Apoptosis In Saos-2 Research Articles

Circular RNA 0086996 regulates growth and migration of osteosarcoma cells via miR-125b-5p

Circular RNAs (CircRNAs) have been found to be critical in tumorigenesis; however, the role of CircRNAs in osteosarcoma is to be further studied. In this study, we preliminarily identified the up-expressed CircRNAs and its downstream microRNA in osteosarcoma and investigated its potential regulation mechanism. Hsa_circ_0086996 (Circ_0086996) was found to upregulated in tumor tissue compared to adjacent tissue. Circ_0086996 was significantly overexpressed in osteosarcoma tissue, as well as in osteosarcoma cell lines of SAOS2 and MG-63. Circ_0086996 knockdown significantly suppressed cell proliferation, migration, and invasion. Circ_0086996 knockdown also induced cell cycle arrest in G0/G1 phaseand promoted cell apoptosis in SAOS2 and MG-63 cells. Bioinformatics analysis revealed that miR-125b-5p might be of complementary binding region with Circ_0086996, which was confirmed by dual-luciferase reporter assay. Moreover, Circ_0086996 could reverse the effect of miR-125b-5p, as knockdown of Circ_0086996 or application of miR-125b-5p both can inhibit cell proliferation, migration, invasion and promote cell apoptosis and cell cycle arrest. Our study discovers that Circ_0086996 acts as miR-125b-5p sponge to mediate the tumorigenicity, which could act as a potential biomarker for the osteosarcoma and provides a novel insight for the mechanism in osteosarcoma.

Long Non-Coding RNA FEZF1-AS1 Modulates CXCR4 to Promote Cell Proliferation, Warburg Effect and Suppress Cell Apoptosis in Osteosarcoma by Sponging miR-144.

BackgroundOsteosarcoma (OS) is a common bone tumor among children, adolescents, and young adults. Long non-coding RNA (lncRNA) FEZF1 antisense RNA 1 (FEZF1-AS1) has been reported as an oncogene in diverse tumors including colorectal cancer, pancreatic cancer and hepatocellular carcinoma, as well as in osteosarcoma. This study focused on the functions and mechanism of lncRNA FEZF1-AS1 in osteosarcoma.MethodsThe levels of FEZF1-AS1, microRNA miR-144 and CXC motif chemokine receptor 4 (CXCR4) in OS tissues and cells (Saos-2 and HOS) were measured by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot assay. The interactions between miR-144 and FEZF1-AS1 or CXCR4 were predicted by DIANA tools online database. Then, the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to validate the interactions. Moreover, the cell viability and apoptotic rate in transferred Saos-2 and HOS cells were assessed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry, respectively. The levels of glucose and lactate productions were measured by glucose uptake and lactate production assay. In addition, the protein levels of Warburg-effect-related protein hexokinase 2 (HK2) and apoptosis-related proteins Bcl-2 or Bax in transferred Saos-2 and HOS cells were detected via Western blot assay.ResultsThe levels of FEZF1-AS1 and CXCR4 were strikingly up-regulated, and miR-144 was notably down-regulated in OS tissues and cells. DIANA tools online database exhibited that miR-144 was a direct target of FEZF1-AS1 and CXCR4 was a direct target of miR-144. Then the interactions were validated by dual-luciferase reporter assay and RIP assay. Functionally, FEZF1-AS1 silencing or miR-144 overexpression inhibited cell viability, the glucose and lactate productions and promoted cell apoptosis in Saos-2 and HOS cells. Furthermore, miR-144 inhibitor mitigated the inhibitory effects on cell viability, the glucose and lactate productions and the promoted effect on cell apoptosis rate in Saos-2 and HOS cells induced by FEZF1-AS1 depletion. Mechanistically, FEZF1-AS1 regulated CXCR4 in Saos-2 and HOS cells by sponging miR-144.ConclusionWe verified that FEZF1-AS1, CXCR4 were up-regulated, and miR-144 was downregulated in OS tissues and cells. Furthermore, FEZF1-AS1 promoted cell proliferation, Warburg effect and suppressed cell apoptosis in osteosarcoma via miR-144/CXCR4 axis, this novel pathway may provide a basis for the further study of osteosarcoma.

Upregulation of miR‑95-3p inhibits growth of osteosarcoma by targeting HDGF

Osteosarcoma is the most common bone malignancy and miR-95-3p plays an important role in multiple cancers. The purpose of this study was to explore the effect and potential mechanism of miR-95-3p on the growth of osteosarcoma. In vitro, the osteosarcoma cell lines, SAOS-2 and U2OS cells, were transfected with miR-95-agomir to assess the role of miR-95-3p in proliferation and apoptosis of osteosarcoma cells. We determined that overexpression of miR-95-3p significantly attenuated cell proliferation but enhanced apoptosis in SAOS-2 and U2OS cells. We also found that overexpression of miR-95-3p in osteosarcoma cells downregulated the expression of hepatoma-derived growth factor (HDGF). Next, knockdown of HDGF by siRNA targeting HDGF clearly inhibited cell proliferation and induced apoptosis in U2OS cells. In vivo, a tumor formation assay in BALB/c nude mice was conducted by injecting the pre-miR-95 or control vector lentivirus-infected U2OS cells to determine the effect of miR-95-3p on the growth of osteosarcoma. Results showed miR-95-3p overexpression inhibited the osteosarcoma growth and downregulated the HDGF expression in xenografted tumor. For mechanism study, we co-transfected HDGF/pcDNA3.1 plasmid and miR-95-agomir to U2OS cells, and we demonstrated that overexpression of HDGF could attenuate the effects of miR-95-3p on U2OS cell proliferation, apoptosis and migration. These findings indicated that miR-95-3p might act as a potential tumor suppressor in osteosarcoma by targeting HDGF. Thus, miR-95-3p may become a potential therapeutic in treatment of osteosarcoma.

Inositol polyphosphate-4-phosphatase type II and rucaparib treatment inhibit the growth of osteosarcoma cells dependent on phosphoinositide 3-kinase/protein kinase B pathway.

Osteosarcoma (OS) is an aggressive malignant tumor of bone, which often occurs in children and adolescents. Currently, the effective method for the treatment of OS is still limited. The study aimed to investigate the synergistic antitumor effect of inositol polyphosphate-4-phosphatase, type-II (INPP4B) and rucaparib on OS cells. The expression levels of INPP4B in OS tissues and OS cell lines were examined by quantitative real-time polymerase chain reaction and Western blot analysis. SaOS2 and U2OS cells were then transfected with overexpression vector of INPP4B or were treated with different concentrations of rucaparib, and cell viability, cell cycle, and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry. Western blot assay uncovered the combined effects of INPP4B and rucaparib on cell cycle, apoptosis and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signal pathway. Further, the tumor formation was examined in vivo. Results showed that INPP4B was low expressed in OS tissues and in OS cell lines. INPP4B overexpression significantly decreased cell viability and induced apoptosis in SaOS2 and U2OS cells. Additionally, rucaparib remarkably reduced cell viability in a dose-dependent and time-dependent manner. Meanwhile, rucaparib suppressed cell cycle progression in the S phase and promoted apoptosis in a dose-dependent manner. Further, combination of INPP4B overexpression and rucaparib declined Myc, cyclin E1 and cyclin D1 expressions, enhanced Bad, Bax, and cleaved-caspase-3 expressions, and blocked PI3K/AKT signal pathway in SaOS2 and U2OS cells. Finally, combination of INPP4B overexpression and rucaparib inhibited tumor formation in vivo. The study demonstrated that INPP4B and rucaparib exhibited synergistic antitumor effect by regulating PI3K/AKT pathway in OS cells.

SIX1 reduces the expression of PTEN via activating PI3K/AKT signal to promote cell proliferation and tumorigenesis in osteosarcoma

ObjectiveOsteosarcoma is the most common form of primary malignant bone cancer which is most prevalent in children and adolescents. Dysregulated expressions of SIX1 and PTEN/PI3K/AKT have been demonstrated in bone malignancies including osteosarcoma. However, the mechanism of SIX1/PTEN/PI3K/AKT on osteosarcoma progression remains unknown. Therefore, this study aims to investigate the molecular mechanism of SIX1 and PTEN/PI3K/AKT on osteosarcoma progression. MethodsIn this study, we first examined the expression of SIX1 and PTEN in human osteosarcoma tissues or blood samples and cell lines by immunohistochemistry, western blot analysis and qPCR. MTT, clone formation assay, wound healing assay, Transwell assay, in vivo tumorigenesis, flow cytometry and western blot were used to determine the function of SIX1/PTEN on cell proliferation, clone formation ability, migration, invasion, tumorigenesis, and cell apoptosis in SAOS2 and U2OS cells, respectively. ResultsResults showed that SIX1 was overexpressed in osteosarcoma tissues, blood samples and cell lines, whereas PTEN expression was reduced. SIX1 promoted cell growth, migration, invasion, and suppressed cell apoptosis. Up-regulation of SIX1 associated with reduced expression of PTEN and activation of PI3K/AKT signaling pathway. Down-regulated the expression of PTEN using gene transfer in U2OS and SAOS2 cells increased cell proliferation and inhibited cell apoptosis through activating PI3K/AKT signaling cascade. In addition, the tumorigenesis of U2OS and SAOS2 cells was suppressed when the cells were stably overexpressed SIX1 and PTEN simultaneously, compared with that in cells stably overexpressed SIX1 only. ConclusionsSIX1 promoted the progression of osteosarcoma via regulating PTEN/PI3K/AKT signaling cascade, which might provide a new potent therapeutic target for osteosarcoma.

MiR-200-3p inhibits tumor cell proliferation and induces apoptosis by upregulation of FOXO1 in osteosarcoma cells

Osteosarcoma has been reported as a severe and worldwide clinical problem, research focusing on osteosarcoma for overall analysis and understanding of the mechanism will be of great importance for theoretical basis and treatment of osteosarcoma. In the present study, we sought to explore the correlation of miR-200-3p with osteosarcoma proliferation, apoptosis and the related signaling pathway. MTT, flow cytometry, qRT-PCR and western blot were separately used to test the influence of miR-200-3p on cells proliferation, cells apopsotis and the FOXO expression. The results shown that miR-200-3p inhibits cell viability, and promotes apoptosis in Saos-2 and U2OS. The wertern blot results proved the overexpression of miR-200-3p can activate the FOXO signaling pathway by upregulating FOXO1 expression.

Inhibition of autophagy and enhancement of endoplasmic reticulum stress increase sensitivity of osteosarcoma Saos-2 cells to cannabinoid receptor agonist WIN55,212-2.

WIN55,212-2, a cannabinoid receptor agonist, can activate cannabinoid receptors, which has proven anti-tumour effects in several tumour types. Studies showed that WIN can inhibit tumour cell proliferation and induce apoptosis in diverse cancers. However, the role and mechanism of WIN in osteosarcoma are still unclear. In this study, we examined the effect of WIN55,212-2 on osteosarcoma cell line Saos-2 in terms of cell viability and apoptosis. Meanwhile, we further explored the role of endoplasmic reticulum stress and autophagy in apoptosis induced by WIN55,212-2. Our results showed that the cell proliferation of Saos-2 was inhibited by WIN55,212-2 in a dose-dependent and time-dependent manner. WIN55,212-2-induced Saos-2 apoptosis through mitochondrial apoptosis pathway. Meanwhile, WIN55,212-2 can induce endoplasmic reticulum stress and autophagy in Saos-2 cells. Inhibition of autophagy and enhancement of endoplasmic reticulum stress increased apoptosis induced by WIN55,212-2 in Saos-2 cells. These findings indicated that WIN55,212-2 in combination with autophagic inhibitor or endoplasmic reticulum stress activator may shed new light on osteosarcoma treatment. Copyright © 2016 John Wiley & Sons, Ltd.

Glucose-Dependent Insulinotropic Peptide Prevents Serum Deprivation-Induced Apoptosis in Human Bone Marrow-Derived Mesenchymal Stem Cells and Osteoblastic Cells.

Human bone marrow-derived mesenchymal stem cells (hBMSC) are able to differentiate into cells of connective tissue lineages, including bone and cartilage. They are therefore considered as a promising tool for the treatment of bone degenerative diseases. One of the major issues in regenerative cell therapy is the biosafety of fetal bovine serum used for cell culture. Therefore, the development of a culture medium devoid of serum but preserving hBMSC viability will be of clinical value. The glucose-dependent insulinotropic peptide (GIP) has an anti-apoptotic action in insulin-producing cells. Interestingly, GIP also exerts beneficial effects on bone turnover by acting on osteoblasts and osteoclasts. We therefore evaluated the ability of GIP to prevent cell death in osteoblastic cells cultured in serum-free conditions. In hBMSC and SaOS-2 cells, activation of the GIP receptor increased intracellular cAMP levels. Serum deprivation induced apoptosis in SaOS-2 and hBMSC that was reduced by 30 and 50 %, respectively, in the presence of GIP. The protective effect of GIP involves activation of the adenylate cyclase pathway and inhibition of caspases 3/7 activation. These findings demonstrate that GIP exerts a protective action against apoptosis in hBMSC and suggest a novel approach to preserve viability of hBMSC cultured in the absence of serum.

MiR-218 expression in osteosarcoma tissues and its effect on cell growth in osteosarcoma cells.

To investigate the expression of miR-218 and its clinical significance in osteosarcoma tissues and explore its effect on proliferation and apoptosis in osteosarcoma cells. miR-218 expression was detected in 76 samples of surgically resected osteosarcoma and matched normal tumor-adjacent tissues using quantitative reverse transcription polymerase chain reaction (qRT-PCR). MiR-218 was over-expressed by exogenous miR-218 plasmids in Saos-2 cells, and then BrdU cell proliferation assay and flow cytometry were used to determine cell proliferation and apoptosis. The expression of miR-218 in osteosarcoma tissues was significantly lower than those in normal tumor-adjacent tissues (t=8.735, P<0.001). MiR-218 expression in tumor tissues was significantly correlated with tumor size (χ(2)=5.380, P=0.020), clinical stage (χ(2)=6.692, P=0.010) and distant metastasis (χ(2)=4.180, P=0.041). MiR-218 was obviously over-expressed by exogenous miR-218 plasmids (t=19.42, P<0.001), and miR-218 overexpression significantly reduced cell proliferation (t=9.045, P<0.001) and induced apoptosis (t=12.38, P<0.001) in Saos-2 cells. The low-expression of miR-218 is correlated with the poor clinicopathological features in osteosarcoma. Moreover, miR-218 overexpression reduces cancer cell proliferation and induces apoptosis in Saos-2 cells, suggesting that miR-218 may play a key role in the progression of human osteosarcoma.

Icaritin suppresses the proliferation of human osteosarcoma cells in vitro by increasing apoptosis and decreasing MMP expression

To explore whether icaritin, a prenylflavonoid derivative of the Chinese tonic herb Epimedium, could suppress the proliferation of human osteosarcoma cells in vitro, and to elucidate the mechanisms of the action. Human osteosarcoma SaOS2 cell line was used in the present study. The proliferation of the cells was examined using MTT assay and immunofluorescence DAPI staining. Cell motility was studied with the scratch assay. Cell apoptosis was determined by Annexin V-FITC and PI double staining using flow cytometry. Western blotting and RT-PCR were used to measure the expression of mRNAs and proteins in the cells. Icaritin (5-15 μmol/L) suppressed the proliferation of SaOS2 cells in vitro in a dose-dependent manner. Furthermore, the cell motility was significantly decreased after exposure to icaritin. Moreover, icaritin (5 μmol/L) time-dependently induced the apoptosis of SaOS2 cells, markedly suppressed MMP-2 and MMP-9 expression, upregulated caspase-3 and caspase-9 expression, and increased the level of cleaved caspase-3 in the cells. Co-exposure to the caspase-3 inhibitor zVAD-fmk (10 μmol/L) compromised the icaritin-induced caspase-3 expression and apoptosis in SaOS2 cells. Icaritin suppresses the proliferation of SaOS2 human osteosarcoma cells by increasing apoptosis and downregulating MMP expression.

Effect of silencing human RRM 2 by specific siRNA on the biological behavior of human osteosarcoma cell

Objective To investigate the effect of small interference RNA(siRNA) targeting RRM2 on the biological behavior of human osteosarcoma cell line Saos-2 and the molecular mechanisms.Methods RRM2 expression was knocked down in human osteosarcama cell line Saos-2 by RRM2 siRNA.The expression of RRM2 mRNA and protein was determined in human osteosarcoma cell line Saos-2 and human osteoblast-like cell line hFOB1.19 by real time-PCR and Western blot.The cell proliferation was detected by CCK-8.The migration was observed by using transwell system.The apoptotic rate was observed by ELISA.The expression of CyclinD1 and Bcl-2 proteins were detected by Western blot.Results The expression of RRM2 mRNA and protein was higher in Saos-2 than in hFOB1.19.siRRM2 could down-regulate the expression of RRM2 in Saos-2 cells in a time-and concentration-dependent manner.CCK-8 assay showed that si-RRM2 could inhibit the proliferation ability of Saos-2 cells in a time-and concentrationdependent manner,but had no effect on the proliferation of hFOB1.19 cells.Transwell assay indicated that si-RRM2 could inhibit the migration of Saos-2 cells.si-RRM2 combined with adriamycin could increase the apoptosis of Saos-2 cells.Western blot showed that the expression of Cyclin D1 and Bcl-2 were decreased by silencing RRM2.Condusion RRM2 overexpression maybe associate with the osteosarcoma cells proliferation and migration and suppression of its function is a potential therapeutic strategy in osteosarcoma. Key words: RNA interference; Gene, RRM2 ; Osteosarcoma

Cadmium Induces Apoptosis in the Human Osteoblast-like Cell Line Saos-2

Human exposure to the heavy metal cadmium has been associated with the development of bone diseases, including osteoporosis and osteomalacia. The mechanisms by which cadmium exerts a direct effect on bone remain unclear. Bone cells go through apoptosis for proper bone remodeling; therefore, it was hypothesized that cadmium disrupts this normal balance by inducing apoptosis. Human osteoblast-like cells (Saos-2) were treated with 10–200 μM cadmium chloride (CdCl2) and evaluated by trypan blue staining and phase-contrast microscopy. Exposure to CdCl2 resulted in decreased cell viability and changes in cell morphology characteristic of apoptosis. The role of apoptosis in cadmium-induced toxicity was further evaluated using the fluorescent marker annexin V, which detects externalization of cell membrane phosphatidylserine. Nuclear changes associated with apoptosis were assessed by Hoechst staining and a DNA fragmentation assay. A significant increase in annexin V-positive cells was observed following CdCl2 treatment. Nuclear changes associated with apoptosis, including marginalization and condensing of chromatin and DNA fragmentation, were also observed following CdCl2 treatment. Cadmium-induced apoptosis in Saos-2 cells was also accompanied by an increase in caspase-3 activity. The addition of the caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO) or the known cadmium chelating agent potassium bis(2-hydroxyethy)dithiocarbamate, (K[bhedtc]), blocked caspase-3 activation induced by cadmium. Collectively, this study has identified a role for apoptosis in cadmium-induced toxicity in bone cells, and provides insight for future studies on mechanisms underlying the disruption of apoptotic signaling cascades in bone and the relationship to bone disease.

Inhibitory effects of a new bisphosphonate, minodronate, on proliferation and invasion of a variety of malignant bone tumor cells

Little is known about the biological effects of bisphosphonates on primary malignant bone tumors. The purpose of this study was to investigate the antitumor effects of newly developed minodronate (MIN) on a variety of human malignant bone tumors. We examined the effects of MIN and clinically relevant incadronate (INC) on the proliferation, apoptosis, and cell cycle of two osteosarcoma (Saos-2, MG-63), two chondrosarcoma (SW1353, OUMS27), and two Ewing's sarcoma (RD-ES, SK-ES-1) cell lines. Furthermore, we investigated the anti-invasion effects of MIN on sarcoma cells and the effects of MIN on tumor growth in nude mice. MIN inhibited the viability of all six cell lines in a dose-dependent manner with IC50 values of 2.7 to 5.0 microM, which were significantly lower than those of INC. Importantly, both bisphosphonates affected the viability of normal bone marrow stromal cells much less than sarcoma cells. Both bisphosphonates induced cell cycle perturbation in all sarcoma cells tested and apoptosis in Saos-2 and SW1353 cells, although they failed to induce apoptosis in RD-ES and SK-ES-1 cells. MIN significantly suppressed invasion, even at a low concentration of 1 microM (p < 0.01). Daily injection of 5 microg of MIN inhibited the growth of SK-ES-1 xenograft sarcoma in nude mice without loss of body weight. These findings suggest that MIN may have a beneficial adjuvant role in the treatment of patients with malignant bone tumors.

Effects of IGF-I and -II, IGF binding protein-3 (IGFBP-3), and transforming growth factor-beta (TGF-beta) on growth and apoptosis of human osteosarcoma Saos-2/B-10 cells: lack of IGF-independent IGFBP-3 effects.

Insulin-like growth factor binding protein-3 (IGFBP-3) inhibits cell growth. Previous reports have suggested the existence of plasma membrane IGFBP-3 receptors that could mediate direct, IGF-independent effects. Thus far, however, the only well-defined putative IGFBP-3 receptor is the type V transforming growth factor-beta (TGF-beta) receptor, a membrane glycoprotein that mediates TGF-beta-induced growth inhibition in selected cells. The aim of the study was to test whether IGFBP-3 and TGF-beta exert short-term effects in an osteosarcoma cell line that produces no IGF but contains type 1 IGF receptors. DNA synthesis and apoptosis in Saos-2/B-10 cells were measured in response to IGF-I, IGF-II, IGFBP-3 and TGF-beta2, and to type 1 IGF receptor ligands with poor affinity for IGFBP-3 ([QAYL]-IGF-I and insulin). IGF-I and IGF-II stimulated thymidine incorporation into DNA and suppressed apoptosis in a dose-dependent manner with maximal effects at 1 and 3 nM respectively. TGF-beta2 slightly increased thymidine incorporation into DNA but had no effect on apoptosis. IGFBP-3 had no effect by itself. Whereas it blocked the above effects of 1 nmol/l IGF-I, it did not block those of 1 nmol/l [QAYL]-IGF-I or 100 nmol/l insulin. IGFBP-3 does not affect DNA synthesis or apoptosis in an IGF-independent manner in IGF-responsive osteosarcoma cells. It therefore appears to act essentially by sequestration of IGF.

Deoxynybomycin is a selective anti-tumor agent inducing apoptosis and inhibiting topoisomerase I.

Deoxynybomycin was identified as an inducer of p21the/WAF1 gene following screening using a reporter, p21/luciferase. The present study examined its anti-proliferative effect on human tumor cell lines. Deoxynybomycin selectively inhibited growth of human osteoblastic sarcoma Saos-2, gastric cancer TMK-1, and monocytic leukemia THP-1 cells, but did not affect survival of normal human fibroblasts at doses up to 5 microg/ml. Results from an assay system using a panel of 39 human cancer cell lines indicated that deoxynybomycin has selective cytotoxic activity against lung carcinoma cell lines. Deoxynybomycin induced apoptosis in Saos-2, TMK-1, and THP-1 cells as revealed by DNA fragmentation and TUNEL assays. It inhibited topoisomerase I but not topoisomerase II. These results suggest that deoxynybomycin may be useful in cancer chemotherapy.

Inhibitors of protein synthesis and RNA synthesis protect against okadaic acid-induced apoptosis in human osteosarcoma cell line MG63 cells but not in Saos-2 cells.

In a previous study, we demonstrated that the protein phosphatase inhibitors, okadaic acid and calyculin A, induced apoptosis in human osteosarcoma cell lines, Saos-2 and MG63 cells. In the present study, to determine if new gene transcription and protein synthesis are required for okadaic acid-induced apoptosis in Saos-2 and MG63 cells, the cells were treated for 48h with varying concentrations of the inhibitors of protein or RNA synthesis, i.e., cycloheximide, actinomycin D, and puromycin, in the presence of a fixed dose of okadaic acid. All these reagents in different concentrations prevented the okadaic acid-induced apoptosis in MG63 cells in a dose-dependent fashion. The same concentrations of cycloheximide, actinomycin D, or puromycin alone did not induce any apoptotic features in MG63 cells. However, not all the aforementioned reagents affected okadaic acid-induced apoptosis in Saos-2 cells. Okadaic acid-induced and cycloheximide-prevented apoptosis was shown by phase-contrast microscopy, WST-1 assay, direct visualization of nuclear condensation and fragmentation of chromatin, and the characteristic DNA ladder formation on agarose gel electrophoresis. The present results indicate that the induction of new cell death genes and ongoing protein synthesis may have a role in okadaic acid-induced apoptosis in MG63 cells and that such proteins are not required in Saos-2 cells.