The fundamental event that causes cells to become malignant is an alteration in their DNA structure which results in unrestrained cellular proliferation.

Most of these alterations involve sequence changes in DNA (i.e., mutations).

They may originate as a consequence of random replication errors, exposure to carcinogens (e.g., radiation), or faulty DNA repair processes. ¹

Cancer is of clonal origin, with a single abnormal cell, often with multiple genetic alterations, multiplying to become a mass of cells forming a tumor. The tissue microenvironment influences the processes that occur.

The exact nature of these influences may vary with cell types involved, intercellular interactions and presence of factors such as paracrine signals, local hypoxia and proinflammatory responses. Carcinogenesis is, thus, a multistep process, ultimately transforming normal cells into malignant ones. Hence, tumors often take from a few to tens of years to develop to macroscopic levels.¹ The transformation from a normal cell into a tumor cell is a multistage process, typically a progression from a precancerous lesion to malignant tumors.

The transformation of normal to neoplastic cells is caused by both endogenous and exogenous factors, including chemical and physical agents, viruses, activation of cancer-promoting genes, and inhibition of cancer-suppressing genes. 

Cancers are named based on their origin: those derived from epithelial tissue are called carcinomas, those derived from mesenchymal tissues are sarcomas, and those derived from hematopoietic tissue are leukemias, lymphomas, and plasma cell dyscrasias (including multiple myeloma).

References

http://www.emedicinehealth.com/slideshow_pictures_cancer_101/article_em.htm#

All tumors have two basic components: (1) neoplastic cells that constitute the tumor parenchyma and (2) reactive stroma made up of connective tissue, blood vessels, and variable numbers of cells of the adaptive and innate immune system.

The classification of tumors and their biologic behavior are based primarily on the parenchymal component, but their growth and spread are critically dependent on their stroma. In some tumors, connective tissue is scant and so the neoplasm is soft and fleshy. In other cases, the parenchymal cells stimulate the formation of an abundant collagenous stroma, referred to as desmoplasia. Some demoplastic tumors—for example, some cancers of the female breast—are stony hard or scirrhous.

The terms benign and malignant correlate to the course of the neoplasm. Benign neoplasms stay localized in one place; malignant neoplasms invade surrounding tissue and, in most cases, can metastasize to distant organs. If the neoplasm is malignant, the cells must also gain the ability to invade the basement membrane and surrounding tissue, enter the blood stream, and spread to and grow within distant organs.

Monoclonal Origin

Cancers begin in a single stem cell and therefore are monoclonal in origin. A wide variety of genetic and epigenetic changes can occur in different cells within malignant tumors over time. Most cancers are characterized by marked heterogeneity in the populations of cells. The vast majority of human cancers are characterized by a multiple-step process involving many genetic abnormalities, each of which contributes to the loss of control of cell proliferation and differentiation and the acquisition of capabilities, such as tissue invasion, the ability to metastasize, and angiogenesis.

Immature Features

Cells that have undergone neoplastic transformation usually express cell surface antigens that may be of normal fetal type, and they may display other signs of apparent immaturity, and may exhibit qualitative or quantitative chromosomal abnormalities, including various translocations and the appearance of amplified gene sequences.

It is now well established that a small subpopulation of cells, referred to as tumor stem cells, reside within a tumor mass. They retain the ability to undergo repeated cycles of proliferation as well as to migrate to distant sites in the body to colonize various organs in the process called metastasis. Such tumor stem cells thus can express clonogenic (colony-forming) capability, and they are characterized by chromosome abnormalities reflecting their genetic instability, which leads to progressive selection of subclones that can survive more readily in the multicellular environment of the host. This genetic instability also allows them to become resistant to chemotherapy and radiotherapy. The invasive and metastatic processes as well as a series of metabolic abnormalities associated with the cancer result in tumor-related symptoms and eventual death of the patient unless the neoplasm can be eradicated with treatment.

Many cancers go through recognizable steps of progressively more abnormal phenotypes: hyperplasia, to adenoma, to dysplasia, to carcinoma in situ, to invasive cancer with the ability to metastasize. For most cancers, these changes occur over a prolonged period of time, usually many years.

This heterogeneity significantly complicates the treatment of most cancers because it is likely that there are subsets of cells that will be resistant to therapy and will therefore survive and proliferate even if the majority of cells are killed. These mutations become ancestors of the entire future tumor cell population. Evolution to more undifferentiated cells reflects high mutation rates. Cancers are characterized by unregulated cell division, avoidance of cell death, tissue invasion, and the ability to metastasize.

In general, during the gain-of-function alteration of a proto-oncogene, only one allele is mutated. In contrast, during the loss-of-function alteration of a tumor suppressor gene both alleles need to be inactivated. In certain cases, loss of one allele can result in reduction of gene expression. For some genes, this gene-dosage reduction is sufficient to permit tumorigenic growth.

In addition to being generated through the mutation of cellular proto-oncogenes, oncogenes can also be acquired through the introduction of foreign genomic material, typically transmitted by viruses. Although virally induced tumors are common in animals, only a few human tumors are directly caused by viral infection. Causative viruses and their associated malignancies are listed in Table 5–3. One such virus, human T-cell leukemia virus, is closely related to HIV and can cause a type of T-cell leukemia as a result of proteins encoded by the viral genome that are able to activate latent human genes. Human papillomavirus has long been linked epidemiologically to cervical cancer, and the serotypes most often linked have been found to encode proteins that can bind and inactivate host tumor suppressor gene products. In this situation, a causative gene is not necessarily introduced by the virus, but the viral genome is able to direct the inactivation of tumor suppressor gene products and thereby favor growth and proliferation as well as malignant potential. The ability of viruses to modulate the host cellular machinery—and in some cases retain altered mammalian genes that are oncogenic—is likely to have developed over the course of mammalian evolution, because an actively proliferating cell provides the optimal conditions for replication of virions and propagation of viral infections.

Key: HTLV, human T-cell leukemia-lymphoma virus; HHV-8, human herpesvirus-8; KSHV, Kaposi’s sarcoma herpesvirus.

Oncogenes and Proto-oncogenes

Molecular evidence suggesting the normal cellular origin of retroviral oncogenes was first obtained by showing that radiolabelled DNA from the avian retroviral oncogene src hybridized specifically to normal uninfected avian cellular DNA as well as to normal mammalian DNA and even normal human cellular DNA. All retroviral oncogenes are now known to have hybridizing homologs, that is, close relatives, in the genomes of virtually all normal vertebrate cells. The normal host cell homologs of v-onc genes are called c-onc genes or proto-oncogenes.

The retroviral homologs found in normal cellular DNA have the usual structure of cellular genes, possessing both exons and introns, whereas the oncogenes in retroviruses do not have introns. It has been concluded that the oncogene analogs found in normal cellular DNA represent native cellular DNA and are not of viral origin. It appears, then, that retroviral oncogenes are copies (allowing for subtle differences in gene sequences) of normal cellular genes which were picked up and transduced into the retroviral genome by pre-existing retroviruses. Nevertheless, retroviral oncogenes usually show structural mutations or changes in expression relative to the corresponding proto-oncogenes. Since proto-oncogenes are highly conserved in vertebrates and are demonstrable in the cellular genomes of virtually all metazoans, their roles in cellular functions are apparently of fundamental importance. The gene products of proto-oncogenes have been identified as proteins (growth factors, growth-factor receptors, signal transducer proteins) with known functions in normal cells.

Direct evidence for the activation of proto-oncogenes to transforming genes in-vivo, independently of any retroviral gene participation, has been obtained by isolating proto-oncogenes, attaching them to promotor or enhancer sequences, and introducing this DNA into one-cell mouse embryos (fertilized eggs) and thus ultimately the germ cells of so-called transgenic mice. The promotor causes a high rate of transcription ("activation") of the introduced proto-oncogene resulting in a high incidence of malignant tumors in some of the progeny mice. In other experiments, "weak" retroviruses which lack a separate viral oncogene have been shown to produce tumors only when, by chance, the proviral DNA is inserted into the cellular genome next to a cellular proto-oncogene, which is then activated through the effect of the inserted viral promoter.

In summary, the oncogene theory postulates that the oncogenes of transforming retroviruses are derived from normal cellular genes (proto-oncogenes); and that an increased expression (activation) of proto-oncogenes or an inappropriate expression of mutated forms of proto-oncogenes, occurring spontaneously or induced by cancer-causing agents, contributes to neoplastic transformation and the development of cancer, including human cancer.

It is now recognized, as you shall learn, that sequential mutations or inappropriate expressions of several different classes of cellular genes [oncogenes, tumor suppressor genes, DNA nucleotide mismatch repair genes, and genes that mediate programmed cell death (apoptosis)] are probably involved in the usual multiple-step process that leads to human cancer.

Oncogenes and Human Cancers

Most of the known retroviral oncogenes have hybridizing homologs in normal human cellular DNA (proto-oncogenes). Oncogenes are activated in somatic cells in many forms of human cancer, including carcinoma, sarcoma, leukemia, and lymphoma. The chief mechanisms of oncogene activation are chromosomal translocation, point mutation, and gene amplification (Table).

Table: Activation of Cellular Proto-oncogenes in Human Cancers

Burkitt's Lymphoma

In the cells of this human B cell lymphoma there is often a translocation between chromosomes 8 and 14, or between 8 and 2 or 22. This change transposes the cellular myc gene, which is normally on chromosome 8, into proximity with a predictably active promotor (in B lymphocytes) involved in immunoglobulin (Ig) synthesis. (Ig heavy chain loci are on chromosome 14, Ig light chain loci are on chromosomes 2 and 22). The translocation interrupts the normal transcriptional control of the myc gene and leads to amplification of the amount of the myc-encoded protein, thereby thrusting the myc proto-oncogene into the role of an oncogene.

Chronic Myelogenous Leukemia (CML)

The leukemia cells of virtually all (90-95%) patients with CML have a characteristic cytogenitic abnormality called Philadelphia chromosome, Ph', (Nowell, P.C., and Humperford, D.A., Science 132:1497, 1960) resulting from a reciprocal translocation between the long arms of chromosomes 9 and 22 (Rowley, J.D., Nature 243:290, 1973).

In this translocation, the proto-oncogene c-abl is translocated from its normal location on chromosome 9 to the break point region in chromosome 22, resulting in the generation of a fusion gene and its protein product (BCR-ABL), a constitutively activated mutant tyrosine kinase with proven oncogenic activity. Clinical trials with a selective inhibitor (imatinib, Gleevec) of BCR-ABL tyrosine kinase in CML provide cytogenetic and hematologic evidence of improvement in patients with the chronic phase of CML (Drucker, B.J., et al., N Eng J Med 344: 1031-37, 2001; Kantarjian, H., et al., ibid. 346:645-52, 2002).

Human Carcinomas

The cellular DNA of tumor cells can be extracted and, following co-precipitation with calcium phosphate, introduced into the nuclei of selected cells (usually the mouse fibroblast cell line NIH 3T3) in vitro, a process termed transfection. Those cells which take up intact segments of DNA that induced the neoplastic state become transformed themselves. One can then isolate and sequence the transforming genes from these cells. In one of the earliest reported studies, such a transforming gene was identified in cells of the human bladder carcinoma cell line T24. When sequenced, this human oncogene in T24 cells was found to be a homolog of the v-H-ras gene (known to induce sarcomas and leukemias in susceptible murine animals). Furthermore, the human oncogene differed from the corresponding normal human proto-oncogene by only a single base substitution (G for T), a point mutation leading to the substitution of a single aminoacid (valine for glycine) at position 12 of the 21,000 m.wt. encoded protein.

Other transfection studies have shown that the transforming genes of a number of other human cancers are also homologs of members of the ras family: for example, the transforming genes of cell lines and primary carcinomas of human lung and colon are homologs of the v-K-ras gene. (as noted in the previous table)

An important finding is that when primary cultures of mouse embryo fibroblasts, rather than established cell lines such as NIH 3T3, are used as the DNA recipients in transfection assays, at least two co-operating oncogenes (often myc and ras) are usually required for complete neoplastic or "tumorigenic" conversion. This observation is consistent with the prevailing view that carcinogenesis is a multiple-step process. At this writing, more than 60 different oncogenes have been characterized in avian and mammalian, including human, species.

Role of Individual Cellular Oncogenes in Neoplastic Transformation

One of the simplest classifications of oncogenes, on the basis of the localization of their encoded proteins either in the cytoplasm or the nucleus, is useful in grouping those with similar roles. The accompanying table indicates the cellular localization of proteins encoded by some cellular proto-oncogenes and, for comparison, transforming proteins encoded by oncogenic DNA viruses.

Table: Proteins Encoded by Proto-oncogenes and DNA Tumor Viruses

 

Localization	Proteins Encoded by
	Cellular Proto-oncogene	DNA Tumor Virus
Nuclear	myc	SV40 large T
	N-myc	Polyoma large T
	myb	Adenovirus E1a
Cytoplasmic	ras	Polyoma middle T
	abl
	src
	erb B
	sis

Most of the nuclear cellular oncogenes, that is, those with nuclear localization of the encoded proteins, appear to have similar, although not identical, roles. The most predictably observed trait which, as oncogenes, they confer on the affected cells is "immortalization", or the ability to be passaged in cell culture continuously without limit. Related traits which appear to be conferred are an incresed plating efficiency and the ability of cells to divide in the presence of low concentrations (0.5%) of serum. Nuclear cellular oncogenes show little ability to confer anchorage independence (growth in the absence of an added support structure) on cells in which they are activated and likewise only minimally induce morphologic changes, such as multilayering, of cultured cells.

Cellular oncogenes with cytoplasmic protein products are relatively "weak" in inducing immortalization of cells in culture, but, in contrast to nuclear oncogenes, are strong in conferring the morphologic changes of transformation as well as in conferring anchorage independence.

Neither nuclear nor cytoplasmic cellular oncogenes, acting alone, appear able to induce full transformation of cells. Their actions in transformation appear to be complementary. Many types of cells have been transformed, and rendered "tumorigenic" in animals, by transfection with collaborating pairs (one nuclear, one cytoplasmic) of cellular oncogenes in various combinations.

Conversion of Cellular Proto-oncogenes to Oncogenes

The known mechanisms of proto-oncogene activation and well-studied examples include:

• translocation: c-myc, c-abl;
• promotor insertion: c-myc, c-erb B;
• point mutation: c-ras;
• deletional mutation: c-erb B;
• amplification: c-myc.

Nuclear oncogenes of cellular origin are converted from proto-oncogenes by processes (translocation, promotor insertion, amplification) which lead to an increased level (or amount) of their encoded proteins. The disordered regulation of c-myc expression related to the 8-14 chromosomal translocation in Burkitt's lymphoma is the best documented example. In this genetic rearrangement, the normal promotor-enhancer regulators for c-myc are removed and replaced by those from predictably active immunoglobulin genes, resulting in amplification of the amount of the myc-encoded protein.

Most of the cytoplasmic oncogenes of cellular origin are derived from proto-oncogenes by mutations which alter the structure of the encoded proteins. The c-H-ras gene whose activation is associated with several human carcinomas is an important example. Point mutations of the proto-oncogene involving aminoacids at residues 12, 13, or 61 induce transformation even when the 21,000 m.w. protein, p21ras, is present at low levels. Further studies of cytoplasmic cellular oncogenes, such as src, erb B, and neu (related to erb B), show that structurally altered protein products cause transformation, while over-expression of the normal protein brings about little change.

An important cytoplasmic proto-oncogene is the abl gene which is present at the site of the 9-22 translocation resulting in the formation of the Philadelphia (Ph') chromosome typically seen in chronic myelogenous leukemia (CML). The abl protein is present in increased amounts in the tumor cells, but, apparently more important, the protein is altered (the translocation removes its amino terminus). Over-expression of the normal intact abl protein seems to cause no effect.

In summary, many cytoplasmic proto-oncogenes appear to be converted to oncogenes by mutations which alter the structure of their encoded proteins. In these circumstances, the amount of the normal protein appears to be of little importance in cellular transformation.

While the genetic changes in Burkitt's lymphoma and chronic myelogenous leukemia occur in cells of hematologic origin, no single genetic abnormality has thus far been identified as the transforming event in common human cancers of epithelial origin.

Cellular Functions of Oncogene-Encoded Proteins

The functions of proteins encoded by selected protooncogenes (and oncogenes) are given in the accompanying table.

Table: Cellular Functions of Proto-oncogene Encoded Proteins

 

Function	Protein	Protoonco- gene	Associated Human Cancers
Growth factor	PDGF	sis	Osteosarcoma
Growth-factor receptor	EGF receptor	erb B	Breast, lung, ovarian cancer
Post-receptor signal transduction	GTP-binding protein	ras	Lung, colon, pancreatic cancer
Nuclear transcription regulator	myc protein	myc	Breast, colon, lung cancer, Burkitt's lymphoma

Most, if not all, of the many proteins encoded by cellular oncogenes are involved in the "growth factor-receptor-response" pathways of transmission of growth stimulatory signals from the cell surface to the nucleus, culminating in the transcription of certain genes and in DNA synthesis.

Aberrations in the normal process of transmembrane signaling involving growth factors (GFs), GF receptors, post-receptor "transducer" proteins, and nuclear controls may be caused by activated oncogenes and result in the abnormal growth characteristics of neoplastic cells.

• Growth factors (GFs) are ubiquitous polypeptides that are produced and secreted by cells locally and that stimulate cell proliferation by binding to specific cell-surface receptors on the same cells (autocrine or autostimulation) or on neighboring cells (paracrine stimulation).
• Conceptually, uncontrolled autostimulation from the persistent over-production of GFs may convert a cell to the neoplastic state. Human platelet-derived growth factor (PDGF), so named because it is released from platelets during blood clotting, is a major growth factor recoverable from serum. PDGF is apparently encoded by c-sis, the normal analog of v-sis which is the transforming gene of simian sarcoma virus (SSV). Cultured cells infected with SSV produce a PDGF-like mitogen which binds to PDGF receptors and stimulates the cells to proliferate in an uncontrolled manner, resulting in transformation.
• Structural changes or amplification of GF receptors may promote neoplastic transformation. If GF receptors are changed in a way that continually presents cells with growth stimulatory signals, even in the absence of GFs, cells may respond as though high levels of GFs were present. One of several examples, all in the group of cytoplasmic oncogenes, can be mentioned. A portion of the human epidermal, growth factor receptor protein (now called Her-2/neu or Her-2) is similar to the proto-oncogene encoded erb B protein. Her-2 protein is over expressed in tissue and serum of ~25-30% of patients with breast cancer. Trastuzumab (Herceptin) is a recombinant humanized monoclonal anitbody that selectively binds to and mediates antibody-dependent cytolytic destruction of Her-2 protein and is approved for use in patients with metastatic breast cancer whose tumors over express Her-2.
• Autonomy to GFs may be produced by changes in post-receptor "transducer" proteins that enable them to transmit growth stimulatory signals without prompting by a GF receptor. Ras-encoded proteins are guanosine triphosphate (GTP)-binding proteins that function as post-receptor signal transducers (cyclic on-off switches) for growth stimulatory signals.. Ras proteins are located in the plasma membrane and mediate the passage of growth signals from outside to inside the cell and to the cell nucleus, thus initiatiating the cell cycle and DNA synthesis. Mutations in ras proteins, potentially associated with a continuous growth signal that cannot be deactivated, are found in about 30% of human cancers (Wittinghofer, F., Nature 394: 317, 1998).
• The cell division cycle is normally monitored at critical check points along the mitogenic signaling pathway. Deregulation of the cell cycle at the critical transition phase from G1 (resting) to S (synthesis of DNA) occurs in many types of human cancer. Transcriptional amplification of myc and related nuclear proteins is also associated with unregulated cell proliferation and transformation. Additionally, the microtubule network is necessary for interphase and mitotic cellular events, and paclitaxel (Taxol) which disrupts microtuble assembly is used for adjuvant therapy in advanced cancers of breast, ovary and lung.

In summary, the activation of proto-oncogenes to cancer-associated oncogenes represents a "gain of gene function" (dominant allele) mutation involving pathways of transmission and transduction of mitogenic signals from cell to cell and from cell surface to nucleus and the activation of certain nuclear genes, culminating in DNA synthesis, unregulated cell division, and neoplastic transformation.

Tumor Suppressor Genes

Most studies of genes and growth factors involved in carcinogenesis have focused on functions that have a positive stimulatory effect on cell proliferation and, hence, neoplastic transformation. While the activation of some genes may result in cancer, it is reasonable to suppose that the activation of other genes may suppress the development of cancer. The existence of tumor suppressor genes, or so-called "anti-oncogenes", in normal cells is suggested by the results of somatic cell hybridization experiments in which fusions of malignant tumor cells, including those with activated oncogenes, with normal diploid fibroblasts were usually found to yield non-malignant hybrid cells. It is possible, therefore, that the loss or inactivation of putative tumor suppressing genes may remove a block to cell proliferation and the development of the neoplastic state. Supporting evidence linking the loss of genes to the development of human cancer comes from studies of heritable cancers, among them retinoblastoma of childhood.

Retinoblastoma

Retinoblastoma, the most common primary malignant eye tumor of children, occurs in both a heritable (autosomally dominant) form characterized by early onset and bilateral eye involvement and a sporadic form with later onset and unilateral involvement.

Sequential deletions or mutational losses of function of both allelic genes ("two-hit" model) at the Rb locus on chromosome 13q14 are required for the development of retinoblastoma of either form. In this genetic model for the two forms of retinoblastoma, first proposed by Knudson and now a paradigm for some other forms of hereditary cancer, the first mutation (first "hit") affecting retinal cells may be one that is inherited at birth through the germ line (heritable form) or that is acquired later by somatic mutation (sporadic form). The second mutation (second "hit") at the same locus is somatic and random in either form, and both allelic (homozygous) mutations are necessary for neoplastic transformation of a retinal cell to retinoblastoma. The sequential mutations necessary for transformation of a retinal cell are much (>1000-fold) more likely to occur in a cell that already has one inherited mutant allele than in a cell that has none, consistent with the clinical characteristics of this and some other forms of hereditary cancer: early onset, multiple primaries, bilateral involvement of paired organs, and Mendelian pattern of inheritance.

Germ line inheritance of a mutated Rb allele also predisposes to the development of a second form of childhood cancer, notably osteosarcoma in adolescence. Somatic mutations (deletions, frameshifts, etc.) of Rb are also found in various forms of adult cancer of lung, breast, prostate, and other sites.

The normal (wild-type) Rb gene on human chromosome 13q14 is a transcription regulator, and its protein product pRb regulates a critical check point in the cell cycle, namely progression from a G1 (resting) phase to the S (DNA-synthetic) phase. Loss of function mutation of both Rb alleles removes this regulatory control.

There are many regulator controls in the cell cycle. Very briefly, cyclin proteins regulate timing mechanisms through CDKs (cyclin-dependent kinases) that enzymatically phosphorylate and activate proteins critical to the cell cycle, particularly at the transition phase from G1 (rest) to S (synthesis of DNA). pRb normally binds to and inactivates at least two protein enzymes that are essential for the synthesis of DNA precursors and the promotion of DNA replication. The hyper-phosphorylation of pRb by CDKs releases and reactivates these enzymes and thus abolishes the inhibitory control of pRb in the cell cycle.

The majority of human cancers appear to have alterations in at least one of the main regulatory controls of the G1-S transition point in the cell cycle (Russo, A.A., et al., Nature 395:237-43, 1998).

Colorectal Cancer

Carcinoma of colon and rectum ranks second to lung cancer as a cause of cancer mortality in males and third (after breast and lung cancer) in females and accounts for about 60,000 deaths annually in the U.S. The majority of colorectal carcinomas arise from adenomatous polyps (adenomas) that develop in the bowel mucosa.

Familial adenomatous polyposis (FAP), an autosomal dominant trait with a prevalence of about 1 in 10,000, predisposes to the development of multiple intestinal polyps and colorectal carcinoma with early age of onset (age 30 or so). Recent molecular genetic studies of FAP and its progression into cancer indicate that normal colonic epithelial cells evolve into polyps and subsequently into cancer through a sequence of mutations and interactions of two kinds of genes: tumor suppressor genes and oncogenes.

The first genetic change in the development of FAP is the mutational inactivation of a tumor suppressor gene called APC (adenomatous polyposis coli) on chromosome 5(q21) of normal colonic epithelial cells (Kinzler, K.W., et al. Identification of FAP locus genes from chromosome 5q21. Science 253:661-664, 1991). The subsequent progression of polyp into colonic cancer involves stepwise changes, beginning with mutational activation of the ras gene on chromosome 12 and followed by deletions of tumor suppressor genes on chromosomes 18 and17, and perhaps others. The observed sequence of mutations involving APC, ras, DCC ("Deleted in Colon Cancer"), and p53 is also found during tumor development in some, but not all, patients with non-familial (sporadic) colorectal cancer, indicating that this is not the only genetic pathway to this common cancer.

p53 Tumor Suppressor Gene

A normal (wild-type) nuclear protein of 53 kilodaltons called p53 protein is the product of the p53 tumor suppressor gene on human chromosome 17(p13). As previously noted with the Rb gene, mutated or deleted tumor suppressor genes such as mutant p53, while heritable as a dominant allele, are recessive to the normal (wild-type) allele in somatic cells, and mutations or deletions in both p53 alleles ("two hits") are required for loss of function. Patients with germline mutations at the p53 locus are at very high risk for cancer development, as seen in the Li-Fraumeni hereditary cancer syndrome characterized by early onset of breast carcinoma, childhood sarcomas, and other tumors.

Somatic mutations at the p53 locus, usually point mutations substituting one amino acid for another and inactivating suppressor activity, are the most common genetic change in human cancers and occur in about 50% of them, including carcinoma of breast, colon, stomach, bladder, and testis, melanoma, and soft-part sarcoma.

Normal p53 protein is a transcription factor (increases gene expression) and mediates several cellular functions: regulation of the cell division cycle, DNA repair, and programmed cell death. In response to various forms of genomic DNA damage (caused by oncogene activation, radiation, cytotoxic drugs, hypoxia, certain viruses), the p53 protein can arrest the cell cycle at the G1 to S transition point, thus affording time for DNA repair and preventing duplication of a mutant cell or, alternatively, failing DNA repair, p53 protein can implement programmed cell death (apoptosis). Accordingly, p53 has been dubbed the "guardian of the genome." The cellular process of DNA replication is initiated by the formation of complexes between proteins called cyclins and enzymes termed cyclin-dependent kinases (CDKs). The formation of cyclin-CDK complexes is inhibited by p53 protein and other cell-growth inhibitors.

As previuosly noted, mutated or deleted tumor suppressor genes, such as mutant p53 or mutant Rb, while heritable as a dominant allele are recessive to the normal (wild-type) allele in somatic cells, and mutations or deletions in both alleles are required for loss of gene function.

DNA Mismatch-Repair Genes

A new class of cancer susceptibility genes was recently identified in hereditary non-polyposis colorectal cancer (HNPCC):a defective hMSH2 gene located on human chromosome 2p and a defective hMLH1 gene located on chromosome 3p. These defective genes are associated with widespread instability of microsatellite DNA and other short repeat sequences in HNPCC cells and with the accumulation of mutations, both germ line and somatic, throughout the genome of some HNPCC patients (Peltomaki,P.,et al., Science 260: 810-812, 1993; Ionov,Y.,et al.,Nature 363:558-561, 1993).Other studies indicate that natural (wild-type) hMSH2 and hMLH1 are human homologs, respectively, of a bacterial gene and a yeast gene that encode a base-binding enzyme involved in DNA mismatch repair.Genetic defects in DNA repair genes not only contribute to the development of HNPCC but may also be involved in other hereditary cancer syndromes as well as in the genetic instability (heterogeneity) commonly shown by cancer cells.

It is estimated that about 0.5% of the general population (~ 1 million Americans) may carry this or other mutator genes, along with a greatly increased risk of colorectal carcinoma or other cancers, such as, ovarian, uterine, and renal.

Apoptosis

Apoptosis (Gr. apo, away; ptosis, falling) is a normal process of non-random cell death that occurs in many biological conditions, among them: embryological development; perinatal thymus selection and deletion of self-responsive T-cells; normal cell turnover throughout life; and in response to abnormal stimuli, such as genomic DNA damage, but without concurrent pathological necrosis and inflammation.

Apoptosis (programmed cell death) is an active, energy-dependent process characterized by the rapid occurrence of distinctive morphological and biochemical changes in the cell. These changes include: the formation of cytoplasmic blebs; chromatin condensation at the nuclear membrane; and cleavage of chromatin by an endonuclease that is exclusively activated in apoptosis and that yields a distinctive 'chromatin ladder' pattern of DNA fragments, as shown by gel electrophoresis. Specialized proteases called caspases (cysteine aspartases), associated with mitochondria and the cytochrome C respiratory pathway, accelerate the cell-death response (Earnshaw, W. C., Nature 397: 387-389, 1999).

As noted elsewhere (see: p53 Tumor suppressor gene), p53 protein expression in response to genomic DNA damage can arrest the cell cycle (at G1) for DNA repair, thus preventing duplication of a mutant cell or, alternatively, failing DNA repair, implement cell suicide through programmed cell death.

If oncogenes are likened to an "accelerator" of cell proliferation or transformation and tumor suppressor genes to a "brake", then apoptosis is a final "suicidal crash". Thus, at least two signals ( and obviously multiple genetic controls) are required for cell proliferation or transformation: one that drives cell proliferation; and one that blocks cell death. Surprisingly, the protein product of the myc proto-oncogene has one domain that mediates cell proliferation and another one that, in the absence of required growth factors, nutrients, or other gene products, induces apoptosis. On the other hand, the bcl-2 proto-oncogene (known to be activated by chromosomal translocation in a variety of B-cell lymphomas) encodes an antiapoptosis protein; the bcl-2 protein product functions as an inhibitor of apoptotic cell death.

Human Cancer Cells Created with Defined Genetic Elements

Sequential mutations or inappropriate expressions of different classes of cellular genes (oncogenes, tumor suppressor genes, mismatch repair genes, and genes that mediate apoptosis) are involved in the usually multiple-step process that leads to human cancer.

This concept was put to critical test in a recent landmark study: normal human epithelial cells (and fibroblasts) in culture were transformed into cancer cells by the insertion of defined genetic elements (Hahn, W.C., Counter, C. M., et al., Nature 400: 464-468, 1999). The three necessary genetic elements were serially inserted and included: a subunit of the telomerase gene, to immortalize the cells by telomere maintenance, i.e., preventing telomere shortening at each cell division (see: Neoplasia VIII. Chromosomal Abnormalities: Telomeres)); an activated ras oncogene, known to be mutated in many kinds of human cancers; and a viral oncoprotein gene (SV 40 large T), known to inhibit the tumor suppressor proteins p53 and pRb.

When injected into immunodeficient mice, the transformed cells formed malignant tumors having similar identifying cellular and molecular genetic characteristics as the injected cells, thus suggesting that the tumorigenic growth was not the consequence of some other rare random event occurring in vivo after inoculation of these cells. Telomere maintenance appeared to be essential for the formation of human cancer cells.

Cancer Susceptibility Genes - Summary

The following table gives only a short list of the ever increasing number of cellular genes that, through inherited or somatic mutations and a gain (dominant allele) or loss (recessive allele) of function, are associated with the development of certain forms of human cancer. Prototype examples of affected genes and associated cancers were previously discussed.

Activating (gain of function) somatic mutations of proto-oncogenes (such as ras, myc, abl, etc.,) are present in a variety of sporadic human cancers (Table). Surprisingly, inherited mutations of proto-oncogenes are not regularly found in hereditary cancers with the following notable exception: inherited germ line mutation in the ret (RET) proto-oncogene confers a genetic predisposition to multiple endocrine neoplasia.

Germ line and subsequently somatic (loss of function) mutations associated with human cancer susceptibility and expression (see Table) mainly involve tumor suppressor genes (established examples include: APC, Rb,& p53 which is mutated in many forms of cancer; BRCA1, BRCA2, NF1, WT1, and DNA repair genes (such as hMSH2).In familial breast cancer, an inherited germ line mutation of the BRCA1 gene located on chromosome 17(q21) confers a predisposition to early-onset (~ premenopausal) breast cancer and ovarian cancer; and an inherited mutation of the BRCA2 gene located on chromosome 13(q12-13 is also linked to early-onset breast cancer. Mutations in BRCA1 and BRCA2 are each estimated to account for less than 5% of the total of all female breast cancer cases (~180,000) occurring annually in the U.S.

Table: Cellular Genes Associated with Human Cancer Susceptibility and Expression

Human Genes Associated with Cancer Susceptibility and Expressions

Tobacco Smoke

Chemical carcinogenesis by tobacco smoke products is a major cause of common lung cancers.

The discovery of chemical carcinogenesis was made by Sir Percival Pott (1713-1788), English surgeon, who related the cause of scrotal skin cancer in a number of his patients to a common history of occupational exposure to large amounts of coal soot as chimney sweepers when they were boys.

The table lists chemical agents that have been established over time as causes of occupational cancers.

Table:

Agent	Occupation	Cancer Site
Polycyclic hydrocarbons in soot, tar, oil	chimney sweepers, manufacturers of coal gas, many other groups of exposed industrial workers	scrotum, skin, bronchus
2-Naphthylamine; 1-naph-thylamine	chemical workers, rubber workers, manufacturers of coal gas	bladder
Benzidine; 4-aminobiphenyl	chemical workers	bladder
Asbestos	asbestos workers, shipyard and insulation workers	bronchus, pleura, and peritoneum
Arsenic	sheep dip manufacturers, gold miners, some vineyard workers and ore smelters	skin and bronchus
Bis(chloromethyl) ether	makers of ion-exchange resins	bronchus
Benzene	workers with glues, varnishes, etc.	marrow (leukemia)
Mustard gas	poison gas makers	bronchus, larynx, nasal sinuses
Vinyl chloride	PVC manufacturers	liver (angio-sarcoma)

Osteogenic sarcoma of femur

An occupational cancer in a radium dial painter. Neoplastic osteocytes embedded in malignant osteoid matrix (the homogeneous substance between tumor cells).

 

 

In addition to occupational exposure to carcinogens, medical treatment with agents such as ionizing radiations and natural exposure to solar ultraviolet radiation were early recognized as causes of human cancers. Occupational cancers comprise a small but preventable part of the worldwide incidence of human cancers. It is paradoxical that, more than two centuries after the discovery of the carcinogenic hazards of coal soot, a smoke product of another source, associated with life-style rather than occupation, is etiologically related to one of the most prevalent human cancers today, namely, bronchogenic carcinoma of tobacco smokers.

 

 

 

Classification of Chemical Carcinogens

Chemical carcinogens are of synthetic ("man made") or natural origin, are extremely diverse in structure without any common feature, and are classified into two categories:

• direct-acting (DNA-reactive, activation independent, genotoxic) carcinogens that bind covalently to cellular genomic DNA and are mutagens;
• procarcinogens (activation dependent) that require metabolic conversion to metabolites ("ultimate carcinogens") capable of transforming cells and inducing tumors.

Procarcinogens are among the most potent chemical carcinogens.

Table: Major Chemical Carcinogens

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Pro-carcinogens (require metabolic activation to "ultimate carcinogens")

Polycyclic aromatic hydrocarbons
• benzanthracene (first pure carcinogen)
• 3,4-benzpyrene (isolated from coal tar)
• 3-methylcholanthrene (prepared from a steroid, deoxycholic acid)
• 7,12-dimethylbenzanthracene (most potent carcinogen)

Aromatic Amines and Azo Dyes

• 2-naphtylamine (produces bladder carcinoma)
• benzidine (produces bladder carcinoma)
• 2-acetylaminofluorene
• 4-dimethylaminoazobenzene (produces liver tumors)

Natural Products

• aflatoxin B1 (potent hepatocarcinogen produced by mold contamination of food)
• mitomycin C

Other

• nitrosamine (can be formed by action of nitrites on foods)
• some insecticides (chlordane and others)
• some metals (chromium and nickel)
• carbon tetrachloride

Direct-Acting Carcinogens (DNA-reactive)

Alkylating agents
• anticancer chemotherapeutic drugs (cyclophosphamide, busulfan, chlorambucil)
• beta-propiolactone
• bis(chloromethyl)ether

Acetylating agents

• 1-acetylimidazole

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Biological Aspects of Chemical Carcinogenesis

Carcinogenesis is a multiple step process. One of the characteristics of chemical or physical carcinogenesis is the usually extended period of time (latent period) between contact with the carcinogen and the appearance of a tumor. The latent periods of occupational cancers may extend from one to several years and commonly to several decades, as noted in the accompanying table.

Table: Latent Periods of Representative Occupational Cancers

			LatentPeriod (years)
Site of Cancer	Type of Cancer	Agent	Average	Range
Skin	Epidermoid and basal cell carcinomas	Arsenic Coal tar & pitch Ionizing radiation Solar radiation	25 20-24 7 20-30	4-46 1-50 1-12 15-40
Lung	Bronchogenic carcinoma	Asbestos Ionizing radiation	18 25-35	15-48 7-50
Bone marrow	Leukemia	Benzene Ionizing radiation		3-19 3-15
Bladder	Squamous cell carcinoma	Aromatic amines	11-15	2-40
Bone	Osteogenic sarcoma	Ionizing radiation		10-25

Biochemical Aspects of Chemical Carcinogenesis

Most chemical carcinogens are , or are metabolically converted into, electrophilic reactants (electron-attracting chemicals) that cause their biological effects by covalent binding with cellular proteins and nucleic acids, particularly chromosomal DNA. The most frequent reaction sites in DNA are with guanine.

The majority of chemical carcinogens are procarcinogens and require metabolic conversion into chemically reactive forms (ultimate carcinogens). Many chemical pathways (oxidation, reduction, hydroxylation, hydrolysis, conjugation, etc.) lead to metabolic conversion of procarcinogens to intermediate metabolites (proximate carcinogens) and finally to ultimate carcinogens which react with cellular DNA to cause neoplastic transformation.

Most procarcinogens are activated by microsomal enzymes in the endoplasmic reticulum. The conversion of polycyclic aromatic hydrocarbons to ultimate carcinogens is initiated by aryl hydrocarbon hydroxylase (AHH). Cytochrome P-450, a terminal component of an electron transport system present in liver microsomes, is also involved in the metabolic activation of procarcinogens.

Tests for mutagenicity indicate that virtually all ultimate carcinogens are mutagenic. Conversely, most mutagens show carcinogenic activity. In the Ames screening test for mutagenicity, a putative carcinogen is incubated with liver microsomes and an indicator microorganism. An increase in the frequency of specific mutants above control levels is scored as a positive result .

Modifying Factors in Carcinogenesis

Host factors (genetics, gender, hormones, aging) and environmental factors may have a modifying role in increasing, or decreasing, the susceptibility to carcinogens. With procarcinogens, activating enzyme systems must be present or inducible in target cells. This genetically determined activity explains the organ and species specificity of some procarcinogens.

Microsomal enzymes in the liver degrade (detoxify) a large part of a procarcinogen to non-carcinogenic products. Enzymes can be induced which accelerate detoxification. A variety of naturally occurring compounds, such as indole, flavones, and related compounds that occur in vegetables (brussels sprouts, cabbage, broccoli, cauliflower) have a protective action in animals exposed to carcinogenic polycyclic hydrocarbons.

Endogenous (and exogenous) sex hormones are important factors apparently in the promotion stage of human carcinomas of breast, endometrium, and prostate. Additionally, a history of exposure in-utero to the synthetic estrogen diethylstilbestrol (DES) is strongly associated with the development of carcinoma of the vagina and cervix in some young women.

Other exogenous factors in human carcinogenesis include dietary excesses and deficiencies and, most notably, tobacco smoking which is a major factor associated with lung carcinoma.

Non-genotoxic Carcinogens

The actions previously described are those of agents which react with cellular DNA and cause genomic alterations. As more and more chemicals are tested for carcinogenicity, a number are now being recognized as "non-genotoxic". These chemicals do not form stable covalent bonds with cellular DNA or other macromolecules. Solid state materials (asbestos) are an example.

DNA Damage and Repair

Ultimate carcinogens are mutagens and cause point mutations (base-pair substitutions) and frame-shift mutations. The activation of ras proto-oncogenes by point mutations is associated with chemical carcinogenesis in experimental animals and with many types of human cancers. Ultraviolet and ionizing radiations are also mutagenic and produce several types of lesions (strand breaks, cross-links, base alterations) in cellular DNA. Ultraviolet radiation also produces dimers between adjacent thymidines.

Excision repair of damaged DNA occurs to a greater or lesser extent and involves the excision of the damaged strand, synthesis of a patch, and rejoining of the strand by a DNA ligase.

Several autosomal recessive disorders associated with an increased incidence of cancer exhibit defects in the repair and maintenance of DNA. Notably, the repair of damage caused by ultraviolet radiation is defective in patients with xeroderma pigmentosa (autosomal recessive) who have a high incidence of skin cancer in areas exposed to sunlight. Bloom's syndrome, a condition characterized by multiple chromosomal breaks and a high incidence of leukemia or intestinal cancer, is associated with a defect in a DNA ligase.

Specific causes of common human cancers - breast, colon, rectum, lymph nodes, uterus, bladder, pancreas, bone marrow, and stomach, remain unknown.