2. Transforming viruses carry oncogenes

28.1 Introduction

Key terms defined in this section

Anchorage dependence describes the need of normal eukaryotic cells for a surface to attach to in order to grow in culture.
Aneuploid chromosome constitution differs from the usual diploid constitution by loss or duplication of chromosomes or chromosomal segments.
Metastasis describes the ability of tumor cells to leave their site of origin and migrate to other locations in the body, where a new colony is established.
Monolayer describes the growth of eukaryotic cells in culture as a layer only one cell deep.
Oncogenes are genes whose products have the ability to transform eukaryotic cells so that they grow in a manner analogous to tumor cells. Oncogenes carried by retroviruses have names of the form v-onc.
Primary cells are eukaryotic cells taken into culture directly from the animal.
Proto-oncogenes are the normal counterparts in the eukaryotic genome to the oncogenes carried by some retroviruses. They are given names of the form c-onc .
Serum dependence describes the need of eukaryotic cells for factors contained in serum in order to grow in culture.
Transformation of bacteria describes the acquisition of new genetic markers by incorporation of added DNA.

A major feature of all higher eukaryotes is the defined life span of the organism, a property that extends to the individual somatic cells, whose growth and division are highly regulated. A notable exception is provided by cancer cells, which arise as variants that have lost their usual growth control. Their ability to grow in inappropriate locations or to propagate indefinitely may be lethal for the individual organism in which they occur.

Figure 28.1 Three types of properties distinguish a cancer cell from a normal cell. Sequential changes in cultured cells can be correlated with changes in tumorigenicity.

Three types of changes that occur when a cell becomes tumorigenic are summarized in Figure 28.1:

Immortalization describes the property of indefinite growth (without any other changes in the phenotype necessarily occurring).
Transformation describes the failure to observe the normal constraints of growth; for example, transformed cells become independent of factors usually needed for cell growth.
Metastasis describes the stage at which the cancer cell gains the ability to invade normal tissue, so that it can move away from the tissue of origin and establish a new colony elsewhere in the body.

To characterize the aberrant events that enable cells to bypass normal control and generate tumors, we need to compare the growth characteristics of normal and transformed cells in vitro. Transformed cells can be grown readily, but it is much more difficult to grow their normal counterparts.

When cells are taken from a vertebrate organism and placed in culture, they grow for several divisions, but then enter a senescent stage, in which growth ceases. This is followed by a crisis, in which most of the cells die. The survivors that emerge are capable of dividing indefinitely, but their properties have changed in the act of emerging from crisis. This comprises the process of immortalization. (The features of crisis depend on both the species and tissue. Typically mouse cells pass through crisis at ~12 generations. Human cells enter crisis at ~40 generations, although it is rare for human cells to emerge from it, and only some types of human cells in fact can do so.)

The limitation of the life span of most cells by crisis restricts us to two options in studying nontransformed cells, neither entirely satisfactory:

Primary cells are the immediate descendants of cells taken directly from the organism. They faithfully mimic the in vivo phenotype, but in most cases survive for only a relatively short period, because the culture dies out at crisis.
Cells that have passed through crisis become established to form a (nontumorigenic) cell line. They can be perpetuated indefinitely, but their properties have changed in passing through crisis, and may indeed continue to change during adaptation to culture. These changes may partly resemble those involved in tumor formation, which reduces the usefulness of the cells.

An established cell line by definition has become immortalized, but usually is not tumorigenic. Nontumorigenic established cell lines display characteristic features similar to those of primary cultures, often including:

Anchorage dependence Xa solid or firm surface is needed for the cells to attach to.
Serum dependence (growth factor dependence) Xserum is needed to provide essential growth factors.
Density-dependent inhibition Xcells grow only to a limited density, because growth is inhibited, perhaps by processes involving cell-cell contacts.
Cytoskeletal organization Xcells are flat and extended on the surface on which they are growing, and have an elongated network of stress fibers (consisting of actin filaments).

The consequence of these properties is that the cells grow as a monolayer (that is, a layer one cell thick) on a substratum.

These properties provide parameters by which the normality of the cell may be judged. Of course, any established cell line provides only an approximation of in vivo control. The need for caution in analyzing the genetic basis for growth control in such lines is emphasized by the fact that almost always they suffer changes in the chromosome complement and are not true diploids. A cell whose chromosomal constitution has changed from the true diploid is said to be aneuploid.

Figure 28.2 Normal fibroblasts grow as a layer of flat, spread-out cells, whereas transformed fibroblasts are rounded up and grow in cell masses. The cultures on the left contain normal cells, those on the right contain transformed cells. The top views are by conventional microscopy, the bottom by scanning electron microscopy. Photographs kindly provided by Hidesaburo Hanafusa and J. Michael Bishop.

Cells cultured from tumors instead of from normal tissues show changes in some or all of these properties. They are said to be transformed. A transformed cell grows in a much less restricted manner. It has reduced serum-dependence, does not need to attach to a solid surface (so that individual cells "round-up" instead of spreading out) and the cells pile up into a thick mass of cells (called a focus) instead of growing as a surface monolayer. Furthermore, the cells may form tumors when injected into appropriate test animals. Figure 28.2 compares a "normal" fibroblast growing in culture with a "transformed" variant.

The joint changes of immortalization and transformation of cells in culture provide a paradigm for the formation of animal tumors. By comparing transformed cell lines with normal cells, we hope to identify the genetic basis for tumor formation and also to understand the phenotypic processes that are involved in the conversion.

Certain events convert normal cells into transformed cells, and provide models for the processes involved in tumor formation. Usually multiple genetic changes are necessary to create a cancer; and sometimes tumors gain increased virulence as the result of a progressive series of changes. The incidence of human cancers with age suggests that typically 6 V7 events are required over a span of 20 V40 years to induce a cancer. In certain (rare) cases, propensity to cancer is inherited as a Mendelian trait, implying that a single genetic change is an important or necessary component (although other changes are also necessary).

A variety of agents increase the frequency with which cells (or animals) are converted to the transformed condition; they are said to be carcinogenic. Sometimes these carcinogens are divided into those that "initiate" and those that "promote" tumor formation, implying the existence of different stages in cancer development. Carcinogens may cause epigenetic changes or (more often) may act, directly or indirectly, to change the genotype of the cell.

There are two classes of genes in which mutations cause transformation:

Oncogenes were initially identified as genes carried by viruses that cause transformation of their target cells. A major class of the viral oncogenes have cellular counterparts that are involved in normal cell functions. The cellular genes are called proto-oncogenes, and in certain cases their mutation or aberrant activation in the cell is associated with tumor formation. About 100 oncogenes have been identified. The oncogenes fall into several groups, representing different types of activities ranging from transmembrane proteins to transcription factors, and the definition of these functions may therefore lead to an understanding of the types of changes that are involved in tumor formation. The generation of an oncogene represents a gain-of-function in which a cellular proto-oncogene is inappropriately activated. This can involve a mutational change in the protein, or constitutive activation, over-expression, or failure to turn off expression at the appropriate time.
Tumor suppressors are detected by deletions (or other inactivating mutations) that are tumorigenic. The most compelling evidence for their nature is provided by certain hereditary cancers, in which patients with the disease develop tumors that have lost both alleles, and therefore lack an active gene. There is also now evidence that changes in these genes may be associated with the progression of a wide range of cancers. About 10 tumor suppressors are known at present. They represent loss-of-function in genes that usually impose some constraint on the cell cycle or cell growth; the release of the constraint is tumorigenic.

In the first part of this chapter, we consider how these two classes of genes are identified, and we ask how oncogenes are activated, and how tumor suppressors are inactivated. Then we consider the molecular basis for these events, and how oncogenes are connected into pathways that extend from signal transduction at the cell surface to activation of transcription factors in the nucleus.