Growth factors and growth factor receptors are thought to play key roles in malignant transformation, including progression to, and maintenance of, the malignant phenotype and in neovascularization. Human brain tumor cells have been shown to express many growth factors and growth factor receptors inappropriately. We have partially purified from serum-free culture medium conditioned by D-54MG cells, a malignant human glioma line, a physiologically active, constitutively produced, high molecular weight transforming growth factor-beta termed HMW-TGFbeta. It has an apparent molecular weight of >200 kD (gel filtration HPLC) and a pI of 6.7; is resistant to heat (100oC, 5 min), >4 freeze-thaw cycles and either acidic (pH 2, 2 hr) or alkaline (pH 11, 2 hr) conditions activities with similar properties have been noted in culture media conditioned by other human glioma cell lines (U-251MG, U-105MG and U-373MG). HMW-TGFbeta was not neutralized by antibodies to TGFalpha, EGF, PDGF, TNFalpha, alpha2Macroglobulin, Hodgkin's HMW-TGFbeta could not be dissociated into <200kD molecular weight active components by gel filtration HPLC in buffers containing 5% isopropanol, 6mM CHAPS and high salt (2-5M). It does not appear to be Latent TGFbeta or a precursor. Our objective will be to identify definitively HMW-TGFbeta using two independent strategies. HMW- TGFbeta will be purified using sequential chromatographic procedures (e.g. gel filtration, anti-TGFbeta affinity, ion-exchange, hydrophobic interaction, reversed-phase) and preparative isoelectric focusing. Purification will be monitored by SDS-PAGE and assays of suppression of Mv1Lu cell mitogenesis. Sequencing of N-terminal amino acids may provide enough information to deduce a degenerative DNA primer sequence (to be used with a poly-T primer) to amplify mRNA by PCR for direct sequencing. If this approach proves unsuccessful, we will generate murine monoclonal antibodies against partially purified HMW-TGFbeta. Monoclonal antibodies will be conjugated to Sepharose beads for affinity purification or used to screen expression libraries. U-251MG and D-54MG glioma cDNA libraries, cloned into the Lambda-Zap expression vector and expressed in E. coli, will be screened with monoclonal or rabbit polyclonal antibodies. DNA from positive clones will be amplified and sequenced. Identification of HMW- TGFbeta should provide the means to study its regulation and interactions that may contribute to increased survival of malignant glial cells. HMW- TGFbeta may be an important autocrine or paracrine factor, perturbing normal cell growth regulation, glioma motility and inappropriate expression of cell adhesion receptors, cell adhesion molecules or specific cytokines. Its relation to elevated TGFbeta-activity in biofluids of brain tumor patients with poorer prognoses will be determined.
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