1188    Chapter 22 : Differentiated Cells and the Maintenance of Tissues

Table 24-1 Cancer Incidence and Cancer Mortality in the United States, 1993

'Nonmelanoma skin cancers are not included in total of all cancers, since almost all are cured easily and many go unrecorded.

In the world as a whole, the five most common cancers are those of the lung, stomach, breast, colon/rectum, and uterine cervix, and the total number of new cancer cases per year is just over 6 million. Note that only about half the number of people who develop cancer die of it. (Data for USA from American Cancer Society, Cancer Facts and Figures, 1993.)

the corresponding type of malignant tumor being an adenocarcinoma (Figure 24-2); a chondroma and a chondrosarcoma are, respectively, benign and malig­nant tumors of cartilage. About 90% of human cancers are carcinomas, perhaps because most of the cell proliferation in the body occurs in epithelia or perhaps because epithelial tissues are most frequently exposed to the various forms of physical and chemical damage that favor the development of cancer.

Each cancer has characteristics that reflect its origin. Thus, for example, the cells of an epidermal basal-cell carcinoma, derived from a keratinocyte stem cell in the skin, will generally continue to synthesize cytokeratin intermediate fila­ments, whereas the cells of a melanoma, derived from a pigment cell in the skin, will often (but not always) continue to make pigment granules. Cancers originat­ing from different cell types are, in general, very different diseases. The basal-cell carcinoma, for example, is only locally invasive and rarely forms metastases, whereas the melanoma is much more malignant and rapidly gives rise to many metastases (behavior that recalls the migratory tendencies of the normal pig­ment-cell precursors during development, discussed in Chapter 21). The basal-cell carcinoma is usually easy to remove by surgery, leading to complete cure; but the malignant melanoma, once it has metastasized, is often impossible to extir­pate and consequently fatal.

Cancer as a Microevolutionary Process

This hastens evolution of the complex set of properties required for neoplasia and malignancy and helps the cancer cells develop resistance to anticancer drugs. At the same time, however, defects ofDNA metabolism underlying such mutability may make the cancer cells uniquely vulnerable to a suitably designed therapeutic attack.

The Molecular Genetics of Cancer ¹⁵

Because cancer is the outcome of a series of random genetic accidents subject to natural selection, no two cases even of the same variety of the disease are likely to be genetically identical. Nevertheless, all cancers can be expected to involve a disruption of the normal restraints on cell proliferation, and for each cell type there is a finite number of ways in which such disruption can occur. In fact, changes in a relatively small set of genes appear to be responsible for much of the derangement of cell behavior in cancer. The identification and characteriza tion of many of these genes has been one of the great triumphs of molecular biology.

Cell proliferation can be regulated directly or indirectly — directly through the mechanism that determines whether a cell passes the restriction point, or "Start," of the cell-division cycle, as discussed in Chapter 17; or indirectly, for example, through regulation of the commitment to terminal differentiation or programmed cell death. In either case the normal regulatory genes can be loosely classified into those whose products help stimulate an increase in cell numbers and those whose products help inhibit it. Correspondingly, there are two mutational routes toward the uncontrolled cell proliferation and invasiveness that are characteristic of cancer.  'The first is to make a stimulatory gene hyperactive: this type of mutation has a dominant effect — only one of the cell's two gene copies need undergo the change — and the altered gene is called an oncogene (the normal allele being a proto-oncogene; from Greek onkos, a tumor). The second is to K make an inhibitory gene inactive: this type of mutation usually has a recessive effect — both the cell's gene copies must be inactivated or deleted to free the cell of the inhibition — and the lost gene is called, for want of a better term, a tumor suppressor gene.

The mutant genes with a dominant effect — that is, the oncogenes — can be identified directly by taking DNA from the tumor cells and searching for fragments of it that, when introduced into normal cells, will cause these cells to behave like tumor cells. Techniques for achieving this feat were first devised in the late 1970s; their development followed earlier studies of a very similar process that occurs naturally, when viruses move their genetic material from cell to cell. | This work paved the way for an explosion of discoveries of oncogenes and proto-4 oncogenes. More recently, progress has been made in the more difficult task of identifying and cloning tumor suppressor genes.

In this section we discuss oncogenes and tumor suppressor genes in turn. We conclude by presenting a case study of one common variety of cancer, where the steps of tumor progression can be related to a series of identified mutations.

Retroviruses Can Act as Vectors for Oncogenes That Transform Cell Behavior ^{16> 17}> ¹⁸

Viruses have played a remarkable part in the search for the genetic causes of human cancer. Although viruses have no role in the majority of common human cancers, they are more prominent as causes of cancer in some animal species, and analysis of animal tumor viruses has provided a key to the mechanisms of cancer in general.

The first animal tumor virus was discovered more than 80 years ago in chick­ens, which are subject to infections that cause connective-tissue tumors, or sar­comas. The infectious agent was characterized as a virus — the Rons sarcoma virus,

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1 Some Changes Commonly Observed When a Normal Tissue-Culture Cell Is transformed by a Tumor Virus

1.  Plasma-membrane-related abnormalities

A.    Enhanced transport of metabolites

B.    Excessive bleeding of plasma membrane

C.    Increased mobility of plasma membrane proteins

2.  Adherence abnormalities

A.    Diminished adhesion to surfaces: therefore able to maintain a rounded

morphology

B.    Failure of actin filaments to organize into stress fibers C.    Reduced external coat of fibronectin D.   High production of plasminogen activator, causing increased extracellular

proteoiysis

3.  Growth and division abnormalities

A. Growth to an unusually high cell density.  B. Lowered requirement for growth factors. C. Less anchorage dependence (can grow even without attachment to rigid surface).  D.   "Immortal" (can continue proliferating indefinitely).  E. Can cause tumors when injected into susceptible animals which we now know to be an RNA virus. Like all the other RNA tumor viruses discovered since, it is a retrovirus. When it infects a cell, its RNA is copied into DNA by reverse transcription and the DNA is inserted into the host genome, where it can persist and be inherited by subsequent generations of cells. Figure 6-82 outlines the life cycle of a retrovirus and shows how its genome undergoes reverse transcription, integration into host DNA, and exit from and entry into host cells.

"But how does the viral infection cause tumors? The solution to this problem, as to so many others in cell biology, depended on the development of a conve­nient assay by which different strains of virus could be rapidly tested for their tumor-causing capacity. The assay system, still widely used, consists simply of fibroblasl cells proliferating in a culture dish. If active tumor virus is added to the culture medium, small colonies of abnormally proliferating transformed cells appear within a few days. Each such colony is a clone derived from a single cell that has been infected with the virus and has stably incorporated the viral genetic material. Released from the social controls on cell division, the transformed cells outgrow normal ones in the culture dish just as in the body and are therefore usually easy to select. The transformed cells commonly show a complex syn­drome of abnormalities (summarized in Table 24-3). They tend not to be con­strained by density-dependent inhibition of cell division (see Figure 17-39), for example, but pile up in layer upon layer as they proliferate (Figure 24-21). In addition, they often do not depend on anchorage for growth and are capable of comaci-inhibited monolayer of normal cells

growth medium multilayer of uninhibited cancer cells.

 Loss of contact inhibition. Canter cells, unlike ™f» normal cells, usually continue 10 fi'»" and pile up on top of one another after they have formed a confluent monolayer.

plastic tissue-culture dish dividing even when held in suspension; they have an altered shape and adhere poorly to the substratum and to other cells, maintaining a rounded appearance reminiscent of a normal cell in mitosis; they may be able to proliferate even in the absence of growth factors; they are immortal and do not undergo senescence in culture; and when they are injected back into a suitable host animal, they can give rise to tumors.

The misbehavior of the transformed cells can be traced to an oncogene dial is carried by the virus but is not necessary for the virus's own survival or repro­duction. This was first demonstrated by the discovery of mutant Rous sarcoma viruses that multiply normally but no longer transform their host cells. The loss of transforming ability could be shown to correspond to loss or inactivation of a particular gene, which was given the name sir (Figure 24-22). This specific gene in the Rous sarcoma virus is responsible for cell transformation in vitro and for tumor formation in vivo, but it is unnecessary baggage from the point of view of the virus's own propagation.

Retroviruses Pick Up Oncogenes by Accident^I6'^{17> l9}

If the viral src gene is bad for the animal and unnecessary to the virus, why is it present and where does it come from? When a radioactive DNA copy of the vi­ral src gene sequence was used as a probe to search for related sequences by DNA-DNA hybridization, it was found that the genomes of normal vertebrate cells contain a sequence that is closely similar, but not identical, to the src gene of the Rous sarcoma virus. This normal cellular counterpart of the viral src gene (v-src) is called c-src {or just src). It is the proto-oncogene corresponding to the oncogene v-src. Evidently, the gene has been picked up accidentally by the retrovirus from the genome of a previous host cell but has undergone mutation in the process (Figure 24-23). The result is a perturbed gene function that leads to cancer and so brings the gene, and the virus that carries it, to the scientist's attention. The retrovirus has, in effect, cloned the gene for us. A large number of other oncogenes have been identified in other retroviruses and analyzed in simi­lar ways (Table 24-4]. Each has led to the discovery of a corresponding proto-oncogene that is present in every normal cell.

A Retrovirus Can Transform a Host Cell by Inserting Its DNA Next to a Proto-oncogene of the Host²⁰

There are two ways in which a proto-oncogene can be converted into an oncogene upon incorporation into a retrovirus: the gene sequence may be altered

figure 24-22 Cell transformation by the Rous sarcoma virus. The

scanning electron micrographs show cells in culture infected with a form of the Rous sarcoma virus that carries a temperature-sensitive mutation in the gene responsible Tor transformation (the v-src oncogene). (A) The cells are transformed and have an abnormal rounded shape at low temperature (34°C), where the oncogene product is functional. IB) The same cells adhere strongly to the culture dish and thereby regain their normal flattened appearance when the oncogene product is inactivated by a shift to higher temperature (39"C). (Courtesy of G. Steven Martin.)

The Molecular Genetics of Cancer

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murine leukemia virus terminal repeats Cap-C 939pol Rous sarcoma virus

CapH__L 5'

(A) host cell DNA pol intron region encoding kinase domain chicken c-src proto-oncogene

(B)

Table 24-4 Some Oncogenes Originally Identified Through Their Presence in Transforming Retroviruses

Oncogene  Proto-oncogene Function      Source of Virus   Virus-induced Tumor

epidermal growth factor (EGF) receptor

fes         protein kinase (tyrosine) cat/chicken

fins        protein kinase (tyrosine): cat macrophage colony-stimulating factor (M-CSF) receptor

fps    \ products associate to form mouse jun   j     AP-1 gene regulatory protein       chicken

kit         protein kinase (tyrosine): cat Steel factor receptor

raf        protein kinase (serine/threonine) chicken/ activated by Ras mouse

myc       gene regulatory protein of chicken the HLH family

H-ras    GTP-binding protein rat K-ras     GTP-binding protein                      rat

rel         gene regulatory protein related      turkey to NFKB

sis         platelet-derived growth factor,       monkey B chain

src         protein kinase (tyrosine) chicken

fibrosarcoma, sarcoma sarcoma, osteosarcoma fibrosarcoma, sarcoma sarcoma

sarcoma; myelocytoma, carcinoma sarcoma; erythroleukemia sarcoma; erythroleukemia reticuloendotheliosis sarcoma sarcoma

 The structure of the Rous sarcoma virus. (A) The organization of the viral genome as compared with that of a more typical retrovirus (murine leukemia virus). Rous sarcoma virus is unusual among the retroviruses that carry oncogenes in that it has retained all the three viral genes required for the ordinary viral life cycle: gag (which produces a polyprotein that is cleaved to generate the capsid proteins), pol (which produces reverse transcriptase and an enzyme involved in integrating the viral chromosome into the host genome), and env (which produces the envelope glycoprotein). In other oncogenic retroviruses one or more of these viral genes are wholly or partly lost in exchange for the acquisition of the transforming oncogene, and therefore infectious particles of the transforming virus can be generated only in a cell that is simultaneously infected with a nondefective, nontransforming helper virus, which supplies the missing functions. (Often the transforming oncogene is fused to a residual fragment of gag, leading to production of a hybrid oncogenic protein that includes part of the Gag sequence.) (B) The relationship between the v-src oncogene and the cellular src proto-oncogene from which it has been derived. The introns present in cellular src have been spliced out of v-src; in addition, v-src contains mutations that alter the amino acid sequence of the protein, making it hyperactive and unregulated as a tyrosine-specific protein kinase. Rous sarcoma virus has been highly selected (by cancer research workers) for its ability to transform cells to neoplasia, and it does this with unusual speed and efficiency.

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