IGFBP5 domains exert distinct inhibitory effects on the tumorigenicity and metastasis of human osteosarcoma.

Osteosarcoma (OS) is the most common primary malignancy of bone. We investigated the roles of insulin-like growth factor binding protein 5 (IGFBP5) domains in modulating OS tumorigenicity and metastasis. The N-terminal (to a lesser extent the C-terminal) domain inhibited cell proliferation and induced apoptosis while the C-terminal domain inhibited cell migration and invasion. The Linker domain had no independent effects. In vivo, the N-terminal domain decreased tumor growth without affecting pulmonary metastases while the C-terminal domain inhibited tumor growth and metastases. In summary, the N- and C-terminal domains modulated OS tumorigenic phenotypes while the C-terminal domain inhibited OS metastatic phenotypes.

BMP9-regulated angiogenic signaling plays an important role in the osteogenic differentiation of mesenchymal progenitor cells.

Mesenchymal stromal progenitor cells (MSCs) are multipotent progenitors that can be isolated from numerous tissues. MSCs can undergo osteogenic differentiation under proper stimuli. We have recently demonstrated that bone morphogenetic protein 9 (BMP9) is one of the most osteogenic BMPs. As one of the least studied BMPs, BMP9 has been shown to regulate angiogenesis in endothelial cells. However, it is unclear whether BMP9-regulated angiogenic signaling plays any important role in the BMP9-initiated osteogenic pathway in MSCs. Here, we investigate the functional role of hypoxia-inducible factor 1α (HIF1α)-mediated angiogenic signaling in BMP9-regulated osteogenic differentiation of MSCs. We find that BMP9 induces HIF1α expression in MSCs through Smad1/5/8 signaling. Exogenous expression of HIF1α potentiates BMP9-induced osteogenic differentiation of MSCs both in vitro and in vivo. siRNA-mediated silencing of HIF1α or HIF1α inhibitor CAY10585 profoundly blunts BMP9-induced osteogenic signaling in MSCs. HIF1α expression regulated by cobalt-induced hypoxia also recapitulates the synergistic effect between HIF1α and BMP9 in osteogenic differentiation. Mechanistically, HIF1α is shown to exert its synergistic effect with BMP9 by inducing both angiogenic signaling and osteogenic signaling in MSCs. Thus, our findings should not only expand our understanding of the molecular basis behind BMP9-regulated osteoblastic lineage-specific differentiation, but also provide an opportunity to harness the BMP9-induced synergy between osteogenic and angiogenic signaling pathways in regenerative medicine.

Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells.

Growth hormone (GH) is usually released by somatotrophs in the anterior pituitary in response to the GH-releasing hormone and plays an important role in skeleton development and postnatal growth. However, it is unclear if extrapituitary GH exerts any effect on murine multilineage cells (MMCs). MMCs are multipotent progenitors that give rise to several lineages, including bone, cartilage, and fat. We have identified bone morphogenic protein 9 (BMP9) as one of the most osteogenic BMPs in MMCs by regulating a distinct set of downstream mediators. In this study, we find that GH is one of the most significantly upregulated genes by BMP9 in mouse MMCs through expression-profiling analysis. We confirm that GH is a direct early target of and upregulated by BMP9 signaling. Exogenous GH synergizes with BMP9 on inducing early and late osteogenic markers in MMCs. Furthermore, BMP9 and GH costimulation leads to a significant expansion of growth plate in cultured limb explants. Although GH alone does not induce de novo bone formation in an ectopic bone formation model, BMP9 and GH costimulated MMCs form more mature bone, which can be inhibited by silencing GH expression. The synergistic osteogenic activity between BMP9 and GH can be significantly blunted by JAK/STAT inhibitors, leading to a decrease in GH-regulated insulin-like growth factor 1 (IGF1) expression in MMCs. Our results strongly suggest that BMP9 may effectively regulate extrapituitary GH expression in MMCs. Thus, it is conceivable that the BMP9-GH-IGF axis may be exploited as an innovative strategy to enhance osteogenesis in regenerative medicine.

Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential.

Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.

Time-course imaging of therapeutic functional tumor vascular normalization by antiangiogenic agents.

We describe here new technology that enables noninvasive imaging of therapeutic functional normalization of tumor blood vessels by antiangiogenic agents. Noninvasive variable-magnification in vivo-fluorescence imaging as well as fluorescence tomography was used to visualize functional vessel normalization. Changes in the same vessel before and after drug treatment were imaged with high resolution in real time. Differences in vascular responses to the mTOR inhibitor rapamycin and to an anti-VEGF antibody were functionally imaged. Tumor vessel normalization was shown by significantly reduced leakiness and subsequent improved tumor delivery of Paclitaxel-BODPY as well as by normalized morphology. The tumor vascular pool agent, AngioSense(750), was retained only in tumors after either anti-VEGF antibody or rapamycin treatment, as visualized by noninvasive fluorescence tomography. The antiangiogenic therapy normalized vessels, which significantly enhanced the antitumor efficacy of paclitaxel because of increased drug penetration throughout the tumor. The optical imaging technology described here is thus a powerful, noninvasive, time-course imaging tool of functional tumor vessel normalization and its therapeutic consequences.

Tetrandrine inhibits Wnt/ß-catenin signaling and suppresses tumor growth of human colorectal cancer.

As one of the most common malignancies, colon cancer is initiated by abnormal activation of the Wnt/ß-catenin pathway. Although the treatment options have increased for some patients, overall progress has been modest. Thus, there is a great need to develop new treatments. We have found that bisbenzylisoquinoline alkaloid tetrandrine (TET) exhibits anticancer activity. TET is used as a calcium channel blocker to treat hypertensive and arrhythmic conditions in Chinese medicine. Here, we investigate the molecular basis underlying TET's anticancer activity. We compare TET with six chemotherapy drugs in eight cancer lines and find that TET exhibits comparable anticancer activities with camptothecin, vincristine, paclitaxel, and doxorubicin, and better than that of 5-fluorouracil (5-FU) and carboplatin. TET IC50 is ≤5 µM in most of the tested cancer lines. TET exhibits synergistic anticancer activity with 5-FU and reduces migration and invasion capabilities of HCT116 cells. Furthermore, TET induces apoptosis and inhibits xenograft tumor growth of colon cancer. TET treatment leads to a decrease in ß-catenin protein level in xenograft tumors, which is confirmed by T-cell factor/lymphocyte enhancer factor and c-Myc reporter assays. It is noteworthy that HCT116 cells with allelic oncogenic ß-catenin deleted are less sensitive to TET-mediated inhibition of proliferation, viability, and xenograft tumor growth. Thus, our findings strongly suggest that the anticancer effect of TET in colon cancer may be at least in part mediated by targeting ß-catenin activity. Therefore, TET may be used alone or in combination as an effective anticancer agent.

Ginsenoside Rg3 inhibits colorectal tumor growth through the down-regulation of Wnt/ß-catenin signaling.

Colorectal cancer (CRC) is one of the most common and deadly malignancies in the world. Most CRCs are initiated by aberrant activation of the Wnt/ß-catenin signaling pathway. Despite the advances in its early diagnosis, optimized surgical approaches, and chemotherapies, the clinical management of advanced CRC requires effective adjuvant agents. Ginsenoside Rg3 is a single compound isolated from American ginseng (Panax quinquefolius L., Araliaceae) and Asian ginseng (Panax ginseng C. A. Meyer). We investigated the anticancer activity of Rg3 on colon cancer cells and its potential molecular mechanism behind Rg3's anticancer activity. We found that Rg3 inhibits cell proliferation and viability of cancer cells in vitro. This inhibitory effect of Rg3 is, at least in part, mediated by blocking nuclear translocation of the ß-catenin protein and hence inhibiting ß-catenin/Tcf transcriptional activity. Allelic deletion of the oncogenic ß-catenin in HCT116 cells renders the cells more sensitive to Rg3-induced growth inhibition. Using the xenograft tumor model of human colon cancer, we have demonstrated that Rg3 effectively inhibits the growth of tumors derived from the human colon cancer cell line HCT116. Histologic examination revealed that Rg3 inhibits cancer cell proliferation, decreases PNCA expression and diminishes nuclear staining intensity of ß-catenin. Taken together, our results strongly suggest that the anticancer activity of Rg3 may be in part caused by blocking the nuclear translocation of ß-catenin in colon cancer cells. This line of investigation may lead to the development of novel therapies in which Rg3 can be used as an effective adjuvant agent for the clinical management of colorectal cancers.

Lysophosphatidic acid acyltransferase ß (LPAATß) promotes the tumor growth of human osteosarcoma.

BACKGROUND: Osteosarcoma is the most common primary malignancy of bone with poorly characterized molecular pathways important in its pathogenesis. Increasing evidence indicates that elevated lipid biosynthesis is a characteristic feature of cancer. We sought to investigate the role of lysophosphatidic acid acyltransferase ß (LPAATß, aka, AGPAT2) in regulating the proliferation and growth of human osteosarcoma cells. LPAATß can generate phosphatidic acid, which plays a key role in lipid biosynthesis as well as in cell proliferation and survival. Although elevated expression of LPAATß has been reported in several types of human tumors, the role of LPAATß in osteosarcoma progression has yet to be elucidated. METHODOLOGY/PRINCIPAL FINDINGS: Endogenous expression of LPAATß in osteosarcoma cell lines is analyzed by using semi-quantitative PCR and immunohistochemical staining. Adenovirus-mediated overexpression of LPAATß and silencing LPAATß expression is employed to determine the effect of LPAATß on osteosarcoma cell proliferation and migration in vitro and osteosarcoma tumor growth in vivo. We have found that expression of LPAATß is readily detected in 8 of the 10 analyzed human osteosarcoma lines. Exogenous expression of LPAATß promotes osteosarcoma cell proliferation and migration, while silencing LPAATß expression inhibits these cellular characteristics. We further demonstrate that exogenous expression of LPAATß effectively promotes tumor growth, while knockdown of LPAATß expression inhibits tumor growth in an orthotopic xenograft model of human osteosarcoma. CONCLUSIONS/SIGNIFICANCE: Our results strongly suggest that LPAATß expression may be associated with the aggressive phenotypes of human osteosarcoma and that LPAATß may play an important role in regulating osteosarcoma cell proliferation and tumor growth. Thus, targeting LPAATß may be exploited as a novel therapeutic strategy for the clinical management of osteosarcoma. This is especially attractive given the availability of selective pharmacological inhibitors.

Pharmacologic inactivation of kinase suppressor of Ras1 sensitizes epidermal growth factor receptor and oncogenic Ras-dependent tumors to ionizing radiation treatment.

Selective enhancement of tumor response to radiation therapy is a highly attractive objective, but it has not been met clinically. Gain-of-function Ras (gf) signaling via hyperactivation of receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR), or via oncogenic mutation of Ras is shown to confer radioresistance and requires the engagement of the Raf/MEK/ERK pathway. However, upstream mediators of such interaction in cancer cells that could be targeted for radiosensitization have not been identified and characterized. Here, we provide original observations both in vitro and in vivo that kinase suppressor of Ras1 (KSR1) is a new target for reversing gf Ras-mediated radioresistance. We employed EGFR-dependent A431 squamous cell carcinoma (SCC) and genetically defined the molecular function of KSR1 in irradiation-induced Raf/MEK/ERK activation. In vitro KSR1 inactivation via genetic inhibition of its expression or kinase function abrogated ionizing radiation-induced activation of the Raf/MEK/ERK2 cascade, enhanced the cytotoxic effect of radiation, and achieved radiosensitization associated with inhibition of DNA damage repair and enhancement of clonogenic death. In vivo pharmacologic inactivation of KSR1 by KSR1 AS-ODN infusion leads to radiosensitization in EGFR-dependent A431 SCC and in oncogenic K-Ras-driven A549 human non-small cell lung carcinoma. These observations collectively establish KSR1 as a novel target for radiosensitization and show the feasibility of using KSR1 AS-ODN as a radiosensitizer for treating gf Ras-dependent human malignancies. Identification of such mediators of gf Ras signaling in response to irradiation holds promises for improving the therapeutic efficacy of radiation therapy and our ability to eradicate tumor.

Retinoic acids potentiate BMP9-induced osteogenic differentiation of mesenchymal progenitor cells.

BACKGROUND: As one of the least studied bone morphogenetic proteins (BMPs), BMP9 is one of the most osteogenic BMPs. Retinoic acid (RA) signaling is known to play an important role in development, differentiation and bone metabolism. In this study, we investigate the effect of RA signaling on BMP9-induced osteogenic differentiation of mesenchymal progenitor cells (MPCs). METHODOLOGY/PRINCIPAL FINDINGS: Both primary MPCs and MPC line are used for BMP9 and RA stimulation. Recombinant adenoviruses are used to deliver BMP9, RARalpha and RXRalpha into MPCs. The in vitro osteogenic differentiation is monitored by determining the early and late osteogenic markers and matrix mineralization. Mouse perinatal limb explants and in vivo MPC implantation experiments are carried out to assess bone formation. We find that both 9CRA and ATRA effectively induce early osteogenic marker, such as alkaline phosphatase (ALP), and late osteogenic markers, such as osteopontin (OPN) and osteocalcin (OC). BMP9-induced osteogenic differentiation and mineralization is synergistically enhanced by 9CRA and ATRA in vitro. 9CRA and ATRA are shown to induce BMP9 expression and activate BMPR Smad-mediated transcription activity. Using mouse perinatal limb explants, we find that BMP9 and RAs act together to promote the expansion of hypertrophic chondrocyte zone at growth plate. Progenitor cell implantation studies reveal that co-expression of BMP9 and RXRalpha or RARalpha significantly increases trabecular bone and osteoid matrix formation. CONCLUSION/SIGNIFICANCE: Our results strongly suggest that retinoid signaling may synergize with BMP9 activity in promoting osteogenic differentiation of MPCs. This knowledge should expand our understanding about how BMP9 cross-talks with other signaling pathways. Furthermore, a combination of BMP9 and retinoic acid (or its agonists) may be explored as effective bone regeneration therapeutics to treat large segmental bony defects, non-union fracture, and/or osteoporotic fracture.

TGFbeta/BMP type I receptors ALK1 and ALK2 are essential for BMP9-induced osteogenic signaling in mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are bone marrow stromal cells that can differentiate into multiple lineages. We previously demonstrated that BMP9 is one of the most potent BMPs to induce osteogenic differentiation of MSCs. BMP9 is one of the least studied BMPs. Whereas ALK1, ALK5, and/or endoglin have recently been reported as potential BMP9 type I receptors in endothelial cells, little is known about type I receptor involvement in BMP9-induced osteogenic differentiation in MSCs. Here, we conduct a comprehensive analysis of the functional role of seven type I receptors in BMP9-induced osteogenic signaling in MSCs. We have found that most of the seven type I receptors are expressed in MSCs. However, using dominant-negative mutants for the seven type I receptors, we demonstrate that only ALK1 and ALK2 mutants effectively inhibit BMP9-induced osteogenic differentiation in vitro and ectopic ossification in MSC implantation assays. Protein fragment complementation assays demonstrate that ALK1 and ALK2 directly interact with BMP9. Likewise, RNAi silencing of ALK1 and ALK2 expression inhibits BMP9-induced BMPR-Smad activity and osteogenic differentiation in MSCs both in vitro and in vivo. Therefore, our results strongly suggest that ALK1 and ALK2 may play an important role in mediating BMP9-induced osteogenic differentiation. These findings should further aid us in understanding the molecular mechanism through which BMP9 regulates osteogenic differentiation of MSCs.

Insulin-like growth factor 2 (IGF-2) potentiates BMP-9-induced osteogenic differentiation and bone formation.

Efficient osteogenic differentiation and bone formation from mesenchymal stem cells (MSCs) should have clinical applications in treating nonunion fracture healing. MSCs are adherent bone marrow stromal cells that can self-renew and differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. We have identified bone morphogenetic protein 9 (BMP-9) as one of the most osteogenic BMPs. Here we investigate the effect of insulin-like growth factor 2 (IGF-2) on BMP-9-induced bone formation. We have found that endogenous IGF-2 expression is low in MSCs. Expression of IGF-2 can potentiate BMP-9-induced early osteogenic marker alkaline phosphatase (ALP) activity and the expression of later markers. IGF-2 has been shown to augment BMP-9-induced ectopic bone formation in the stem cell implantation assay. In perinatal limb explant culture assay, IGF-2 enhances BMP-9-induced endochondral ossification, whereas IGF-2 itself can promote the expansion of the hypertropic chondrocyte zone of the cultured limb explants. Expression of the IGF antagonists IGFBP3 and IGFBP4 leads to inhibition of the IGF-2 effect on BMP-9-induced ALP activity and matrix mineralization. Mechanistically, IGF-2 is further shown to enhance the BMP-9-induced BMPR-Smad reporter activity and Smad1/5/8 nuclear translocation. PI3-kinase (PI3K) inhibitor LY294002 abolishes the IGF-2 potentiation effect on BMP-9-mediated osteogenic signaling and can directly inhibit BMP-9 activity. These results demonstrate that BMP-9 crosstalks with IGF-2 through PI3K/AKT signaling pathway during osteogenic differentiation of MSCs. Taken together, our findings suggest that a combination of BMP-9 and IGF-2 may be explored as an effective bone-regeneration agent to treat large segmental bony defects, nonunion fracture, and/or osteoporotic fracture.

Synergistic antitumor effect of the activated PPARgamma and retinoid receptors on human osteosarcoma.

PURPOSE: Osteosarcoma is the most common primary malignancy of bone. The long-term survival of osteosarcoma patients hinges on our ability to prevent and/or treat recurrent and metastatic lesions. Here, we investigated the activation of peroxisome proliferator-activated receptor gamma (PPARgamma) and retinoid receptors as a means of differentiation therapy for human osteosarcoma. EXPERIMENTAL DESIGN: We examined the endogenous expression of PPARgamma and retinoid receptors in a panel of osteosarcoma cells. Ligands or adenovirus-mediated overexpression of these receptors were tested to inhibit proliferation and induce apoptosis of osteosarcoma cells. Osteosarcoma cells overexpressing the receptors were introduced into an orthotopic tumor model. The effect of these ligands on osteoblastic differentiation was further investigated. RESULTS: Endogenous expression of PPARgamma and isotypes of retinoic acid receptor (RAR) and retinoid X receptor (RXR) is detected in most osteosarcoma cells. Troglitazone, 9-cis retinoic acid (RA), and all-trans RA, as well as overexpression of PPARgamma, RARalpha, and RXRalpha, inhibit osteosarcoma cell proliferation and induce apoptosis. A synergistic inhibitory effect on osteosarcoma cell proliferation is observed between troglitazone and retinoids, as well as with the overexpression pairs of PPARgamma/RARalpha, or PPARgamma/RXRalpha. Overexpression of PPARgamma, RARalpha, RXRalpha, or in combinations inhibits osteosarcoma tumor growth and cell proliferation in vivo. Retinoids (and to a lesser extent, troglitazone) are shown to promote osteogenic differentiation of osteosarcoma cells and mesenchymal stem cells. CONCLUSIONS: Activation of PPARgamma, RARalpha, and RXRalpha may act synergistically on inhibiting osteosarcoma cell proliferation and tumor growth, which is at least partially mediated by promoting osteoblastic differentiation of osteosarcoma cells.

Human breast cancer cell lines co-express neuronal, epithelial, and melanocytic differentiation markers in vitro and in vivo.

Differentiation programs are aberrant in cancer cells allowing them to express differentiation markers in addition to their tissue of origin. In the present study, we demonstrate the multi-lineage differentiation potential of breast cancer cell lines to express multiple neuronal/glial lineage-specific markers as well as mammary epithelial and melanocytic-specific markers. Multilineage expression was detected in luminal (MCF-7 and SKBR3) and basal (MDA-MB-231) types of human breast cancer cell lines. We also observed comparable co-expression of these three cell lineage markers in MDA-MB-435 cells in vitro, in MDA-MB-435 primary tumors derived from parental and single cell clones and in lung metastases in vivo. Furthermore, ectoderm multi-lineage transdifferentiation was also found in human melanoma (Ul-MeL) and glioblastoma cell lines (U87 and D54). These observations indicate that aberrant multi-lineage transdifferentiation or lineage infidelity may be a wide spread phenomenon in cancer.

The role of the intravascular microenvironment in spontaneous metastasis development.

Metastasis is primarily responsible for the morbidity and mortality of cancer. Improved therapeutic outcomes and prognosis depend on improved understanding of mechanisms regulating the establishment of early metastasis. In this study, use of green fluorescent protein (GFP)-expressing PC-3 orthotopic model of human prostate cancer and two complementary fluorescence in vivo imaging systems (Olympus OV100 and VisEn FMT) allowed for the first time real-time characterization of cancer cell-endothelium interactions during spontaneous metastatic colonization of the liver and lung in live mice. We observed that prior to the detection of extra-vascular metastases, GFP-expressing PC-3 cancer cells resided initially inside the blood vessels of the liver and the lung, where they proliferated and expressed Ki-67 and exhibited matrix metalloprotenases (MMP) activity. Thus, the intravascular cancer cells produced their own microenvironment, where they could continue to proliferate. Extravasation occurred earlier in the lung than in the liver. Our results demonstrate that the intravascular microenvironment is a critical staging area for the development of metastasis that later can invade the parenchyma. Intravascular tumor cells may represent a therapeutic target to inhibit the development of extravascular metastases. Therefore, this imageable model of intravascular metastasis may be used for evaluation of novel anti-metastatic agents.

Elevated vitamin D receptor levels in genetic hypercalciuric stone-forming rats are associated with downregulation of Snail.

Patients with idiopathic hypercalciuria (IH) and genetic hypercalciuric stone-forming (GHS) rats, an animal model of IH, are both characterized by normal serum Ca, hypercalciuria, Ca nephrolithiasis, reduced renal Ca reabsorption, and increased bone resorption. Serum 1,25-dihydroxyvitamin D [1,25(OH)(2)D] levels are elevated or normal in IH and are normal in GHS rats. In GHS rats, vitamin D receptor (VDR) protein levels are elevated in intestinal, kidney, and bone cells, and in IH, peripheral blood monocyte VDR levels are high. The high VDR is thought to amplify the target-tissue actions of normal circulating 1,25(OH)(2)D levels to increase Ca transport. The aim of this study was to elucidate the molecular mechanisms whereby Snail may contribute to the high VDR levels in GHS rats. In the study, Snail gene expression and protein levels were lower in GHS rat tissues and inversely correlated with VDR gene expression and protein levels in intestine and kidney cells. In human kidney and colon cell lines, ChIP assays revealed endogenous Snail binding close to specific E-box sequences within the human VDR promoter region, whereas only one E-box specifically bound Snail in the rat promoter. Snail binding to rat VDR promoter E-box regions was reduced in GHS compared with normal control intestine and was accompanied by hyperacetylation of histone H(3). These results provide evidence that elevated VDR in GHS rats likely occurs because of derepression resulting from reduced Snail binding to the VDR promoter and hyperacetylation of histone H(3).

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells.

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the alpha-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARalpha and/or RXRalpha is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.

Establishment and characterization of a new highly metastatic human osteosarcoma cell line.

Osteosarcoma is the most common primary malignancy of bone in children and young adults. There is a paucity of tumorigenic and highly metastatic human osteosarcoma cell lines that have not been further transformed by exogenous means. Here we establish and characterize a highly metastatic human osteosarcoma cell line that is derived from a poorly metastatic MG63 line through serial passage in nude mice via intratibial injections. The occasional pulmonary metastases developed from MG63 were harvested and repassaged in mice until a highly metastatic subline (MG63.2) was established. The parental MG63 and highly metastatic MG63.2 cells were further characterized in vitro and in vivo. MG63.2 cells demonstrated increased cell migration and invasion compared to the parental MG63 cells. Conversely, cell adhesion was significantly greater in MG63 cells when compared to the MG63.2 cells. MG63.2 cells grew at a slightly slower rate than that of the parental cells. When injected into nude mice, MG63.2 cells had a greater than 200-fold increase in developing pulmonary metastases compared to the parental MG63 cells. MG63.2 cells also formed larger primary tumors when compared to the parental MG63 cells. Further analysis revealed that ezrin expression was up-regulated in the metastatic MG63.2 cells. Interestingly, expressions of MMP-2 and MMP-9 were down-regulated, and expression of TIMP-2 was up-regulated in the MG63.2 cells. Taken together, we have established and characterized a highly metastatic human osteosarcoma cell line that should serve as a valuable tool for future investigations on the pathogenesis, metastasis, and potential treatments of human osteosarcoma.