Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion.     

mTOR Signal Transduction Pathways Contribute to TN-C FNIII A1 Overexpression by Mechanical Stress in Osteosarcoma Cells

Osteosarcoma is the most common primary malignant bone tumor with a very poor prognosis. Treating osteosarcoma remains a challenge due to its high transitivity. Tenascin-C, with large molecular weight variants including different combinations of its alternative spliced FNIII repeats, is specifically over expressed in tumor tissues. This study examined the expression of Tenascin-C FNIIIA1 in osteosarcoma tissues, and estimated the effect of mechanical stimulation on A1 expression in MG-63 cells. Through immunohistochemical analysis, we found that the A1 protein was expressed at a higher level in osteosarcoma tissues than in adjacent normal tissues. By cell migration assay, we observed that there was a significant correlation between A1 expression and MG-63 cell migra-tion. The relation is that Tenascin-C FNIIIA1 can promote MG-63 cell migration. According to our further study into the effect of mechanical stimulation on A1 expression in MG-63 cells, the mRNA and protein levels of A1 were significantly up-regulated under mechanical stress with the mTOR molecule proving indispensable. Meanwhile, 4E-BP1 and S6K1 (downstream molecule of mTOR) are necessary for A1 normal expression in MG-63 cells whether or not mechanical stress has been encountered. We found that Tenascin-C FNIIIA1 is over-expressed in osteosar-coma tissues and can promote MG-63 cell migration. Furthermore, mechanical stress can facilitate MG-63 cell migration though facilitating A1 overexpression with the necessary molecules (mTOR, 4E-BP1 and S6K1). In con-clusion, high expression of A1 may promote the meta-stasis of osteosarcoma by facilitating MG-63 cell migration. Tenascin-C FNIIIA1 could be used as an indicator in metastatic osteosarcoma patients.     

褪黑素对骨肉瘤MG-63细胞的增殖和抑制作用的体外实验研究

目的：研究褪黑素对人成骨肉瘤细胞株MG-63增殖和抑制作用。方法：不同浓度的褪黑素作用于体外培养的MG-63细胞,倒置显微镜下观察MG-63细胞的形态学变化,采用四甲基偶氮唑蓝（MTT）法检测不同浓度褪黑素对MG-63存活率,细胞计数试剂8（CCK-8）法检测褪黑素对MG-63细胞增殖抑制能力,AnnexinV／PI双染法标记检测细胞凋亡情况以及黏附实验测定细胞黏附能力和培养液中LDH释放量的检测。结果：MTT法检测经褪黑素作用后的MG-63细胞存活率以及细胞黏附能力明显下降,LDH的增加提示MG-63细胞死亡率上升;褪黑素可抑制骨肉瘤细胞株MG-63细胞增殖,5mmol／L的褪黑素对MG-63细胞诱导凋亡作用最明显,不同浓度的褪黑素之间实验结果具有统计学意义。结论：褪黑素能够诱导MG-63细胞凋亡,降低存活率,抑制细胞增殖,是治疗骨肉瘤潜在的靶向药物。     

Isolation of stem-like cells from human MG-63 osteosarcoma cells using limiting dilution in combination with vincristine selection

To isolate stem-like cells from the human MG-63 osteosarcoma cell line, different subpopulations of MG-63 cells were cloned by limiting dilution and passaged to obtain different sublines. The subline with highest clonogenicity was identified using a proliferation assay, cell-cycle analysis, and soft-agar colony-forming assay. The sublines were further selected in serum-free medium containing 20 ng/ml vincristine to identify cells that could form suspended sarcospheres. Identified cells were then characterized based on morphology, cell surface markers, adipogenic and osteogenic differentiation, and tumorigenicity in nude mice. A total of 19 holoclones that could be stably passaged were obtained. Sublines A1, A3, and D1 were markedly different from other sublines and the parental cell line. Subline D1 not only had a higher colony-forming efficiency and formed larger colonies, but also possessed a shorter latency of tumorigenesis in vivo. After subline D1 was cultured in suspension in medium containing vincristine, a highly enriched subpopulation of cells that could form sarcospheres and be stably passaged were obtained. These cells, designated as MG-63-M expressed multiple markers of multipotent or embryonic stem cells and possessed the capacity for self-renewal, multilineage differentiation, and significant multi-drug resistance. Thus, our results suggest that a subpopulation of stem-like cells can be isolated from human MG-63 osteosarcoma cell line.     

基于JAK2/STAT3信号通路探讨白藜芦醇对骨肉瘤MG-63细胞凋亡及迁移的影响

摘要 目的：研究白藜芦醇(RES)通过蛋白酪氨酸激酶2/信号转导子与激活子3（JAK2/STAT3）信号通路对人骨肉瘤体外细胞株MG-63细胞凋亡、侵袭和迁移的影响。方法：体外培养MG-63细胞,以不同浓度的RES作用于MG-63细胞。Annexin V-FITC/PI双染流式细胞术检测不同时间和不同浓度的RES对MG-63细胞凋亡的影响。划痕实验和Transwell实验检测不同时间和不同浓度的RES对MG-63细胞侵袭和迁移能力的影响。免疫印迹实验检测不同时间和不同浓度的RES对MG-63细胞磷酸化蛋白酪氨酸激酶2（p-JAK2）、磷酸化信号转导子与激活子3（p-STAT3）、凋亡相关蛋白B淋巴细胞瘤-2（Bcl-2）、Bcl-2家族促凋亡蛋白（Bax）及基质金属蛋白酶（MMP）-2、MMP-9表达的影响。结果：RES浓度越高,时间越久,MG-63细胞凋亡率越高（P<0.05）。RES浓度越高,MG-63细胞迁移和侵袭能力越弱(P<0.05)。RES处理MG-63细胞后其p-JAK2、p-STAT3、Bcl-2以及MMP-2、MMP-9的表达明显降低,而Bax蛋白表达明显升高,且p-JAK2、p-STAT3、Bax、Bcl-2以及MMP-2、MMP-9的表达水平变化具有RES浓度依赖性（P<0.05）。结论：RES可能通过调控JAK2/STAT3信号通路促使人骨肉瘤MG-63细胞凋亡,并抑制MG-63细胞侵袭和迁移。     

Sanguinarine induces apoptosis of human osteosarcoma cells through the extrinsic and intrinsic pathways

The quaternary benzo[c]phenanthridine alkaloid sanguinarine inhibits the proliferation of cancerous cells from different origins, including lung, breast, pancreatic and colon, but nothing is known of its effects on osteosarcoma, a primary malignant bone tumour. We have found that sanguinarine alters the morphology and reduces the viability of MG-63 and SaOS-2 human osteosarcoma cell lines in concentration- and time-dependent manner. Incubation with 1 μmol/L sanguinarine for 4 and 24 h killed more efficiently MG-63 cells than SaOS-2 cells, while incubation with 5 μmol/L sanguinarine killed almost 100% of both cell populations within 24 h. This treatment also changed the mitochondrial membrane potential in both MG-63 and SaOS-2 cells within 1 h, caused chromatin condensation and the formation of apoptotic bodies. It activated multicaspases, and increased the activities of caspase-8 and caspase-9 in both MG-63 and SaOS-2 cells. These data highlight sanguinarine as a novel potential agent for bone cancer therapy.     

不同浓度白花丹素对骨肉瘤细胞MG-63凋亡迁移、基质金属蛋白酶及Bcl-2、Bax、Ezrin蛋白表达的影响

目的:研究不同浓度白花丹素对骨肉瘤细胞MG-63凋亡迁移、基质金属蛋白酶(MMP)及Bcl-2、Bax、Ezrin蛋白表达的影响。方法:取对数生长期的骨肉瘤MG-63细胞,传代培养成细胞株后以随机法分成对照组、低剂量组、中剂量组、高剂量组。其中对照组加入到0.1%浓度的DMSO完全培养基中培养,低剂量组、中剂量组、高剂量组分别加入到浓度为5、10、20μmol/L的白花丹素的有关培养基中培养。培养24 h后,采用Transwell法检测MG-63细胞迁移率、Hoechst33342染色法检测MG-63细胞凋亡率、Western blot法检测四组MG-63细胞的MMP-2、MMP-9、Bcl-2、Bax、Ezrin蛋白表达水平。结果:培养24 h后,低剂量组、中剂量组、高剂量组的骨肉瘤细胞MG-63凋亡率及Bax蛋白表达水平均较对照组升高(P<0.05),且随白花丹素浓度的增加而升高(P<0.05);骨肉瘤细胞MG-63的细胞迁移率、MMP-2、MMP-9、Bcl-2及Ezrin蛋白表达水平较对照组降低(P<0.05),且随白花丹素浓度的增加而降低(P<0.05)。结论:白花丹素对骨肉瘤细胞MG-63凋亡的促进作用以及迁移的抑制作用明显,其作用机制可能与抑制骨肉瘤细胞MG-63中的MMP-2、MMP-9、Bcl-2、Ezrin蛋白表达及促进Bax蛋白表达有关,且浓度越高,抑制或促进作用越明显。     

NALP1 基因与骨肉瘤细胞的相关研究

目的:探讨NALP1基因在骨肉瘤细胞株MG-63、U-2OS中的表达,以及高表达NALP1基因对于骨肉瘤细胞体外凋亡的影响。方法:使用RT-PCR、Western-blot法检测骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平并与人成骨细胞株hFOB1.19比较。将NALP1基因转染质粒PcDNA3.1,将重组质粒转染骨肉瘤细胞,分成高表达基因组、空质粒组及对照组,加入抗肿瘤药物顺铂及甲氨蝶呤促使肿瘤细胞凋亡,使用流式细胞仪测定各组肿瘤细胞凋亡率。结果:通过统计分析,骨肉瘤细胞株MG-63、U-2OS中的mRNA及蛋白表达水平均低于人成骨细胞株hFOB1.19(P<0.05),NALP1高表达组的肿瘤细胞凋亡率明显高于空白质粒组及对照组。结论:上调骨肉瘤细胞株MG-63、U-2OS中的NALP1的表达量可以促进肿瘤细胞凋亡。     

Decorin-induced growth inhibition is overcome through protracted expression and activation of epidermal growth factor receptors in osteosarcoma cells

Decorin is an established natural oncosuppressive factor whose action is being studied in detail. Recently, decorin gene therapy formulations using adenoviral vectors have been shown in several animal models with very promising results. The present study describes the first exception to the established oncosuppression model using human osteosarcoma cells. MG-63 osteosarcoma cells were found to constitutively produce decorin, and furthermore, to be resistant to decorin-induced growth arrest. On the contrary, decorin seemed to be beneficial to osteosarcoma cells because it was necessary for MG-63 cell migration and acted as a mediator, counteracting the transforming growth factor-beta2-induced cytostatic function. Efforts to determine how MG-63 cells could overcome the decorin-induced cytostatic effect established that decorin in MG-63 cells does not induce p21 expression nor does it cause protracted retraction and inactivation of the epidermal growth factor receptor. Conversely, epidermal growth factor receptor seemed to be overexpressed and continuously phosphorylated. In view of the proposed design of decorin-based anticancer therapeutic strategies, our study provides new data on pathways that cancer cells might employ to overcome the established decorin-induced growth suppression.     

Apoptosis and autophagy induced by pyropheophorbide-α methyl ester-mediated photodynamic therapy in human osteosarcoma MG-63 cells

Pyropheophorbide-α methyl ester (MPPa) was a second-generation photosensitizer with many potential applications. Here, we explored the impact of MPPa-mediated photodynamic therapy (MPPa-PDT) on the apoptosis and autophagy of human osteosarcoma (MG-63) cells as well as the relationships between apoptosis and autophagy of the cells, and investigated the related molecular mechanisms. We found that MPPa-PDT demonstrated the ability to inhibit MG-63 cell viability in an MPPa concentration- and light dose-dependent manner, and to induce apoptosis via the mitochondrial apoptosis pathway. Additionally, MPPa-PDT could also induce autophagy of MG-63 cell. Meanwhile, the ROS scavenger N-acetyl-l-cysteine (NAC) and the Jnk inhibitor SP600125 were found to inhibit the MPPa-PDT-induced autophagy, and NAC could also inhibit Jnk phosphorylation. Furthermore, pretreatment with the autophagy inhibitor 3-methyladenine or chloroquine showed the potential in reducing the apoptosis rate induced by MPPa-PDT in MG-63 cells. Our results indicated that the mitochondrial pathway was involved in MPPa-PDT-induced apoptosis of MG-63 cells. Meanwhile the ROS-Jnk signaling pathway was involved in MPPa-PDT-induced autophagy, which further promoted the apoptosis in MG-63 cells.     

Mechanosensitivity of human osteosarcoma cells and phospholipase C beta2 expression

Bone adapts to mechanical load by osteosynthesis, suggesting that osteoblasts might respond to mechanical stimuli. We therefore investigated cell proliferation and phospholipase C (PLC) expression in osteoblasts. One Hertz uniaxial stretching at 4000 microstrains significantly increased the proliferation rates of human osteoblast-like osteosarcoma cell line MG-63 and primary human osteoblasts. However, U-2/OS, SaOS-2, OST, and MNNG/HOS cells showed no significant changes in proliferation rate. We investigated the expression pattern of different isoforms of PLC in these cell lines. We were able to detect PLC beta1, beta3, gamma1, gamma2, and delta1 in all cells, but PLC beta2 was only detectable in the mechanosensitive cells. We therefore investigated the possible role of PLC beta2 in mechanotransduction. Inducible antisense expression for 24h inhibited the translation of PLC beta1 in U-2/OS cells by 35% and PLC beta2 in MG-63 by 29%. Fluid shear flow experiments with MG-63 lacking PLC beta2 revealed a significantly higher level of cells losing attachment to coverslips and a significantly lower number of cells increasing intracellular free calcium.     

Fucoidan Induces Cell Aggregation and Apoptosis in Osteosarcoma MG-63 Cells

Fucoidan, a sulfated polysaccharide found in brown algae, possesses various biological activities including anti-inflammatory and anti-cancer effects. In this study, we investigated the effects of fucoidan on the cell growth and morphology of human osteosarcoma MG-63 cells and found that fucoidan induces cell aggregation and apoptosis in osteosarcoma cells when the cells are treated with fucoidan upon seeding before becoming stably attached to the plates. Typical characteristics of apoptotic cells such as chromatin condensation and nuclear fragmentation were observed in fucoidan-treated cells. The number of annexin V-stained cells was increased by fucoidan treatment in a dose-dependent manner. Consistent with these results, cell viability and growth rate were decreased and cell spreading was inhibited in the presence of fucoidan, leading to rounded morphological changes of the cells. Our results demonstrate that fucoidan treatment results in cell aggregation through F-actin accumulation at the rounded cell cortex and apoptosis in osteosarcoma MG-63 cells.     

STIM1调控细胞运动促进骨肉瘤转移的研究

目的:明确STIM1是否参与调控细胞运动促进骨肉瘤的转移。方法:应用靶向STIM1的si RNA沉默MG-63骨肉瘤细胞中STIM1的表达,然后用侵袭实验、迁移实验以及黏附实验检测骨肉瘤细胞侵袭、迁移与黏附能力的变化,采用Western Blot检测细胞FAK和paxillin的表达及Rac1和RhoA信号通路的活性。结果:转染靶向STIM1的si RNA后,MG-63骨肉瘤细胞中STIM1的蛋白表达和m RNA表达均明显降低(P0.05),细胞的侵袭、迁移与黏附能力均显著下降(P0.05),细胞伪足与细胞骨架的重要组分FAK和paxillin的表达及调控细胞运动的Rac1和RhoA信号通路活性均显著降低(P0.05)。结论:STIM1可能通过激活RhoA和Rac1的信号通路,增加FAK和paxillin的表达,从而调控骨肉瘤细胞运动,促进骨肉瘤转移。     

Rhaponticin suppresses osteosarcoma through the inhibition of PI3K-Akt-mTOR pathway

Osteosarcoma is the frequent pediatric bone cancer where pediatric osteosarcoma incidences are more than 10% within the population. Most of the patients with osteosarcoma fall within the age of 15–30 years. Therefore, in this research, we examined the anticancer effect of Rhaponticin against the human osteosarcoma (MG-63) cells. The cytotoxicity of Rhaponticin on the MC3T3-E1 and MG-63 cells was examined through the MTT assay. The intracellular ROS accumulation, cell nuclear morphological alterations, apoptotic cell death and nuclear damages, and MMP status of Rhaponticin administered MG-63 cells were inspected by fluorescent staining techniques. The cell migration was assessed through scratch assay. The mRNA expressions of PI3K-Akt-mTOR signaling proteins were studied by RT-PCR analysis. Rhaponticin showed potent cytotoxicity, substantially inhibited the MG-63 cell growth, and displayed morphological alterations. However, rhaponticin did not affect the MC3T3-E1 cell viability. Rhaponticin administered MG-63 cells demonstrated augmented intracellular ROS accretion, weakened MMP, increased nuclear damages, and increased apoptosis. Rhaponticin effectively down-regulated the PI3K-Akt-mTOR signaling cascade in the MG-63 cells. These outcomes proved that the Rhaponticin can be a hopeful chemotherapeutic agent in the future to treat human osteosarcoma.     

No effects of pulsed electromagnetic fields on expression of cell adhesion molecules (integrin, CD44) and matrix metalloproteinase-2/9 in osteosarcoma cell lines

Pulsed electromagnetic fields (PEMF) could enhance the cytocidal effects of chemotherapeutic drugs on malignant tumor cell lines, but metastasis effects of PEMF on tumor cells have not been investigated. We investigated the effects of PEMF exposure on the expression levels of some metastasis-related molecules, including integrin α subunits (α1, α2, α3, α4, α5, α6, αv), integrin β subunits (β1, β2, β3, β4), CD44, and matrix metalloproteinase-2/9 (MMP-2/9) in four human osteosarcoma cell lines (HOS, MG-63, SAOS-2, NY) and two mouse osteosarcoma cell lines (DOS, LM8) by using FACScan analysis, gelatin zymography, and Western blot analysis. Our results indicate that PEMF exposure has no effect on the expression of some molecules that are associated with tumor cell invasion and metastasis, and therefore suggest that PEMF exposure may be safely applied to chemotherapy for osteosarcoma.     

Inhibition of MG-63 cell proliferation and PDGF-stimulated cellular processes by inhibitors of phosphatidylinositol 3-kinase

Studies on a platelet-derived growth factor (PDGF) responsive osteosarcoma cell line, MG-63, were initiated to determine the effects of phosphatidylinositol (Ptdlns) 3-kinase inhibitors on serum-stimulated cell proliferation and PDGF-stimulated DNA replication, actin rearrangements, or Ptdlns 3-kinase activity. In a dose-dependent manner, the fungal metabolite wortmannin and a quercetin derivative, LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), inhibited serum-stimulated MG-63 cell proliferation. The mitogenic effects of PDGF on MG-63 cells, as determined by incorporation of [³H]-thymidine, were also substantially inhibited in the presence of 0.10 μM wortmannin or 10 μM LY294002. Furthermore, MG-63 cells stimulated by PDGF form distinct actin-rich, finger-like membrane projections which are completely inhibited by either 0.10 μM wortmannin or 10 μM LY294002. At these same concentrations, wortmannin and LY294002 were also effective at reducing levels of phosphatidylinositol 3-phosphate in PDGF-stimulated MG-63 cells. Treatment of these cells with increasing concentrations of wortmannin reduced the level of PDGF stimulated tyrosine phosphorylation of the PDGF receptor but did not significantly affect the amount of the Ptdlns 3-kinase regulatory subunit, p85, associated with the receptor. Additionally, pretreatment of cells with 0.250 μM wortmannin followed by stimulation with PDGF resulted in a slightly reduced level of receptor autokinase activity; however, similar treatment with 50 μM LY294002 did not affect the level of autokinase activity. These results demonstrate the effects of two different Ptdlns 3-kinase inhibitors on serum- and PDGF-stimulated MG-63 cell proliferation and PDGF-stimulated morphological changes and suggest a greater role for Ptdlns 3-kinase in these processes. J. Cell. Biochem. 64:182–195. © 1997 Wiley-Liss, Inc.     

Effects of lysophosphatidic acid (LPA) signaling via LPA receptors on cellular functions associated with ATP reduction in osteosarcoma cells treated with ethidium bromide

Lysophosphatidic acid (LPA) signaling via LPA receptors (LPA₁ to LPA₆) exhibits a variety of malignant properties in cancer cells. Intracellular ATP depletion leads to the development of necrosis and apoptosis. The present study aimed to evaluate the effects of LPA receptor-mediated signaling on the regulation of cancer cell functions associated with ATP reduction. Long-term ethidium bromide (EtBr) treated (MG63-EtBr) cells were established from osteosarcoma MG-63 cells. The intracellular ATP levels of MG63-EtBr cells were significantly lower than that of MG-63 cells. LPAR2, LPAR3, LPAR4 and LPAR6 gene expressions were elevated in MG63-EtBr cells. The cell motile and invasive activities of MG63-EtBr cells were markedly higher than those of MG-63 cells. The cell motile activity of MG-63 cells was increased by LPA₄ and LPA₆ knockdowns. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 3 days. The cell survival to CDDP of MG63-EtBr cells was lower than that of MG-63 cells. LPA₂ knockdown decreased the cell survival to CDDP of MG-63 cells. The cell survival to CDDP of MG-63 cells was inhibited by (2 S)-OMPT (LPA₃ agonist). Moreover, the cell survival to CDDP of MG-63 cells was enhanced by LPA₄ and LPA₆ knockdowns. These results indicate that LPA signaling via LPA receptors is involved in the regulation of cellular functions associated with ATP reduction in MG-63 cells treated with EtBr.

     

Insulin-like growth factor I activates insulin receptor substrate 1 and Ras in human osteosarcoma cells.

Insulin-like growth factor I (IGF-I) stimulates multiplication of the human osteosarcoma cell line, MG-63, by acting through IGF-I receptor. We have characterized IGF-I stimulated phosphorylation of IRS-1, activation of Ras cycle and phosphorylation of c-Jun in this cell line. Serum starved MG-63 cells were (1) IGF-I stimulated and lysates were immunoprecipitated with polyclonal IRS-1 antibody or (2) metabolically labeled with [32P]orthophosphoric acid and then cells were treated with IGF-I. Cell lysates were immunoprecipitated with p21Ras antibody (Y13-259) and bound nucleotides were analysed by thin-layer chromatography. We demonstrated tyrosine phosphorylation of IRS-1/2 immunoprecipitated from MG-63 cells stimulated with IGF-I. We also showed an increased level of GTP in p21Ras immunoprecipitates from IGF-I treated cells. Nuclear extracts prepared from 32P-labeled cells before and after addition of IGF-I were immunoprecipitated with c-Jun antibody. After electrophoresis and autoradiography, phosphorylation of the c-Jun band was seen to be IGF-I independent. Phosphoamino acid analysis of the c-Jun band showed that phosphoserine was the major species.     

PDGF induces c-myc mRNA expression in MG-63 human osteosarcoma cells but does not stimulate cell replication

The platelet-derived growth factor (PDGF) modulated growth response of the MG-63 human osteosarcoma cell line, which neither expresses c-sis mRNA nor secretes a PDGF analogue, was characterized. Scatchard analysis demonstrated that the MG-63 cells have 23,000 receptors per cell with a Kd of 5 X 10(-11) M. The receptor became phosphorylated, in a PDGF concentration-dependent manner, when 32P-orthophosphate-labeled cells were treated with PDGF for 3 h at 4 degrees C. The phosphorylated receptor was identified by autoradiography and gel electrophoresis after isolation of the 32P-labeled receptor using a solid-phase monoclonal antibody directed against phosphotyrosine. Binding of the receptor to the antibody was inhibited by 5 mM phenyl phosphate, further suggesting that PDGF stimulated tyrosine-specific receptor autophosphorylation. In addition, treatment of MG-63 cells with PDGF for 3 h at 37 degrees C induced a 7.5-fold increase in c-myc mRNA accumulation as analyzed on Northern gels. However, MG-63 cells grew equally well in either serum-(which contains PDGF) or plasma-(which does not) supplemented medium. Furthermore, PDGF did not stimulate DNA synthesis in growth arrested MG-63 cells, nor did it potentiate DNA synthesis modulated by somatomedin C. Thus MG-63 cells are a naturally occurring cell variant in which PDGF stimulates c-myc expression but does not modulate mitogenesis.