Upregulation of interleukin 6 and granulocyte colony-stimulating factor receptors by transcription factor CCAAT enhancer binding protein alpha (C/EBP alpha) is critical for granulopoiesis.

Cytokines stimulate granulopoiesis through signaling via receptors whose expression is controlled by lineage-specific transcription factors. Previously, we demonstrated that granulocyte colony-stimulating factor (G-CSF) receptor mRNA was undetectable and granulocyte maturation blocked in CCAAT enhancer binding protein alpha (C/EBPalpha)-deficient mice. This phenotype is distinct from that of G-CSF receptor-/- mice, suggesting that other genes are likely to be adversely affected by loss of C/EBPalpha. Here we demonstrate loss of interleukin 6 (IL-6) receptor and IL-6-responsive colony-forming units (CFU-IL6) in C/EBPalpha-/- mice. The observed failure of granulopoiesis could be rescued by the addition of soluble IL-6 receptor and IL-6 or by retroviral transduction of G-CSF receptors, demonstrating that loss of both of these receptors contributes to the absolute block in granulocyte maturation observed in C/EBPalpha-deficient hematopoietic cells. The results of these and other studies suggest that additional C/EBPalpha target genes, possibly other cytokine receptors, are also important for the block in granulocyte differentiation observed in vivo in C/EBPalpha-deficient mice.

Cooperative interaction between ETS1 and GFI1 transcription factors in the repression of Bax gene expression.

The proto-oncoproteins ETS1 and growth factor independent-1 (GFI1) are implicated in cell growth and differentiation in various types of cells, and their deregulated expression is involved in malignant transformation. Here, we report that ETS1 and GFI1 interact and affect gene expression through their cross-talk. Co-immunoprecipitation analyses and glutathione-S-transferase pull-down assays revealed that ETS1 bound directly to GFI1 via its Ets domain, and GFI1 bound to ETS1 via its zinc-finger domain. Luciferase (Luc) assays using artificial reporters showed that GFI1 repressed ETS1-mediated transcriptional activation and ETS1 repressed GFI1-mediated transcriptional activation, in a dose-dependent manner. However, in the Bax promoter where the Ets- and Gfi-binding sites (EBS and GBS) are adjacent, ETS1 and GFI1 cooperatively reduced activation. Site-directed mutagenesis on the EBS and GBS of the Bax promoter showed that both binding sites were necessary for full repression. Chromatin immunoprecipitation analyses confirmed that an ETS1-GFI1 complex formed on the Bax promoter even when either EBS or GBS was mutated. Introduction of small interfering RNA against ETS1 and/or GFI1 enhanced endogenous Bax gene expression. Our results suggest that the interaction between ETS1 and GFI1 facilitates their binding to specific sites on the Bax promoter and represses Bax expression in vivo.

Dual inhibition of EZH1/2 breaks the quiescence of leukemia stem cells in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an aggressive and lethal blood cancer originating from rare populations of leukemia stem cells (LSCs). AML relapse after conventional chemotherapy is caused by a remaining population of drug-resistant LSCs. Selective targeting of the chemoresistant population is a promising strategy for preventing and treating AML relapse. Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to maintain the stemness of LSCs. Here, we show that quiescent LSCs expressed the highest levels of enhancer of zeste (EZH) 1 and EZH2, the PRC2 catalytic subunits, in the AML hierarchy, and that dual inactivation of EZH1/2 eradicated quiescent LSCs to cure AML. Genetic deletion of Ezh1/2 in a mouse AML model induced cell cycle progression of quiescent LSCs and differentiation to LSCs, eventually eradicating AML LSCs. Quiescent LSCs showed PRC2-mediated suppression of Cyclin D, and Cyclin D-overexpressing AML was more sensitive to chemotherapy. We have developed a novel EZH1/2 dual inhibitor with potent inhibitory activity against both EZH1/2. In AML mouse models and patient-derived xenograft models, the inhibitor reduced the number of LSCs, impaired leukemia progression, and prolonged survival. Taken together, these results show that dual inhibition of EZH1/2 is an effective strategy for eliminating AML LSCs.

Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization.

Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor alpha promoter through activation and repression domains.

Interleukin-5 (IL-5) plays a central role in the differentiation, proliferation, and functional activation of eosinophils. The specific action of IL-5 on eosinophils and hematopoietically related basophils is regulated by the restricted expression of IL-5 receptor alpha (IL-5Ralpha), a subunit of high-affinity IL-5R, on these cells. We have previously identified an enhancer-like cis element in the IL-5Ralpha promoter that is important for both full promoter function and lineage-specific activity. Here, we demonstrate by yeast one-hybrid screening that RFX2 protein specifically binds to this cis element. RFX2 belongs to the RFX DNA-binding protein family, the biological role of which remains obscure. Using an electrophoretic mobility shift assay, we further show that RFX1, RFX2, and RFX3 homodimers and heterodimers specifically bind to the cis element of the IL-5Ralpha promoter. The mRNA expression of RFX1, RFX2, and RFX3 was detected ubiquitously, but in transient-transfection assays, multimerized RFX binding sites in front of a basal promoter efficiently functioned in a tissue- and lineage-specific manner. To further investigate RFX functions on transcription, full-length and deletion mutants of RFX1 were targeted to DNA through fusion to the GAL4 DNA binding domain. Tissue- and lineage-specific transcriptional activation with the full-length RFX1 fusion plasmid on a reporter controlled by GAL4 binding sites was observed. Distinct activation and repression domains within the RFX1 protein were further mapped. Our findings suggest that RFX proteins are transcription factors that contribute to the activity and lineage specificity of the IL-5Ralpha promoter by directly binding to a target cis element and cooperating with other tissue- and lineage-specific cofactors.

A novel ubiquitin-specific protease, UBP43, cloned from leukemia fusion protein AML1-ETO-expressing mice, functions in hematopoietic cell differentiation.

Using PCR-coupled subtractive screening-representational difference analysis, we have cloned a novel gene from AML1-ETO knockin mice. This gene is highly expressed in the yolk sac and fetal liver of the knockin mice. Nucleotide sequence analysis indicates that its cDNA contains an 1,107-bp open reading frame encoding a 368-amino-acid polypeptide. Further protein sequence and protein translation analysis shows that it belongs to a family of ubiquitin-specific proteases (UBP), and its molecular mass is 43 kDa. Therefore, we have named this gene UBP43. Like other ubiquitin proteases, the UBP43 protein has deubiquitinating enzyme activity. Protein ubiquitination has been implicated in many important cellular events. In wild-type adult mice, UBP43 is highly expressed in the thymus and in peritoneal macrophages. Among nine different murine hematopoietic cell lines analyzed, UBP43 expression is detectable only in cell lines related to the monocytic lineage. Furthermore, its expression is regulated during cytokine-induced monocytic cell differentiation. We have investigated its function in the hematopoietic myeloid cell line M1. UBP43 was introduced into M1 cells by retroviral gene transfer, and several high-expressing UBP43 clones were obtained for further study. Morphologic and cell surface marker examination of UBP43/M1 cells reveals that overexpression of UBP43 blocks cytokine-induced terminal differentiation of monocytic cells. These data suggest that UBP43 plays an important role in hematopoiesis by modulating either the ubiquitin-dependent proteolytic pathway or the ubiquitination state of another regulatory factor(s) during myeloid cell differentiation.

c-Myc is a critical target for c/EBPalpha in granulopoiesis.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is an integral factor in the granulocytic developmental pathway, as myeloblasts from C/EBPalpha-null mice exhibit an early block in differentiation. Since mice deficient for known C/EBPalpha target genes do not exhibit the same block in granulocyte maturation, we sought to identify additional C/EBPalpha target genes essential for myeloid cell development. To identify such genes, we used both representational difference analysis and oligonucleotide array analysis with RNA derived from a C/EBPalpha-inducible myeloid cell line. From each of these independent screens, we identified c-Myc as a C/EBPalpha negatively regulated gene. We mapped an E2F binding site in the c-Myc promoter as the cis-acting element critical for C/EBPalpha negative regulation. The identification of c-Myc as a C/EBPalpha target gene is intriguing, as it has been previously shown that down-regulation of c-Myc can induce myeloid differentiation. Here we show that stable expression of c-Myc from an exogenous promoter not responsive to C/EBPalpha-mediated down-regulation forces myeloblasts to remain in an undifferentiated state. Therefore, C/EBPalpha negative regulation of c-Myc is critical for allowing early myeloid precursors to enter a differentiation pathway. This is the first report to demonstrate that C/EBPalpha directly affects the level of c-Myc expression and, thus, the decision of myeloid blasts to enter into the granulocytic differentiation pathway.

Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome.

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

The novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces p53-independent mitochondrial apoptosis in acute myeloid leukemia progenitor cells.

Disease recurrence is the major problem in the treatment of acute myeloid leukemia (AML). Relapse is driven by leukemia stem cells, a chemoresistant subpopulation capable of re-establishing disease. Patients with p53 mutant AML are at an extremely high risk of relapse. B-cell-specific Moloney murine leukemia virus integration site 1 (BMI-1) is required for the self-renewal and maintenance of AML stem cells. Here we studied the effects of a novel small molecule inhibitor of BMI-1, PTC596, in AML cells. Treatment with PTC596 reduced MCL-1 expression and triggered several molecular events consistent with induction of mitochondrial apoptosis: loss of mitochondrial membrane potential, BAX conformational change, caspase-3 cleavage and phosphatidylserine externalization. PTC596 induced apoptosis in a p53-independent manner. PTC596 induced apoptosis along with the reduction of MCL-1 and phosphorylated AKT in patient-derived CD34+CD38low/- stem/progenitor cells. Mouse xenograft models demonstrated in vivo anti-leukemia activity of PTC596, which inhibited leukemia cell growth in vivo while sparing normal hematopoietic cells. Our results indicate that PTC596 deserves further evaluation in clinical trials for refractory or relapsed AML patients, especially for those with unfavorable complex karyotype or therapy-related AML that are frequently associated with p53 mutations.

Frequent somatic mutations in epigenetic regulators in newly diagnosed chronic myeloid leukemia.

Although tyrosine kinase inhibitors (TKIs) have significantly improved the prognosis of chronic myeloid leukemia (CML), the ability of TKIs to eradicate CML remains uncertain and patients must continue TKI therapy for indefinite periods. In this study, we performed whole-exome sequencing to identify somatic mutations in 24 patients with newly diagnosed chronic phase CML who were registered in the JALSG CML212 study. We identified 191 somatic mutations other than the BCR-ABL1 fusion gene (median 8, range 1-17). Age, hemoglobin concentration and white blood cell counts were correlated with the number of mutations. Patients with mutations ⩾6 showed higher rate of achieving major molecular response than those<6 (P=0.0381). Mutations in epigenetic regulator, ASXL1, TET2, TET3, KDM1A and MSH6 were found in 25% of patients. TET2 or TET3, AKT1 and RUNX1 were mutated in one patient each. ASXL1 was mutated within exon 12 in three cases. Mutated genes were significantly enriched with cell signaling and cell division pathways. Furthermore, DNA copy number analysis showed that 2 of 24 patients had uniparental disomy of chromosome 1p or 3q, which disappeared major molecular response was achieved. These mutations may play significant roles in CML pathogenesis in addition to the strong driver mutation BCR-ABL1.

Characterization of mouse non-receptor tyrosine kinase gene, HYL.

We previously reported a novel human non-receptor tyrosine kinase gene, HYL (hematopoietic consensus tyrosine-lacking kinase) (Sakano et al., 1994), which consists of each of the SH2 (src homology 2), SH3 and tyrosine kinase catalytic domains. HYL has unique structural features shared with CSK (C-terminal Src kinase). Recently it has also been reported that matk (Bennett et al., 1994) and Ctk (Klages et al., 1994) are isolated as novel kinases with structural similarity to CSK. Comparisons of cDNA sequence indicate the HYL, matk and Ctk are the same gene. We further characterized the mouse HYL genomic structure and HYL mRNA expression in mouse brain. The mouse HYL gene is distributed over 5.8 kb and is composed of 12 exons. The exon-intron organization is almost identical with that of human CSK. The mouse HYL gene was assigned to the R-positive C1 band of chromosome 10 by fluorescent in situ hybridization. RNA in situ hybridization demonstrated the broad distribution of HYL mRNA expression in various neuronal cells. Especially, strong signals were detected in Purkinje cells, pyramidal cells in the hippocampus, granule cells in the dentate gyrus, and mitral cells in the olfactory bulb, indicating that mRNA expression of HYL in brain is very similar to that of SRC-family kinases. These findings establish close relationship between the HYL and CSK genes and also suggest that HYL may play an important role in signal transduction through SRC-family kinases in the central nervous system.

Molecular cloning of a novel non-receptor tyrosine kinase, HYL (hematopoietic consensus tyrosine-lacking kinase).

We identified a novel non-receptor tyrosine kinase from a human megakaryoblastic cell line, UT-7, by means of a PCR-based cloning method. The HYL gene contained a SH2 and SH3 domain and a tyrosine kinase catalytic domain. The deduced amino acid sequence of the protein encoded by this gene was most homologous to CSK (c-src kinase). This gene and CSK shared some unique structural properties such as the absence of a myristylation signal and phosphorylation sites of tyrosine residues corresponding to tyrosines 416 and 527 of chicken p60c-src. Unlike CSK, the SH3 domain of HYL was unique since the ALYDY motif was absent. Northern blot analysis revealed a 2.2 kb transcript in various myeloid cell lines but not in adult tissues except for the brain and the lung, whereas CSK mRNA was ubiquitously expressed. The expression of HYL was upregulated when these myeloid cells were differentiated by induction with phorbol myristate acetate. We named this gene, hematopoietic consensus tyrosine-lacking kinase, HYL. The HYL gene was assigned to chromosome 19 at band p13. It is suggested that HYL plays a significant role in the signal transduction of hematopoietic cells.

Characterization of a ligand for receptor protein-tyrosine kinase HTK expressed in immature hematopoietic cells.

HTK is a receptor tyrosine kinase that belongs to the Eph subfamily. An extensive screening using BIAcore system revealed that a colon cancer cell line, C-1, expressed the ligand for HTK. From the conditioned medium of C-1 cells, a soluble form of ligand was purified by receptor affinity chromatography, and the isolation of full-length cDNA revealed that this ligand is identical to the human HTK ligand (HTKL) previously reported. HTK receptor tyrosine phosphorylation was induced by membrane-bound or clustered soluble HTKL but not by unclustered soluble HTKL, indicating that HTKL requires cell-to-cell interaction for receptor activation. Binding analysis demonstrated that HTKL binds to HTK with a much higher affinity (Kd: 1.23 nM) than the other transmembrane-type ligand for Eph family, LERK-2/ELKL (Kd: 135 nM). The expression of HTK in cord blood cells was upregulated after the culture in the presence of stem cell factor. Clustered soluble HTKL stimulated the proliferation of sorted HTK+ cord blood cells and a hematopoietic cell line, UT-7/EPO from which HTK was isolated. These findings suggest the involvement of HTK-HTKL system in the proliferation of HTK+ hematopoietic progenitor cells in the hematopoietic environment.

The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneurons in the spinal cord during development.

Receptor tyrosine kinases (RTKs) play important roles in cellular proliferation, differentiation, and survival. We performed reverse transcriptase-polymerase chain reactions (RT-PCR) from enriched embryonic day 5 (E5) chick motoneurons by panning to identify RTKs involved in the early development of motoneuron. In situ hybridization revealed that Cek8, a member of the eph family, was specifically expressed on motoneurons at the brachial and lumbar segments of the spinal cord which innervate limb muscles, and disappeared after the naturally occurring cell death period (E6-E11). Immunohistochemistry using an anti-Cek8 monoclonal antibody showed the localization of Cek8 protein at the cell bodies and axonal fibers of motoneurons and muscles. The unique expression of Cek8 suggests its involvement in cellular survival or cell-cell interactions for specific subpopulations of developing motoneurons.

Receptor tyrosine kinases involved in hematopoietic progenitor cells.

To analyze the molecular mechanisms of the proliferation and differentiation of hematopoietic cells, we have cloned PTKs from sorted stem cells. We discuss the expression and function of receptor tyrosine kinases, STK/RON, TIE, TEK and HTK which have been cloned from these cells. STK and its ligand, MSP contributed to the motility and phagocytosis of peritoneal macrophages and bone absorption of osteoclasts. Apoptosis was induced in an erythroid cell line by the binding of MSP(MacrophageStimulating Protein). TIE, TEK and HTK were interestingly expressed in the subpopulations of stem cells and related to the myeloid differentiation. These study will indicate the heterogeneity of stem cells and their diverse differentiation.

Polycomb gene product Bmi-1 regulates stem cell self-renewal.

The Polycomb group (PcG) gene Bmi-1 has recently been implicated in the maintenance of hematopoietic stem cells (HSCs). However, the role of each component of PcG complex in HSCs and the impact of forced expression of PcG genes on stem cell self-renewal remain to be elucidated. To address these issues, we performed both loss-of-function and gain-of-function analysis on various PcG proteins. Expression analysis revealed that not only Bmi-1 but also other PcG genes are predominantly expressed in HSCs. Loss-of-function analyses, however, demonstrated that absence of Bmi-1 is preferentially linked with a profound defect in HSC self-renewal, indicating a central role for Bmi-1, but not the other components, in the maintenance of HSC self-renewal. Over-expression analysis of PcG genes also confirmed an important role of Bmi-1 in HSC self-renewal. Our findings indicate that the expression level of Bmi-1 is the critical determinant for the self-renewal capacity of HSCs. These findings uncover novel aspects of stem cell regulation exerted through epigenetic modifications by the PcG proteins.

Macrophage-stimulating protein (MSP) and its receptor, RON, stimulate human osteoclast activity but not proliferation: effect of MSP distinct from that of hepatocyte growth factor.

Stem cell-derived tyrosine kinase (STK) is a member of the hepatocyte growth factor (HGF) receptor family. The ligand for STK, macrophage-stimulating protein (MSP), is a serum protein activated by members of the coagulation cascade. The RON gene is a human homolog of the murine STK. In this study we examined the role of MSP-RON in the signal pathway of human osteoclasts. Using anti-RON antibody, we detected RON expressed in multinucleated osteoclast-like cells (OCLs) formed in cultures of human bone marrow cells. To determine bone resorption, we placed OCLs on thin films of ceramic calcium phosphate formed on quartz plate-coated slides (Millenium Biologix) and measured pit formation. MSP stimulated pit formation by OCLs in a dose-dependent manner. MSP (50 ng/mL) caused a fourfold increase in pit area compared with the control. Furthermore, we examined the effects of MSP and HGF on OCL formation by purified populations of hematopoietic progenitors. OCLs were phenotypically identified by their cross-reactivity with 23c6, a monoclonal antibody that preferentially binds to osteoclasts. HGF (50 ng/mL) stimulated the differentiation of progenitors to 23c6-positive OCLs but did not enhance bone absorption. In contrast, MSP did not affect proliferation of osteoclast precursors but stimulated bone resorption by OCLs. We conclude that the MSP signal transduction pathway plays a role in bone resorption that is distinct from that of HGF.

The effect of high sugar intake on the development of periradicular lesions in rats with type 2 diabetes.

Diabetes mellitus is associated with depression of natural defenses against infection and increases the risk of periodontal disease. However, the effects of diabetes on periradicular tissue, which differs structurally from periodontal tissue, are not known. In this study, we evaluated the effects of type 2 diabetes on the development of periradicular lesions after exposure of the pulp in the left mandibular first molar through the occlusal surface in rats. GK rats with spontaneous non-insulin-dependent diabetes mellitus and Wistar rats (controls) received a normal laboratory diet and either water or a 30% sucrose solution. At both 2 and 4 weeks after pulp exposure, histologic analysis showed that alveolar bone resorption was most severe and the periradicular lesions were largest in diabetic rats given the sucrose solution. These results suggest that the metabolic conditions produced by type 2 diabetes enhance the development of periradicular lesions in rats.

Age-dependent changes related to reproductive development in the odor preference of blowflies, Phormia regina, and fleshflies, Boetteherisca peregrina.

We investigated the preference for the odor of decaying meat in a T-maze using female and male blowflies, Phormia regina, with one of two nutritional histories (protein-containing or protein-free diet), and compared them with fleshflies, Boetteherisca peregrina. The preference showed age-dependent changes, which differed according to sex and nutritional history. Females in both nutrition groups showed a high preference for the odor. The preference of protein-fed females showed cyclic variations corresponding to the ovarian cycle, while the protein-deficient group, which never oviposited having immature eggs, did not exhibit cyclic changes and their preference remained high. These results suggest that the preference for the odor may be associated with the ovarian cycle. In males, both nutrition groups showed significant changes with age. The preference of protein-deficient males changed similarly to that of females, whereas protein-fed males were not attracted by the odor. This also suggests that the preference for the odor may be related to reproductive development. With B. peregrina, which has different reproductive natures from P. regina, the age-dependent changes in the preference did not differ so markedly with sex and nutritional history as with P. regina.

Cytotoxic effect of endodontic bacteria on periapical fibroblasts.

This study was conducted to investigate the effects of sonicated bacterial extracts (SBEs) from anaerobic Gram-negative bacteria on periapical fibroblast obtained from the apical portion of human periodontal ligaments. Porphyromonas endodontalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum were chosen from among the endodontic bacteria isolated from root canals having a periapical lesion and compared in terms of their cytotoxicity. The purpose of this study was to examine which bacteria are involved in the development of periapical inflammation. The anaerobes were cultured under strict anaerobic conditions, and the bacterial cells were then harvested by centrifugation after incubation. The concentrated cell suspensions were sonicated and subsequently centrifuged. An SBE was made of each of the filtered supernatants. Each SBE was added to cultures of periapical fibroblasts. The cell growth and proliferation were measured by the MTT method after 3, 5, and 7 days. The SBEs from P. endodontalis, P. gingivalis, and F. nucleatum inhibited the growth of the fibroblasts, whereas the SBE from P. intermedia did not inhibit it. The SBEs from P. gingivalis and F. nucleatum inhibited the fibroblast growth more strongly than did the P. endodontalis, P. gingivalis, and F. nucleatum may participate in the development of periapical lesions.