Vascular endothelial growth factor (VEGF) is a multifunctional cytokine with important roles in angiogenesis. VEGF is overexpressed in human cancers, including highly vascularized and infiltrative brain tumors. In our previous study of seven glioma cell lines, VEGF expression levels correlated with blood vessel density and tumorigenicity, and U251 MG and NG-1 cells were recognized as low-tumorigenic glioma cell lines. We hypothesized that low-tumorigenic cells can become highly tumorigenic when high levels of VEGF are expressed. To test this hypothesis, we constructed VEGF expression vectors containing 564 bp or 696 bp of VEGF(121) or VEGF(165) cDNA, respectively, and transfected them into U251 MG and NG-1 cells. In comparison to parental cells, the 20 VEGF-expressing clones examined had on average 8-10-fold more VEGF mRNA and 12-88-fold more secreted VEGF proteins. Four VEGF-overexpressing clones (U251 MG/V121-C2, U251 MG/V165-C3, NG-1/V121-C6, and NG-1/V165-C3) were selected for additional study. As VEGF production increased with population growth, U251 MG/V121-C2 and U251 MG/V165-C3 cells accumulated 47.9 and 22.0 ng of VEGF during a 5-day culture of 10(4) cells, a 313- and 144-fold overexpression when compared with that in parental U251 MG cells. NG-1/V121-C6 and NG-1/V165-C3 cells secreted 30.4 and 9.4 ng of VEGF, respectively, or 138- and 43-fold more than did the parental NG-1 cells. Subcutaneous implantation of the VEGF-overexpressing U251 MG cells into nude mice caused huge, soft hemorrhagic tumors to form, whereas controls maintained very small tumors. Intracranial implantation of the VEGF-overexpressing cell lines significantly shortened survival of the mice when compared with controls, and it caused formation of solid brain tumors with variable sized hemorrhages, whereas the controls had no apparent brain tumors. Tumorigenicity of U251 MG cells was synergized by co-overexpression of VEGF(121) and VEGF(165). In addition, VEGF(165) seemed to be more potent to the brain endothelium than was VEGF(121). More interestingly, except when an admixture of cells was implanted s.c., VEGF overexpression in NG-1 cells did not promote hemorrhagic tumor formation. These data suggested that a switch from a phenotype of low tumorigenicity to one of high tumorigenicity is possible when VEGF overexpression occurs, although other factors may also be required.