vHMEC expressing Ha-ras are resistant to proliferative arrest and display increased numbers of chromosomal abnormalities. (a) Immunoblot analysis demonstrating Ha-rasV12 expression in HMEC and vHMEC following retroviral transduction with pLXSP3-Ha-rasV12 (r) or the control pLXSP3 vector (v). Constructs were expressed in HMEC and vHMEC derived from five different individuals. A representative blot is shown along with actin as a loading control. (b) Growth curves demonstrating that HMEC underwent a proliferative arrest in response to oncogenic ras (orange line, left graph), while vHMEC continued to proliferate (orange line, right graph). (c) Cell cycle analysis of HMEC and vHMEC expressing Ha-rasV12 or control vector demonstrating that the number of cells in S-phase dropped from 33.8% to 8.8% following Ha-rasV12 expression in HMEC, but remained the same in vHMEC (37.9% and 37.6%, respectively). (d) Chromosomal analysis of vHMEC-vector and vHMEC-ras cells. Control vHMEC (vector) and vHMEC expressing oncogenic Ha-RasV12 (ras) were harvested at different passages (P+1, P+5, and P+8), as indicated, and processed for metaphase analysis. Standard G-banding karyotypic analysis was performed on at least fifty metaphase spreads for each cell population. Aneuploidy refers to additions or deletions of whole chromosomes. Structural abnormalities include all deletions, duplications, rings, marker chromosomes, chromatid exchanges and translocations. The total number of abnormalities includes all structural abnormalities and telomeric associations, not including numerical abnormalities.