Establishment and characterization of OS 99-1, a cell line derived from a highly aggressive primary human osteosarcoma.

Osteosarcoma is the most common form of primary bone cancer. In this study, we established a human osteosarcoma cell line (OS 99-1) from a highly aggressive primary tumor. G-banding karyotype analysis demonstrated a large number of clonal abnormalities, as well as extensive intercellular heterogeneity. Through the use of immunologic, molecular, and biochemical analyses, we characterized protein and gene expression profiles confirming the osteogenic nature of the cells. Further evaluation indicated that OS 99-1 cells maintain the capacity to differentiate in an in vitro mineralization assay as well as form tumors in the in vivo chicken embryo model. This cell line provides a useful tool to investigate the molecular mechanisms contributing to osteosarcoma and may have the potential to serve as a culture system for studies involving bone physiology.

Increased filamentous growth of Candida albicans in simulated microgravity.

Knowledge of simulated microgravity (SMG)-induced changes in the pathogenicity of microorganisms is important for success of long-term spaceflight. In a previous study using the high aspect ratio vessel bioreactor, we showed that the yeast species Saccharomyces cerevisiae underwent a significant phenotypic response when grown in modeled microgravity, which was reflected in the analysis of gene expression profiles. In this study, we establish that Candida albicans responds to SMG in a similar fashion, demonstrating that there is a conserved response among yeast to this environmental stress. We also report that the growth of C. albicans in SMG results in a morphogenic switch that is consistent with enhanced pathogenicity. Specifically, we observed an increase in filamentous forms of the organism and accompanying changes in the expression of two genes associated with the yeast-hyphal transition. The morphological response may have significant implications for astronauts' safety, as the fungal pathogen may become more virulent during spaceflight.

Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration.

Spaceflight enhances cell aggregation and random budding in Candida albicans.

This study presents the first global transcriptional profiling and phenotypic characterization of the major human opportunistic fungal pathogen, Candida albicans, grown in spaceflight conditions. Microarray analysis revealed that C. albicans subjected to short-term spaceflight culture differentially regulated 452 genes compared to synchronous ground controls, which represented 8.3% of the analyzed ORFs. Spaceflight-cultured C. albicans-induced genes involved in cell aggregation (similar to flocculation), which was validated by microscopic and flow cytometry analysis. We also observed enhanced random budding of spaceflight-cultured cells as opposed to bipolar budding patterns for ground samples, in accordance with the gene expression data. Furthermore, genes involved in antifungal agent and stress resistance were differentially regulated in spaceflight, including induction of ABC transporters and members of the major facilitator family, downregulation of ergosterol-encoding genes, and upregulation of genes involved in oxidative stress resistance. Finally, downregulation of genes involved in actin cytoskeleton was observed. Interestingly, the transcriptional regulator Cap1 and over 30% of the Cap1 regulon was differentially expressed in spaceflight-cultured C. albicans. A potential role for Cap1 in the spaceflight response of C. albicans is suggested, as this regulator is involved in random budding, cell aggregation, and oxidative stress resistance; all related to observed spaceflight-associated changes of C. albicans. While culture of C. albicans in microgravity potentiates a global change in gene expression that could induce a virulence-related phenotype, no increased virulence in a murine intraperitoneal (i.p.) infection model was observed under the conditions of this study. Collectively, our data represent an important basis for the assessment of the risk that commensal flora could play during human spaceflight missions. Furthermore, since the low fluid-shear environment of microgravity is relevant to physical forces encountered by pathogens during the infection process, insights gained from this study could identify novel infectious disease mechanisms, with downstream benefits for the general public.

Modeled microgravity increases filamentation, biofilm formation, phenotypic switching, and antimicrobial resistance in Candida albicans.

Candida albicans is an opportunistic fungal pathogen responsible for a variety of cutaneous and systemic human infections. Virulence of C. albicans increases upon exposure to some environmental stresses; therefore, we explored phenotypic responses of C. albicans following exposure to the environmental stress of low-shear modeled microgravity. Upon long-term (12-day) exposure to low-shear modeled microgravity, C. albicans transitioned from yeast to filamentous forms at a higher rate than observed under control conditions. Consistently, genes associated with cellular morphology were differentially expressed in a time-dependent manner. Biofilm communities, credited with enhanced resistance to environmental stress, formed in the modeled microgravity bioreactor and had a more complex structure than those formed in control conditions. In addition, cells exposed to low-shear modeled microgravity displayed phenotypic switching, observed as a near complete transition from smooth to "hyper" irregular wrinkle colony morphology. Consistent with the presence of biofilm communities and increased rates of phenotypic switching, cells exposed to modeled microgravity were significantly more resistant to the antifungal agent Amphotericin B. Together, these data indicate that C. albicans adapts to the environmental stress of low-shear modeled microgravity by demonstrating virulence-associated phenotypes.

The role of annexin 2 in osteoblastic mineralization.

While the basic cellular contributions to bone differentiation and mineralization are widely accepted, the regulation of these processes at the intracellular level remains inadequately understood. Our laboratory recently identified annexin 2 as a protein involved in osteoblastic mineralization. Annexin 2 was overexpressed twofold in SaOSLM2 osteoblastic cells as a fusion protein with green fluorescent protein. The overexpression of annexin 2 led to an increase in alkaline phosphatase activity as well as an increase in mineralization. Our data suggest that the increase in alkaline phosphatase activity does not result from increased alkaline phosphatase transcript or protein levels; therefore we evaluated mechanism of action. We determined that both annexin 2 and alkaline phosphatase activity were localized to membrane microdomains called lipid rafts in osteoblastic cells. Annexin 2 overexpression resulted in an increase in alkaline phosphatase activity that was associated with lipid microdomains in a cholesterol-dependent manner. Furthermore, disruption of lipid rafts with a cholesterol sequestering agent or reduction of annexin 2 expression by specific antisense oligonucleotides each resulted in diminished mineralization. Therefore, intact lipid rafts containing annexin 2 appear to be important for alkaline phosphatase activity and may facilitate the osteoblastic mineralization process.

Annexin 2 expression is reduced in human osteosarcoma metastases.

Osteosarcoma is an aggressive primary bone cancer affecting primarily children and young adults. The development of valuable diagnostic indicators and therapeutic agents will be enhanced by the identification and characterization of genes that contribute to its aggressive behavior. We used representational difference analysis to isolate genes differentially expressed between primary human osteosarcoma tumors and subsequent metastatic lung lesions to identify genes potentially involved in metastatic potential. Several genes were differentially expressed between the two tumor populations, including annexin2. The levels of annexin2 mRNA and protein inversely correlated with metastatic potential in a subset of human osteosarcoma tumor specimens, as well as in a human osteosarcoma cell line selected for increased metastatic potential. Annexin2 has been described in several cellular localizations with various functional implications, many of which may be relevant to metastatic potential. Therefore, the subcellular localization of endogenous annexin2 protein was evaluated biochemically by subcellular fractionation and immunologically by flow cytometry and immunofluorescence in osteoblastic cells. Annexin2 was localized to the cytoplasm and intracellular aspect of the plasma membrane, excluded from the nucleus and undetectable on the cell surface or in the conditioned medium. Overexpression of annexin2 in osteosarcoma cells did not alter several in vitro phenotypes often used to assess metastatic potential including motility, adhesion, and proliferation. However, our previous data have implicated annexin2 in the mineralization process of osteoblastic cells in vitro. Consistent with an increase in differentiation-induced mineralization, there was diminished tumorigenicity and experimental metastatic potential of osteosarcoma cells overexpressing annexin2. These data suggest that annexin2 may downregulate osteosarcoma aggressiveness by inducing a more differentiated state in osteoblastic cells.

Role of PTEN and Akt in the regulation of growth and apoptosis in human osteoblastic cells.

Cancer cells are characterized by either an increased ability to proliferate or a diminished capacity to undergo programmed cell death. PTEN is instrumental in regulating the balance between growth and death in several cell types and has been described as a tumor suppressor. The chromosome arm on which PTEN is located is deleted in a subset of human osteosarcoma tumors. Therefore, we predicted that the loss of PTEN expression was contributing to increased Akt activation and the subsequent growth and survival of osteosarcoma tumor cells. Immunoblot analyses of several human osteosarcoma cell lines and normal osteoblasts revealed relatively abundant levels of PTEN. Furthermore, stimulation of cell growth or induction of apoptosis in osteosarcoma cells failed to affect PTEN expression or activity. Therefore, routine regulation of osteosarcoma cell growth and survival appears to be independent of changes in PTEN. Subsequently, the activation of a downstream target of PTEN activity, the survival factor Akt, was analyzed. Inappropriate activation of Akt could bypass the negative regulation by PTEN. Analyses of Akt expression in several osteosarcoma cell lines and normal osteoblasts revealed uniformly low basal levels of phosphorylated Akt. The levels of phosphorylated Akt did not increase following growth stimulation. In addition, osteosarcoma cell growth was unaffected by inhibitors of phosphatidylinositol-3 kinase, an upstream activator of the Akt signaling pathway. These data further suggest that the Akt pathway is not the predominant signaling cascade required for osteoblastic growth. However, inhibition of PTEN activity resulted in increased levels of Akt phosphorylation and enhanced cell proliferation. These data suggest that while abundant levels of PTEN normally maintain Akt in an inactive form in osteoblastic cells, the Akt signaling pathway is intact and functional.