometry revealed a high bone turnover situation in Notch2+/HCS mice, since osteoblast and osteoclast indices were significantly increased compared with wild-type littermates. Whereas ex vivo cultures failed to uncover cell-autonomous gain-of-functions within the osteoclast or osteoblast lineage, an unbiased RNA sequencing approach identified Tnfsf11 and Il6 as Notch-signaling target genes in bone marrow cells cultured under osteogenic conditions. Because we further observed that the high-turnover pathology of Notch2+/HCS mice was fully normalized by alendronate treatment, our results demonstrate that mutational activation of Notch2 does not directly control osteoblast activity but favors a pro-osteoclastic gene expression pattern, which in turn triggers high bone turnover. © 2017 American Society for Bone and Mineral Research.

Oncogenic JAK2V617F causes PD-L1 expression, mediating immune escape in myeloproliferative neoplasms.

Recent evidence has revealed that oncogenic mutations may confer immune escape. A better understanding of how an oncogenic mutation affects immunosuppressive programmed death ligand 1 (PD-L1) expression may help in developing new therapeutic strategies. We show that oncogenic JAK2 (Janus kinase 2) activity caused STAT3 (signal transducer and activator of transcription 3) and STAT5 phosphorylation, which enhanced PD-L1 promoter activity and PD-L1 protein expression in JAK2V617F-mutant cells, whereas blockade of JAK2 reduced PD-L1 expression in myeloid JAK2V617F-mutant cells. PD-L1 expression was higher on primary cells isolated from patients with JAK2V617F-myeloproliferative neoplasms (MPNs) compared to healthy individuals and declined upon JAK2 inhibition. JAK2V617F mutational burden, pSTAT3, and PD-L1 expression were highest in primary MPN patient-derived monocytes, megakaryocytes, and platelets. PD-1 (programmed death receptor 1) inhibition prolonged survival in human MPN xenograft and primary murine MPN models. This effect was dependent on T cells. Mechanistically, PD-L1 surface expression in JAK2V617F-mutant cells affected metabolism and cell cycle progression of T cells. In summary, we report that in MPN, constitutive JAK2/STAT3/STAT5 activation, mainly in monocytes, megakaryocytes, and platelets, caused PD-L1-mediated immune escape by reducing T cell activation, metabolic activity, and cell cycle progression. The susceptibility of JAK2V617F-mutant MPN to PD-1 targeting paves the way for immunomodulatory approaches relying on PD-1 inhibition.

Serial 18F-FDG PET for Monitoring Treatment Response After Allogeneic Stem Cell Transplantation for Myelofibrosis.

Our objective was to assess the feasibility of 18F-FDG PET/CT for noninvasive monitoring of treatment response after allogeneic stem cell transplantation (SCT) for myelofibrosis. METHODS: Twelve patients with myelofibrosis underwent 18F-FDG PET/CT before and after SCT. Bone marrow uptake, spleen uptake, and spleen size were assessed before and after SCT and compared with hematologic response criteria and bone marrow biopsies. RESULTS: All patients who did not achieve complete remission remained PET-positive (P = 0.02). Extent of disease, bone marrow metabolism, spleen metabolism, and spleen volume decreased significantly in patients with complete remission (P = 0.03). PET/CT after SCT had a sensitivity of 1.0 (95% confidence interval [CI], 0.54-1.0), a specificity of 0.83 (95% CI, 0.36-1.0), a negative predictive value of 1.0 (95% CI, 0.48-1.0), and a positive predictive value of 0.86 (95% CI, 0.42-1.0) for diagnosis of residual disease. CONCLUSION: 18F-FDG PET/CT is feasible for noninvasive monitoring of treatment response after allogeneic SCT for myelofibrosis.

Activation of Prn-p gene and stable transfection of Prn-p cDNA in leukemia MEL and neuroblastoma N2a cells increased production of PrP(C) but not prevented DNA fragmentation initiated by serum deprivation.

Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed.

Patterns of cell death and cell cycle profiles of cultured WEHI 13 var fibroblasts exposed to eluates of composite resins used for direct and indirect restorations.

Previous studies have shown that in vitro exposure to single compounds released from composite resins may induce cell death. In the present study the effects of eluates from commercially available composite resins used for direct or indirect restorations were evaluated on the cell cycle progression and type of cell death of cultured WEHI 13 var fibroblasts. Cells exposed to eluates of the materials were assessed for cytotoxicity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell death, for cell cycle profiles by flow cytometry, for caspase-3 biochemically and by immunocytochemistry, and for morphological changes by fluorescence microscopy with acridine orange. The direct composite resin eluates induced extensive apoptosis, followed by secondary necrosis. This was accompanied by cell enlargement, micromultinucleation, chromatin disintegration, cell cycle arrest at different phases, and caspase-3 activation. The composites for indirect restorations were much less cytotoxic at all biological end-points investigated. The findings suggest that composite resins used for direct and indirect dental restorations differ in their cytotoxic potential and their ability to affect basic cellular functions. This underlines the impact of improved polymerization with respect to their biologic behavior.

In vitro assessment of cytotoxicity of resin-based dental restorative materials on WEHI 13 var fibroblasts.

PURPOSE: The aim of this study was to assess the cytotoxicity of 4 resin composites' eluates on WEHI 13 Var fibroblasts as they aged in a biologic medium. MATERIALS AND METHODS: Cytotoxicity was determined by counting the number of viable cells by trypan blue exclusion. Morphologic changes attributable to cytotoxicity were observed by May-Grunwald-Giemsa cytologic staining and microscopic examination. DNA gel electrophoresis was performed to detect possible genotoxicity and DNA damage. RESULTS: All resin composite eluates, except that for Targis, caused a pronounced cytotoxicity during the first 72 hours that gradually decreased after 2 weeks of aging. Severe morphologic alterations and pronounced DNA damage were also observed. CONCLUSION: These findings indicate that resin-based dental restorative materials release agents cytotoxic and genotoxic to fibroblasts. Cytotoxicity is gradually decreased as the composite resins age in a biologic-relevant medium.