Identification of the RAS Oncogene
When DNA from human bladder cancer cells is transfected into cultured 3T3 cells, about one cell in a million incorporates a particular segment of the exogenous DNA that causes a phenotypic change. The progeny of the affected cell are more rounded and less adherent to one another and to the culture dish than are the normal surrounding cells, forming a three-dimensional cluster of cells (a focus) that can be recognized under the microscope. These transformed cells have properties similar to those of malignant tumor cells, including changes in cell morphology, ability to grow unattached to an extracellular matrix, reduced requirement for growth factors, secretion of plasminogen activator, and loss of actin microfilaments.
Figure 1 outlines the procedure for transforming 3T3 cells with DNA from a human bladder cancer and cloning the specific DNA segment that causes the transformation. It was remarkable that a single small piece of DNA had this capability; if more than one incorporated DNA fragment had been needed to induce transformation, the experiment would have failed. Subsequent studies showed that the cloned segment included a mutant version of the cellular RAS gene, in which the glycine normally found in position 12 had been replaced with a valine. This mutant was designated RASD, where the D stands for “dominant.” The mutation is genetically dominant because the active protein has an effect even in the presence of the other, normal RAS allele. Normal RAS protein, which participates in many intracellular signal transduction pathways activated by growth factors (see Chapter 16), cycles between an inactive, “off” state with bound GDP and an active, “on” state with bound GTP. The mutated RASD protein hydrolyzes bound GTP very slowly and therefore accumulates in the active state, sending a growth-promoting signal to the nucleus even in the absence of the hormones normally required to activate its signaling function.
FIGURE 1 Transformation of mouse cells with DNA from a human cancer cell led to the identification and cloning of the RASD oncogene. Addition of DNA from a human bladder cancer to a culture of mouse 3T3 cells caused about one cell in a million to divide abnormally and form a focus, or clone, of transformed cells. To clone the oncogene responsible for the transformation, advantage was taken of the fact that most human genes are located close to repetitive DNA sequences called Alu sequences. DNA from the initial focus of transformed mouse cells was isolated, and the oncogene was separated from adventitious human DNA (human DNA that had no effect on cell transformation but just happened to end up in a cell that also contained the active oncogene) by secondary transfer to mouse cells. The total DNA from a secondarily transfected mouse cell was then cloned into bacteriophage l; only the phage that received human DNA hybridized with an Alu probe. The hybridizing phage should also contain part of or all the transforming oncogene, which was indeed the case.
The production and constitutive activity of RASD protein is not sufficient to cause transformation of normal cells in a primary (fresh) culture of human, rat, or mouse fibroblasts. Unlike cells in a primary culture, however, cultured 3T3 cells already carry several mutations, including loss-of-function mutations in the p19ARF or p53 genes, which are regulators of the cell cycle and cell survival. These mutations allow 3T3 cells to grow for an unlimited time in culture if periodically diluted and supplied with nutrients, which normal nonmutated cells cannot do (see Figure 4-1b). These immortal 3T3 cells are transformed into full-blown tumor cells only when they produce a constitutively active RAS protein or other oncoproteins. For this reason, transfection with the RASD gene can transform 3T3 cells, but not normal cultured primary fibroblast cells, into tumor cells. A mutant RAS gene is found in most human colon, bladder, pancreatic, and other cancers, but not in normal human DNA; thus it must arise as the result of a somatic mutation in one of the tumor progenitor cells.