Plate phage on E. coli

have failed. Subsequent studies showed that the cloned segment included a mutant version of the cellular ras gene, designated rasD. Normal Ras protein, which participates in many intracellular signal-transduction pathways activated by growth factors, 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.

The production and constitutive activation of RasD protein are 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 have undergone a loss-of-function mutation in the p16 gene, which encodes a cyclin-kinase inhibitor that restricts progression through the cell cycle. Such cells can grow for an unlimited time in culture if periodically diluted and supplied with nutrients, which normal cells cannot (see Figure 6-37b). These immortal 3T3 cells are transformed into full-blown tumor cells only when they produce a constitu-tively active Ras protein. 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, 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. Any gene, such as rasP , that encodes a protein capable of transforming cells in culture or inducing cancer in animals is referred to as an oncogene. The normal cellular gene from which it arises is called a proto-oncogene. The oncogenes carried by viruses that cause tumors in animals are often derived from proto-oncogenes that were hijacked from the host genome and altered to be oncogenic. When this was first discovered, it was startling to find that these dangerous viruses were turning the animal's own genes against them.

A Multi-hit Model of Cancer Induction Is Supported by Several Lines of Evidence

As noted earlier and illustrated by the oncogenic transformation of 3T3 cells, multiple mutations usually are required to convert a normal body cell into a malignant one. According to this "multi-hit" model, evolutionary (or "survival of the fittest") cancers arise by a process of clonal selection not unlike the selection of individual animals in a large population. A mutation in one cell would give it a slight growth advantage. One of the progeny cells would then undergo a second mutation that would allow its descendants to grow more uncontrollably and form a small benign tumor; a third mutation in a cell within this tumor would allow it to outgrow the others and overcome constraints imposed by the tumor microenvironment, and its progeny would form a mass of cells, each of which would have these three mutations. An additional mutation in one of these cells would allow its progeny to escape into the blood and establish daughter colonies at other sites, the hallmark of metastatic cancer. This model makes two easily testable predictions.

First, all the cells in a given tumor should contain at least some genetic alterations in common. Systematic analysis of cells from individual human tumors supports the prediction that all the cells are derived from a single progenitor. Recall that during the fetal life of a human female each cell inactivates one of the two X chromosomes. A woman is a genetic mosaic; half the cells have one X inactivated, and the remainder have the other X inactivated. If a tumor did not arise from a single progenitor, it would be composed of a mix of cells with one or the other X inactivated. In fact, the cells from a woman's tumor have the same inactive X chromosome. Different tumors can be composed of cells with either the maternal or the paternal X inactive. Second, cancer incidence should increase with age because it can take decades for the required multiple mutations to occur. Assuming that the rate of mutation is roughly constant during a lifetime, then the incidence of most types of cancer would be independent of age if only one mutation were required to convert a normal cell into a malignant one. As the data in Figure 23-5 show, the incidence of many types of human cancer does indeed increase drastically with age.

More direct evidence that multiple mutations are required for tumor induction comes from transgenic mice

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