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Treatment of acute myeloid leukemia (AML) largely depends on chemotherapy, but current regimens have been unsatisfactory for long-term remission. Although differentiation induction therapy utilizing 1,25(OH) 2 D 3 (VD3) has shown great promise for the improvement of AML treatment efficacy, severe side effects caused by its supraphysiological dose limit its clinical application. Here we investigated the combinatorial effect of l-asparaginase (ASNase)-mediated amino acid depletion and the latent alternation of VD3 activity on the induction of myeloid differentiation. ASNase treatment enhanced VD3-driven phenotypic and functional differentiation of three-different AML cell lines into monocyte/macrophages, along with c-Myc downregulation. Using gene silencing with shRNA and a chemical blocker, we found that reduced c-Myc is a critical factor for improving VD3 efficacy. c-Myc-dependent inhibition of mTORC1 signaling and induction of autophagy were involved in the enhanced AML cell differentiation. In addition, in a postculture of AML cells after each treatment, ASNase supports the antileukemic effect of VD3 by inhibiting cell growth and inducing apoptosis. Finally, we confirmed that the administration of ASNase significantly improved VD3 efficacy in the prolongation of survival time in mice bearing tumor xenograft. Our results are the first to demonstrate the extended application of ASNase, which is currently used for acute lymphoid leukemia, in VD3-mediated differentiation induction therapy for AML, and suggest that this drug combination may be a promising novel strategy for curing AML., (© 2017 UICC.)

For a substance to be used as a drug delivery carrier and tissue inducible material for a target disease, its drug release rate and physical properties should be optimized to facilitate the healing process. We developed multi-tunable hydrogel systems with various physical properties and release behaviors to determine the optimal conditions for bone regeneration. Five injectable poly(phosphazene) hydrogels were developed with different types and amounts of anionic side-chains. The five polymer hydrogels showed considerably different in vitro and in vivo performances for sol-gel phase transition, dissolution/degradation, water uptake, and pore size. Furthermore, bone morphogenetic protein-2 (BMP-2) was loaded into the polymer hydrogels by forming nano-sized ionic-complexes with each polymer. The five types of nanocomplex hydrogels showed completely different BMP-2 release rates. By administering each nanocomplex hydrogel to mouse calvarial, we identified the most adapted nanocomplex hydrogel system for effective bone regeneration. The BMP-2 release rate was the most important factor in effective bone regeneration. Finally, the bone regeneration effect of the optimized hydrogel system was investigated in a critical-sized calvarial defect model., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Vascularization of tissue engineering constructs (TECs) in vitro is of critical importance for ensuring effective and satisfactory clinical outcomes upon implantation of TECs. Biomechanical properties of TECs have remarkable influence on the in vitro vascularization of TECs. This work utilized in vitro experiments and finite element analysis to investigate endothelial patterns in hybrid constructs of soft collagen gels and rigid macroporous poly(ε-caprolactone)-β-tricalcium phosphate (PCL-β-TCP) scaffold seeded/embedded with human umbilical vein endothelial cells (HUVECs) for bone tissue engineering applications. We first fabricated and characterized well-defined porous PCL-β-TCP scaffolds with identical pore size (500µm) but different strut sizes (200 and 400µm) using additive manufacturing (AM) technology, and then assessed the HUVEC׳s proliferation and morphogenesis within collagen, PCL-β-TCP scaffold, and the collagen-scaffold hybrid construct. Results showed that, in the hybrid construct, the cell population in the collagen component dropped by day 7 but then increased by day 14. Also, cells migrated onto the struts of the scaffold component, proliferated over time, and formed networks on the thinner struts (i.e., 200µm). Also, the thinner struts resulted in formation of long linear cellular cords structures within the pores. Finite element simulation demonstrated principal stress patterns similar to the observed cell-network pattern. It is probable that the scaffold component modulated patterns of principal stresses in the collagen component as biomechanical cues for reorganization of cell network patterns. Also, the scaffold component significantly improved the mechanical integrity of hydrogel component in the hybrid construct for weight-bearing applications. These results have collectively indicated that the manipulation of micro-architecture of scaffold could be an effective means to further regulate and guide desired cellular response in hybrid constructs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Localized and continuous osteogenic stimulation to defected sites is required for effective bone regeneration. Here, we suggest an injectable and sustained bone morphogenetic protein-2 (BMP-2) release system using thermosensitive polymeric nanoparticles bearing dual interacting forces with BMP-2. For sustained BMP-2 release, hydrophobic and ionic interactions were introduced to thermosensitive poly(phosphazene). Hydrophobic isoleucine ethyl ester and hydrophilic poly-ethylene glycol were mainly substituted to the poly(phosphazene) back bone for amphiphilicity and hydrophobic interaction with BMP-2. Carboxylic acid moiety was additionally substituted to the back bone for ionic interaction with BMP-2. These dual interacting polymeric nanoparticles (D-NPs) formed compact nanocomplexes with BMP-2. The aqueous solution of BMP-2/D-NP nanocomplexes was transformed to hydrogel when the temperature of the solution increased. Loaded BMP-2 was sustain-released for three weeks from the BMP-2/D-NP nanocomplex hydrogel. The extended BMP-2 exposure caused higher osteocalcin secretion in C2C12 cells. Significant bone generations were observed at the target site by single injection of BMP-2/D-NP nanocomplexes in vivo., (Copyright © 2015. Published by Elsevier B.V.)

Introduction: Despite good physical and biological properties, mineral trioxide aggregate (MTA) has a long setting time. A hydration accelerator could decrease the setting time of MTA. This study assessed the biocompatibility of MTA mixed with hydration accelerators (calcium chloride and low-dose citric acid) and investigated the effect of these materials on osteoblast differentiation., Methods: Cell viability was evaluated by the EZ-Cytox assay kit (Daeil Lab Service, Seoul, Korea). The gene expressions of osteocalcin and bone sialoprotein were detected by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction. The mineralization behavior was evaluated with alizarin red staining., Results: There was no statistically significant difference in cell viability between experimental groups. The messenger RNA level of osteogenic genes significantly increased in MTA mixed with hydration accelerators compared with the control and MTA mixed with water. MTA mixed with the hydration accelerators resulted in similar mineralization compared with MTA mixed with water., Conclusions: Hydration accelerators increase the osteogenic effect and show a similar effect on the mineralization of MTA, which may have clinical applications., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Because a root-end filling material comes into contact with the surrounding cells or tissues, understanding the cell-material interfacial activity is important. Thus, the purpose of this study was to assess the biocompatibility of 3 endodontic bioactive cements (MTA [Dentsply, Tulsa, OK], Bioaggregate [BA; Innovative Bioceramix, Vancouver, BC, Canada], and Biodentine [BD; Septodont, St Maur des Fosses, France]) and to investigate the effect of cements on the differentiation of mesenchymal stem cells., Methods: Cell viability, mineralization, and differentiation were evaluated using an 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay and alkaline phosphatase (ALP) staining. The expressions of ALP, osteocalcin, and bone sialoprotein at the gene level were detected by reverse-transcription polymerase chain reaction and real-time polymerase chain reaction., Results: Cell viability of BD in concentrations of 1, 1/2, and 1/4 was significantly lower than MTA and BA (P < .05). There was no statistically significant difference in cell viability between materials in concentrations of 1/10 and 1/50 (P < .05). The messenger RNA level of osteogenic genes increased significantly in the MTA and BA groups compared with controls (P < .05). However, although the messenger RNA level of osteogenic genes increased in the BD group, there was no statistically significant difference compared with controls. MTA, BA, and BD led to an increase in ALP staining compared with controls., Conclusions: In conclusion, MTA, BA, and BD have effects on osteoblast differentiation in mesenchymal stem cells, suggesting that these cements may be useful for root-end filling material., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Small heterodimer partner interacting leucine zipper protein (SMILE) is an orphan nuclear receptor and a member of the bZIP family of proteins. Several recent studies have suggested that SMILE is a novel co-repressor that is involved in nuclear receptor signaling; however, the role of SMILE in osteoblast differentiation has not yet been elucidated. This study demonstrates that SMILE inhibits osteoblast differentiation by regulating the activity of Runt-related transcription factor-2 (RUNX2). Tunicamycin, an inducer of endoplasmic reticulum stress, stimulated SMILE expression. Bone morphogenetic protein-2-induced expression of alkaline phosphatase and osteocalcin, both of which are osteogenic genes, was suppressed by SMILE. The molecular mechanism by which SMILE affects osteocalcin expression was also determined. An immunoprecipitation assay revealed a physical interaction between SMILE and RUNX2 that significantly impaired the RUNX2-dependent activation of the osteocalcin gene. A ChIP assay revealed that SMILE repressed the ability of RUNX2 to bind to the osteocalcin gene promoter. Taken together, these findings demonstrate that SMILE negatively regulates osteocalcin via a direct interaction with RUNX2., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Conventional root canal treatment is the treatment of choice for the irreversible pulpitis caused by bacterial infection. More recently, vital pulp therapy has been proposed as an alternative for management of inflamed dental pulp. Ketoprofen is an anti-inflammatory agent commonly used as a component of mouth rinse for oral lesions. Here, we examined the effect and mechanisms of action of ketoprofen on the expression of inflammatory mediators induced by the lipopolysaccharide (LPS) in dental pulp cells., Methods: Human dental pulp cells were exposed to LPS or LPS + ketoprofen, and reverse-transcription polymerase chain reaction was used to detect interleukin-1β and tumor necrosis factor α. The effect of these treatments on mitogen-activated protein kinase pathways was assessed by Western blots for extracellular signal-regulated kinase and c-Jun N-terminal kinase., Results: LPS induced interleukin-1β and tumor necrosis factor α in dental pulp cells. Ketoprofen effectively inhibited interleukin-1β and tumor necrosis factor α production in LPS-stimulated dental pulp cells. Notably, ketoprofen inhibited phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase., Conclusions: Ketoprofen inhibited expression inflammatory mediators in dental pulp cells stimulated with LPS. The inhibitory effect of ketoprofen on inflammatory cytokines is associated with inhibition of the mitogen-activated protein kinase pathway., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The aim of this study was to evaluate the biocompatibility of mineral trioxide aggregate mixed with selective hydration accelerators such as calcium chloride (CaCl2), citric acid (CA), and calcium lactate gluconate solution (CLG)., Methods: Inductively coupled plasma-atomic emission spectrometry analysis was used to measure calcium ions in the extracts of test materials. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay was performed using MG-63 cells to examine the cytotoxicity of the test materials. The surface of each sample and the growth pattern of the attached cells were observed using scanning electron microscopy (SEM)., Results: MTA mixed with 10 wt% CaCl2 and MTA mixed with 43.4 wt% CLG released a higher amount of calcium ions than the other groups. The 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay revealed that the cell viability of MTA mixed with 0.1 wt% CA was significantly higher than pure MTA on 7-day extract (P < .05). MTA mixed with 43.4 wt% CLG showed significantly higher cell viability than the other groups on 1-day extract (P < .05). MTA mixed with 10 wt% CaCl2 in all groups showed the lowest cell viability at all time points (P < .05). Under SEM, elongated and confluent cells were observed in all samples except in samples of MTA mixed with 10 wt% CaCl2., Conclusions: MTA mixed with 0.1 wt% CA showed good biocompatibility. MTA mixed with 43.4 wt% CLG showed favorable biocompatibility on 1 day. MTA mixed with 10 wt% CaCl2 in all groups showed the lowest cell viability at every time point and poor cell attachment under SEM., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Aims: MicroRNAs (miRNA) are involved in various biological processes including cellular differentiation. However, the role of miR-433 in osteoblast differentiation remains poorly understood. The objective of this study was to investigate the effect of miR-433 on BMP2-induced osteoblast differentiation., Main Methods: The expression of mature miR-433 in cells was detected by real-time PCR. RT-PCR or real-time PCR was used to confirm the expression of osteogenic genes. For the activation or inhibition of miR-433 expression, we used a precursor form of miR-433 or anti-miR-433. Functional activity of miR-433 and Runx2 was evaluated by promoter study. Osteoblast differentiation was evaluated by analyzing alkaline phosphatase (ALP) activity., Key Finding: ERRγ increased miR-433 expression in the mesenchymal stem cell lineage C3H10T1/2. During the BMP2-induction of osteoblastic differentiation of C3H10T1/2, ERRγ and miR433 expression decreased. In addition, during the osteoblastic differentiation, overexpression of ERRγ or miR-433 inhibited the expression of osteogenic marker genes such as Runx2 and ALP. A computer-based prediction algorithm led to the identification of three miR-433 binding sites [S1 (114-145 bp), S2 (3735-3766 bp) and S3 (3828-3860 bp)] on the 3'-UTR of Runx2 mRNA. Furthermore, miR-433 directly targeted S1 and S2, and decreased the level of Runx2 transcript. In addition, miR-433 inhibited BMP2-induced 6×OSE-Luc activities. Anti-miR-433 recovered ERRγ-suppressed Runx2 expression and ALP activity., Significance: These results demonstrated that miR-433 suppressed BMP2-indcued osteoblast differentiation by decreasing the level of Runx2 transcript., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Introduction: Various materials have been advocated for use as root-end filling materials. The purpose of the present in vitro study was to compare the cytotoxicity of 4 root-end filling materials: glass ionomer cement (GIC; Fuji II, GC Corp, Tokyo, Japan), reinforced zinc oxide-eugenol cement (IRM; Dentsply Tulsa Dental, Tulsa, OK), and 2 types of mineral trioxide aggregate., Methods: This study used MG-63 cells derived from a human osteosarcoma. To quantitatively evaluate the cytotoxicity of test materials, the 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay was used. The cells were exposed to the extracts and incubated. Cell viability was recorded by measuring the optical density of each test well in reference to controls. Each specimen was examined by scanning electron microscopy for the observation of cell morphology., Results: The XTT assay showed that the cell viability of ProRoot MTA (Dentsply Tulsa Dental) was higher than that of GIC and Ortho MTA (BioMTA, Seoul, Republic of Korea) at all time points. IRM showed significantly lower cell viability than the other groups. The scanning electron microscopic analysis revealed that elongated, dense, and almost confluent cells were observed in the cultures of GIC, Ortho MTA, and ProRoot MTA specimens. In contrast, cells on the surface of IRM were rounded in shape, and the numbers and the density of the cells were smaller than that in the other groups., Conclusions: ProRoot MTA and GIC showed good biocompatibility in this study. However, Ortho MTA showed lower biocompatibility compared with ProRoot MTA and GIC., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: To develop a hard coating for stainless surfaces based on silver (Ag)-platinum (Pt) alloys., Materials and Methods: Ag-Pt alloys, which have high degree of biocompatibility, excellent resistance to sterilization conditions, and antibacterial properties to different bacteria, are associated with long-term antibacterial efficiency. Approximately 1.03-µm to 2.34-µm-thick coatings, as determined by scanning electron microscopy, were deposited on stainless surfaces by the simultaneous vaporization of both metals (Ag and Pt) in an inert argon atmosphere. The coating was done by physical vapor deposition. Microorganisms and eukaryotic culture cells were grown on these surfaces., Results: The coatings released sufficient Ag ions when immersed in phosphate-buffered saline and showed significant antimicrobial potency against Streptococcus mutans and Aggregatibacter actinomycetemcomitans strains. At the same time, human gingival fibroblast cells were not adversely affected., Conclusion: Ag-Pt coatings on load-bearing orthodontic bracket surfaces can provide suitable antimicrobial activity during active orthodontic treatment.

Metformin is an oral anti-diabetic drug of the biguanide class that is commonly used to treat type 2 diabetes mellitus. This study examined the molecular mechanism for the action of metformin on osteoblast differentiation. Metformin-induced mRNA expression of the osteogenic genes and small heterodimer partner (SHP) in MC3T3E1 cells were determined by RT-PCR and real-time PCR. Metformin increased significantly the expression of the key osteogenic genes, such as alkaline phosphatase (ALP), osteocalcin (OC) and bone sialoprotein (BSP) as well as SHP. Transient transfection assays were performed in MC3T3E1 cells to confirm the effects of metformin on SHP, OC and Runx2 promoter activities. Metformin increased the transcription of the SHP and OC genes, and the metformin effect was inhibited by dominant negative form of AMPK (DN-AMPK) or compound C (an inhibitor of AMPK). The adenoviral overexpression of SHP increased significantly the level of ALP staining and OC production. However, metformin did not have any significant effect on osteogenic gene expression, ALP staining and activity, and OC production in SHP null (SHP-/-) primary calvarial cells. Moreover, upstream stimulatory factor-1 (USF-1) specifically mediated metformin-induced SHP gene expression. In addition, metformin-induced AMPK activation increased the level of Runx2 mRNA and protein. However, USF-1 and SHP were not involved in metformin-induced Runx2 expression. Transient transfection and chromatin immunoprecipitation assays confirmed that metformin-induced SHP interacts physically and forms a complex with Runx2 on the osteocalcin gene promoter in MC3T3E1 cells. These results suggest that metformin may stimulate osteoblast differentiation through the transactivation of Runx2 via AMPK/USF-1/SHP regulatory cascade in mouse calvaria-derived cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Introduction: An experimental Portland cement was manufactured with pure raw materials under controlled laboratory conditions. The aim of this study was to compare the chemical constitution, physical properties, and biocompatibility of experimentally manufactured Portland cement with those of mineral trioxide aggregate (MTA) and Portland cement., Methods: The composition of the cements was determined by scanning electron microscopy (SEM) and energy-dispersive x-ray analysis (EDAX). The setting time and compressive strength were tested. The biocompatibility was evaluated by using SEM and XTT assay., Results: SEM and EDAX revealed the experimental Portland cement to have a similar composition to Portland cement. The setting time of the experimental Portland cement was significantly shorter than that of MTA and Portland cement. The compressive strength of the experimental Portland cement was lower than that of MTA and Portland cement. The experimental Portland cement showed a similar biocompatibility to MTA., Conclusions: The experimental Portland cement might be considered as a possible substitute for MTA in clinical usage after further testing., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

The orphan nuclear receptor small heterodimer partner (SHP; NR0B2) interacts with a diverse array of transcription factors and regulates a variety of cellular events such as cell proliferation, differentiation, and metabolism. However, the role of SHP in bone formation has not yet been elucidated. SHP expression is significantly increased during osteoblast differentiation, and its expression is partially regulated by bone morphogenetic protein 2 (BMP-2), which plays an important role in bone formation. In our study, inhibition of SHP expression significantly repressed BMP-2-induced osteoblast differentiation and ectopic bone formation. In accordance with these in vitro and in vivo results, osteoblast differentiation in SHP(-/-) mice primary osteoblasts was significantly repressed, and the mice showed decreased bone mass resulting from decreased numbers of osteoblasts. Finally, SHP physically interacts and forms a complex with runt-related transcription factor 2 (Runx2) on the osteocalcin gene promoter, and overexpression of SHP increased Runx2 transactivity via competition with histone deacetylase 4 (HDAC4), an enzyme that inhibits DNA binding of Runx2 to its target genes. Taken together, these results indicate that SHP acts as a novel positive regulator of bone formation by augmenting osteoblast differentiation through regulation of the transcriptional activity of Runx2., (Copyright 2010 American Society for Bone and Mineral Research.)

Estrogen receptor-related receptor gamma (ERRgamma/ERR3/NR3B3) is a member of the orphan nuclear receptor with important functions in development and homeostasis. Recently it has been reported that ERRalpha is involved in osteoblast differentiation and bone formation. In the present study we examined the role of ERRgamma in osteoblast differentiation. Here, we showed that ERRgamma is expressed in osteoblast progenitors and primary osteoblasts, and its expression is increased temporarily by BMP2. Overexpression of ERRgamma reduced BMP2-induced alkaline phosphatase activity and osteocalcin production as well as calcified nodule formation, whereas inhibition of ERRgamma expression significantly enhanced BMP2-induced osteogenic differentiation and mineralization, suggesting that endogenous ERRgamma plays an important role in osteoblast differentiation. In addition, ERRgamma significantly repressed Runx2 transactivity on osteocalcin and bone sialoprotein promoters. We also observed that ERRgamma physically interacts with Runx2 in vitro and in vivo and competes with p300 to repress Runx2 transactivity. Notably, intramuscular injection of ERRgamma strongly inhibited BMP2-induced ectopic bone formation in a dose-dependent manner. Taken together, these results suggest that ERRgamma is a novel negative regulator of osteoblast differentiation and bone formation via its regulation of Runx2 transactivity.

Purpose: The purpose of this study was to compare properties of roughened and polished titanium with respect to their ability to attach to cells and bind to protein as well as their cell spreading behavior., Materials and Methods: Three different titanium surface treatments were compared for their ability to support cell attachment and spreading: sandblasted and acid-etched, resorbable blast media, and machine-polished titanium. The surface of the materials was characterized for surface roughness, surface energy, and surface chemistry. Osteoblast-like MG-63 cells were tested for in vitro attachment and spreading in the presence of serum proteins. Cell attachment was assessed by direct counting, dye binding, and microculture titanium assays. Cell spreading was determined by measuring area/cell in phalloidin-AlexaFluor 488 stained cells. Absorption of bovine serum albumin was determined by assay., Results: Scanning electron micrography and x-ray diffractometry confirmed increased surface roughness of the roughened materials. All 3 materials had similar albumin binding kinetics. Three different methods confirmed that roughened surfaces enhance early cell attachment to titanium in the presence of serum. Cells spread better on smoother machined surfaces than on the roughened surfaces., Conclusion: Roughened titanium surfaces exhibited better early cell attachment than smooth surfaces in the presence of serum. The cells attached to roughened titanium were less spread than those attached to machined titanium. Although albumin binding was not different for roughened surfaces, it is possible that roughened surfaces preferentially bound to serum adhesive proteins to promote early cell attachment.

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.

In Saccharomyces cerevisiae, Rad50 is reported to participate in the repair of double-stranded DNA breaks, and most rad50 mutants are unable to repair gamma-ray-induced DNA damage. In this study, we examined whether human RAD50 is involved in the repair of DNA damage induced by gamma radiation, radiomimetic alkylating agents, or UVB radiation in cultured human cells. Because homozygous null RAD50 mutant cells could not be isolated, human 293 embryonic kidney cells and A431 epithelial tumor cells were transfected with antisense RAD50 cDNA to obtain viable cell lines which expressed reduced RAD50. Selected individual clones were subjected to PCR-Southern and Western blot analyses to confirm the integrity of the antisense RAD50 construct and the reduced RAD50 expression levels. The cells engineered to express reduced RAD50 levels showed significantly increased sensitivity to gamma radiation, mitomycin C and methylmethane sulfonate compared with control cells that were transfected with the vector alone. However, there were no differences in viability of cells with reduced RAD50 levels and control cells treated with UVB radiation. These results indicate that human RAD50 is involved in the repair of DNA damage induced by gamma radiation and alkylating agents in mammalian cells and suggest the possible application of antisense RAD50 cDNA transfection as a radiation sensitizer in radiation oncology.