Transforming Sequences Associated with Atherosclerotic Plaque DNA

Abstract

Proliferation of smooth muscle cells is considered to be essential to the development of atherosclerotic plaques. The stimulus to proliferation is unknown. According to the monoclonal hypothesis, which equates plaques with benign smooth muscle cell tumors. plaques arise via mutational or viral events rather than in response to tissue injury. Here, we provide direct evidence that molecular events heretofore associated only with tumor cells are common to plaque cells as well. We employed a DNA-mediated gene transfer assay that relies on the incorporation and expression of dominant, transforming DNA sequences by NIH 3T3 fibroblasts. Three distinct groups of human coronary artery plaque (hCAP) DNA samples transfected into NIH 3T3 cells gave rise to transformed foci. Each focus represents the clonal outgrowth of a single transformed cell. Foci were scored double blind by at least two investigators 24 days after transfection. Foci were picked individually from the primary transfectants and grown in mass culture. The DNA from cloned foci was employed successfully in a second round of transfection. The efficiency of focus formation with hCAP DNA was one-third to one-half of that for the positive control. T24 DNA. Focus DNA hybridized to nick-translated Ki-ras or Ha-ras probes failed to detect human fragments of these genes. For each of nine clones, primary focus cells were injected into male nu - /nu - mice (5 × 106 cells/mouse; five mice/group). Large oval-round tumors (>15 mm diameter) representing five different clones arose in 6/42 mice. These results are significantly different (χ2 = 4.67. p < 0.05) from those (0/30) obtained in control mice injected with untreated NIH 3T3 cells. DNA extracted from the tumors hybridized to the repetitive human ALU sequence. In summary, our results, which provide direct experimental support for the monoclonal hypothesis, demonstrate that hCAP DNA contains transforming sequences(s) that can be transmitted serially in vitro and are associated with tumor formation in vivo. DNA sequences that behave this way in cancer cells are called oncogenes. By analogy, we propose that one or more atherogenes plays a similar role in plaque cells.