Read this essay to examine the nature of tumor suppressor genes and the ways in which their loss can lead to cancer. Also learn about the roles played by all the types of gene mutations, along with non-mutational changes, in converting normal cells into cancer cells.
1. Essay on Tumor Suppressor Genes: (Around 4000 Words)
Roles in Cell Proliferation and Cell Death:
By definition, tumor suppressors are genes whose loss or inactivation can lead to cancer, a condition characterized by increased cell proliferation and decreased cell death. It is therefore logical to suspect that the normal function of a tumor suppressor gene would be the opposite—namely, to inhibit cell proliferation or promote cell death—and so the loss of such functions would cause increased cell proliferation or decreased cell death.
i. Cell Fusion Experiments Provided the First Evidence for the Existence of Tumor Suppressor Genes:
The first indication that cells might contain genes whose loss is associated with the development of cancer came from experiments using a technique called cell fusion. In 1960, a research team in Paris headed by Georges Barski discovered that cells of two different types grown in culture will occasionally fuse together to form hybrid cells containing the chromosomes of both original cell types.
Shortly thereafter Henry Harris reported that cell fusion can be artificially induced by treating cells with inactivated forms of a particular type of virus called Sendai virus. Treatment with the virus causes the plasma membranes of two cells to fuse with each other, creating a combined cell in which the nuclei of the two original cells share the same cytoplasm.
When the cell subsequently divides, the two separate nuclei break down and a single new nucleus is formed that contains chromosomes derived from both of the original cells. Such a cell, containing a nucleus with chromosomes derived from two different cells, is called a hybrid cell.
Experiments in which cancer cells were fused with normal cells provided some important early insights into the genetic basis for the abnormal behavior of cancer cells. Based on our current understanding of oncogenes, you might expect that the hybrid cells created by fusing cancer cells with normal cells would have acquired oncogenes from the original cancer cell and would therefore exhibit uncontrolled proliferation, just like a cancer cell.
In fact, that is not what usually happens; the fusion of cancer cells with normal cells almost always yields hybrid cells that initially behave like the normal parent and do not form tumors (Figure 1). Such results, first reported in the late 1960s, provided the earliest evidence that normal cells contain genes that can suppress tumor growth and reestablish normal controls on cell proliferation.
Although fusing cancer cells with normal cells generally yields hybrid cells that lack the ability to form tumors, it does not mean that these cells are normal.
When they are allowed to grow for extended periods in culture, the hybrid cells often revert back to the malignant, uncontrolled behavior of the original cancer cells. Reversion to malignant behavior is associated with the loss of certain chromosomes, suggesting that these particular chromosomes contain genes that had been suppressing the ability to form tumors. Such observations eventually led to the naming of the lost genes as “tumor suppressor genes.”
As long as hybrid cells retain both sets of original chromosomes—that is, chromosomes derived from both the cancer cells and the normal cells—the ability to form tumors is suppressed. Tumor suppression is even observed when the original cancer cells possess an oncogene, such as a mutant RAS gene, that is actively expressed in the hybrid cells.
This means that tumor suppressor genes located in the chromosomes of normal cells are able to overcome the effects of a RAS oncogene present in a cancer cell chromosome. The ability to form tumors only reappears after the hybrid cell loses a chromosome containing a critical tumor suppressor gene.
ii. Studies of Inherited Chromosomal Defects and Loss of Heterozygosity have Led to the Identification of Several Dozen Tumor Suppressor Genes:
Although cell fusion experiments provided early evidence for the existence of tumor suppressor genes, identifying these genes did not turn out to be a simple task. By definition, the existence of a tumor suppressor gene only becomes evident after its function has been lost. How do scientists go about identifying something whose very existence is unknown until it disappears?
One approach is based on the fact that defects in tumor suppressors are responsible for several hereditary cancer syndromes. Members of cancer-prone families often inherit a defective tumor suppressor gene from one parent, thereby elevating their cancer risk because a single mutation in the other copy of that tumor suppressor gene can then lead to cancer.
Microscopic examination of cells obtained from individuals in such families sometimes reveals the existence of gross chromosomal defects. For example, certain individuals with familial retinoblastoma exhibit a deleted segment in a specific region of one copy of chromosome 13, not just in cancer cells but in all cells of the body.
To determine whether a tumor suppressor is located in the region that has undergone deletion, scientists have simply examined retinoblastoma cells to see which gene has become mutated in the comparable region of the second copy of chromosome 13.
The loss of tumor suppressor genes is not restricted to hereditary cancers. These genes may also be lost or inactivated through random mutations that strike a particular target tissue, leading to the mutation or loss of both copies of the same gene.
You might think that the most straightforward way for that to happen would be through two independent mutations randomly occurring in sequence. However, the mutation rate for any given gene is about one in a million per cell division, so the chance of two independent mutations affecting two copies of the same gene is extremely remote.
After a single copy of a tumor suppressor gene has undergone mutation, a more efficient approach for disrupting the remaining normal copy is through a phenomenon known as loss of heterozygosity, so named because the initial state, in which one abnormal and one normal gene copy are present, is called the heterozygous state.
Getting rid of the remaining normal copy therefore causes the heterozygous state to be lost. Loss of heterozygosity is more common than you might expect; whereas individual gene mutations arise at a rate of one in a million per gene per cell division, loss of heterozygosity is as frequent as once in a thousand cell divisions and tends to affect large regions of DNA encompassing hundreds of different genes.
Figure 2 illustrates several ways in which loss of heterozygosity may arise. In one mechanism, called mitotic nondisjunction, the two duplicated copies of a given chromosome fail to separate (disjoin) at the time of mitosis, so both copies go to one daughter cell and the other daughter cell receives no copies.
As seen in Figure 2b, the latter cell will no longer be heterozygous for any genes contained on the missing chromosome. A second mechanism involves mitotic recombination, in which homologous chromosomes exchange DNA sequences when they line up during the process of mitosis. Figure 2c shows how such an exchange could lead to loss of heterozygosity.
A third mechanism, called gene conversion, occurs when the DNA molecules from two homologous chromosomes line up next to each other and copy base sequence information from one to the other.
In this way, a DNA region that was originally present in two different versions in the two members of a homologous pair of chromosomes can be made identical by copying DNA sequence information from one chromosome to the other chromosome (Figure 2d).
The existence of the preceding mechanisms means that if a cell happens to acquire a random mutation that inactivates one copy of a tumor suppressor gene, loss of heterozygosity might either replace the normal copy with the defective version or remove it entirely. Loss of heterozygosity usually affects hundreds of neighboring genes simultaneously, making it relatively easy to detect.
You simply analyze a large number of known genes, searching for those that are present in two different versions in the normal cells of a cancer patient but are present in only one version in the same person’s cancer cells. When genes exhibiting this behavior are detected, it is likely that they lie near a tumor suppressor gene whose loss of heterozygosity is actually responsible for the cancerous growth.
Geneticists have performed thousands of searches looking for chromosomal regions that exhibit loss of heterozygosity in cancer cells. This approach, along with the study of chromosomal defects associated with hereditary cancer syndromes, has led to the identification of several dozen tumor suppressor genes.
iii. The RB Tumor Suppressor Gene Produces a Protein that Restrains Passage through the Restriction Point:
The first tumor suppressor gene to be isolated and characterized was the RB gene. The protein produced by the RB gene, called the Rb protein (or simply Rb), restrains cell proliferation in the absence of growth factors. The Rb protein normally exerts this action by halting the cell cycle at the restriction point.
In cells that have been exposed to an appropriate growth factor, however, signaling pathways trigger the production of Cdk-cyclin complexes that catalyze the phosphorylation of Rb. Phosphorylated Rb can no longer exert its inhibitory effects and so the cells are free to pass through the restriction point and into S phase.
The molecular mechanism by which Rb exerts this control over the restriction point is summarized in Figure 3. Prior to phosphorylation, Rb binds to the E2F transcription factor, a protein that (in the absence of bound Rb) activates the transcription of genes coding for enzymes and other proteins required for initiating DNA replication.
As long as the Rb protein remains bound to E2F, the E2F molecule is inactive and these genes stay silent, thereby preventing cells from entering into S phase. However, in a cell that has been stimulated to divide (e.g., by the addition of growth factors), the activation of growth signaling pathways leads to the production of Cdk-cyclin complexes that catalyze the phosphorylation of Rb. Phosphorylation abolishes the ability of Rb to bind to E2F, thus allowing E2F to activate the transcription of genes whose products are required for entry into S phase.
Because the normal purpose of Rb is to halt the cell cycle in the absence of growth factors, RB mutations that lead to the loss or inactivation of the Rb protein remove this restraining influence on the cell cycle and lead to excessive proliferation. Such mutations leading to a loss of Rb function are observed in some hereditary as well as environmentally caused forms of cancer. Certain cancer viruses also disrupt Rb function.
For example, the human papillomavirus (HPV), has an oncogene that codes for the E7 on co-protein, which binds to Rb. When bound to E7, the Rb protein cannot perform its normal function of restraining passage through the restriction point and cell proliferation therefore proceeds unchecked, even in the absence of growth factors. Cancers triggered by a loss of Rb function can thus arise in two fundamentally different ways- through mutations that delete or disrupt both copies of the RB gene and through the action of viral oncoproteins that bind to and inactivate the Rb protein.
The p53 Tumor Suppressor Gene Produces a Protein that Prevents Cells with Damaged DNA from Proliferating:
Since the discovery of the RB gene in the mid-1980s, dozens of additional tumor suppressor genes have been identified (Table 1). One of the most important is the p53 gene (also called TP53 in humans), which produces the p53 protein. The p53 gene is mutated in a broad spectrum of different tumor types, and almost half of the close to the ten million people diagnosed worldwide with cancer each year will have p53 mutations, making it the most commonly mutated gene in human cancers (Figure 4).
The p53 protein is sometimes called the “guardian of the genome” because of the central role that it plays in protecting cells from the effects of DNA damage. Figure 5 illustrates how this function is performed.
When cells are exposed to DNA-damaging agents, such as ionizing radiation or toxic chemicals, the damaged DNA triggers the activation of an enzyme called ATM kinase, which catalyzes the phosphorylation of p53 and several other target proteins. Phosphorylation of p53 by the ATM kinase prevents it from interacting with Mdm2, a protein that would otherwise mark p53 for destruction by linking it to a small protein called ubiquitin.
Mdm2 is one of numerous proteins in the cell, called ubiquitin ligases that attach ubiquitin molecules to a specific set of proteins. As shown in Figure 6, the normal function of ubiquitin is to direct molecules to the proteasome, the cell’s main protein destruction machine.
After p53 has been phosphorylated by ATM in response to DNA damage, the Mdm2 ubiquitin ligase can no longer attach ubiquitin chains to p53. As a result, the p53 protein accumulates in cells containing damaged DNA rather than being degraded by the ubiquitin-mediated proteasome pathway.
The accumulating p53 in turn activates two types of events- cell cycle arrest and cell death. Both responses are based on the ability of p53 to act as a transcription factor that binds to DNA and activates specific genes. Among the targeted genes is the gene coding for the p21 protein, a member of a class of molecules called Cdk inhibitors because they block the activity of Cdk-cyclin complexes.
The p21 protein inhibits the Cdk-cyclin complex that would normally phosphorylate Rb, thereby halting the cell cycle at the restriction point and providing time for the DNA damage to be repaired. At the same time, p53 also activates the production of DNA repair enzymes.
If the damage cannot be successfully corrected, p53 then activates genes that produce proteins involved in triggering cell death by apoptosis. A key protein in this pathway, called Puma (“p53 up-regulated modulator of apoptosis”), promotes apoptosis by binding to and inactivating the Bcl2 protein, a normally occurring inhibitor of apoptosis.
By triggering cell cycle arrest or cell death in response to DNA damage, the p53 protein prevents genetically altered cells from proliferating and passing the damage on to future cell generations. Mutations that disrupt p53 function therefore increase cancer risk because they permit cells with damaged DNA to survive and reproduce.
For example, individuals who inherit a mutant p53 gene from one parent have an elevated risk of developing cancer because they only require one additional mutation to inactivate the second copy of the gene. This high-risk hereditary condition is called the Li-Fraumeni syndrome.
Most p53 mutations, however, are not inherited; they are caused by exposure to DNA-damaging chemicals and radiation. To cite but two examples, carcinogenic chemicals in tobacco smoke have been found to trigger point mutations in the p53 gene of lung cells, and the ultraviolet radiation in sunlight has been shown to cause p53 mutations in skin cells.
When exposure to carcinogenic chemicals or radiation creates mutations in the p53 gene, you might expect that both copies of the gene would need to be inactivated before functional p53 protein would be lost. In some cases, however, mutation of one copy of the p53 gene may be sufficient to disrupt the p53 protein, even when the other copy of the gene is normal.
The apparent explanation is that the p53 molecule is constructed from four protein chains bound together to form a tetramer. As shown in Figure 7, the presence of even one mutant chain in such a tetramer can be enough to prevent the p53 protein from functioning normally. When a mutation in one copy of the p53 gene causes the p53 protein to be inactivated in this way, even in the presence of a normal copy of the gene, it is called a dominant negative mutation.
Mutating the p53 gene is not the only mechanism for disrupting p53 function; the p53 protein can also be targeted directly by certain viruses. For example, human papillomavirus—whose E7 oncoprotein inactivates the Rb protein—produces another molecule, called the E6 oncoprotein, which binds to and targets the p53 protein for destruction.
The ability of human papillomavirus to cause cancer is therefore linked to its capacity to block the action of proteins produced by both the RB and p53 tumor suppressor genes.
The APC Tumor Suppressor Gene Codes for a Protein that Inhibits the Wnt Signaling Pathway:
The next tumor suppressor to be discussed is, like the p53 gene, a frequent target for cancer-causing mutations; in this case, however, cancers arise mainly in one organ, namely the colon. The gene in question, called the APC gene, is the tumor suppressor. Individuals with this condition inherit a defective APC gene that causes thousands of polyps to grow in the colon and imparts a nearly 100% risk of developing colon cancer for individuals who live to the age of 60.
Although familial adenomatous polyposis is quite rare, accounting for less than 1% of all colon cancers, APC mutations are also associated with the more common forms of colon cancer that arise in people with no family history of the disease. In fact, recent studies suggest that roughly two-thirds of all colon cancers involve APC mutations.
The APC gene codes for a protein involved in the Wnt pathway, a signaling mechanism that plays a prominent role in activating cell proliferation during embryonic development. As shown in Figure 8, the central component of the Wnt pathway is a protein called β-catenin. Normally, β-catenin is prevented from functioning by a multi-protein destruction complex that consists of the APC protein combined with the proteins axin and glycogen synthase kinase 3 (GSK3).
When assembled in such an APC-axin-GSK3 complex, GSK3 catalyzes the phosphorylation of β- catenin. The phosphorylated β-catenin then becomes a target for a ubiquitin ligase that attaches it to ubiquitin, thereby marking the phosphorylated β-catenin for degradation by proteasomes. The net result is a low concentration of β-catenin, which makes the Wnt pathway inactive.
The Wnt pathway is turned on by signaling molecules called Wnt proteins, which bind to and activate cell surface Wnt receptors. The activated receptors stimulate a group of proteins that inhibit the axin-APC-GSK3 destruction complex and thereby prevent the degradation of β-catenin. The accumulating β-catenin then enters the nucleus and interacts with transcription factors that activate a variety of genes, including some that stimulate cell proliferation.
Mutations causing abnormal activation of the Wnt pathway have been detected in numerous cancers. Most of them are loss-of-function mutations in the APC gene that are either inherited or, more commonly, triggered by environmental carcinogens. The resulting absence of functional APC protein prevents the axin-APC-GSK3 complex from assembling and β-catenin therefore accumulates, locking the Wnt pathway in the on position and sending the cell a persistent signal to divide.
iv. The PTEN Tumor Suppressor Gene Codes for a Protein that Inhibits the PI3K-Akt Signaling Pathway:
Cell proliferation is controlled through an interconnected network of pathways with numerous branches and shared components. A good example is provided by growth factors that activate the Ras-MAPK pathway. When a growth factor binds to a receptor that activates Ras-MAPK signaling, the receptor usually activates several other pathways at the same time.
One of these additional pathways, called the PI3K-Akt pathway, involves an enzyme called phosphatidylinositol 3-kinase (abbreviated as PI 3-kinase or PI3K). As shown in Figure 9, PI 3-kinase undergoes activation when it binds to phosphorylated tyrosines found in receptors that have been stimulated by growth factor binding.
A similar mechanism is involved in triggering the Ras-MAPK pathway. PI 3-kinase then catalyzes the addition of a phosphate group to a plasma membrane lipid called PIP2 (phosphatidylinositol-4, 5-bisphosphate), which converts PIP2 into PIP3 (phosphatidylinositol-3, 4, 5-trisphosphate).
PIP3 in turn recruits protein kinases to the inner surface of the plasma membrane, leading to phosphorylation and activation of a protein kinase called Akt. Through its ability to catalyze the phosphorylation of several key target proteins, Akt suppresses apoptosis and inhibits cell cycle arrest. The net effect of the PI3K-Akt signaling pathway is therefore to promote cell survival and proliferation.
Dysfunctions in PI3K-Akt signaling have been detected in a number of different cancers. For example, AKT gene amplification occurs in some ovarian and pancreatic cancers, and a v-akt oncogene coding for a mutant Akt protein is present in an animal retrovirus that causes thymus cancers in mice. In such cases, excessive production or activity of the Akt protein leads to hyperactivity of the PI3K-Akt pathway and hence an enhancement of cell proliferation and survival.
Conversely, inhibitors of PI3K-Akt signaling can function as tumor suppressors. A prominent example is PTEN, an enzyme that removes a phosphate group from PIP3 and thus abolishes its ability to activate Akt. In cells that are not being stimulated by growth factors, the intracellular concentration of PIP3 is kept low by the action of PTEN and the PI3K-Akt pathway is therefore inactive.
When loss-of-function mutations disrupt the ability to produce PTEN, the cell cannot degrade PIP3 efficiently and its concentration rises. The accumulating PIP3 in turn activates Akt, thereby leading to enhanced cell proliferation and survival (even in the absence of growth factors). Mutations that reduce PTEN activity are found in up to 50% of prostate cancers and glioblastomas, 35% of uterine endometrial cancers, and to varying extents in ovarian, breast, liver, lung, kidney, thyroid, and lymphoid cancers.
Growth factors are usually thought of as being molecules that stimulate cell proliferation, but some growth factors have the opposite effect: They inhibit cell proliferation. An example is transforming growth factor β (TGFβ), a protein that may either stimulate or inhibit cell proliferation, depending on the cell type and context. TGFβ is especially relevant for tumor development because it is a potent inhibitor of epithelial cell proliferation, and roughly 90% of human cancers are carcinomas—that is, cancers of epithelial origin.
TGFβ exerts its inhibitory effects on cell proliferation through the TGFβ-Smad pathway illustrated in Figure 10. The first step in this pathway is the binding of TGFβ to a cell surface receptor. Like many other growth factor receptors, the receptors for TGFβ catalyze protein phosphorylation reactions, although in this case the amino acids serine and threonine rather than tyrosine are phosphorylated.
TGFβ binds to two types of receptors, called type I and type II receptors, located on the surface of its target cells. Upon binding of TGFβ, type II receptors phosphorylate type I receptors. The type I receptors then phosphorylate a class of proteins known as Smads, which bind to an additional protein (a “co- Smad”) and move into the nucleus.
Once inside the nucleus, the Smad complex activates the expression of genes that inhibit cell proliferation. Two key genes produce the p15 protein and the p21 protein, which both function as Cdk inhibitors.
The p15 and p21 proteins halt progression through the cell cycle by inhibiting the Cdk-cyclin complexes whose actions are required for passing through key transition points in the cycle.
Components of the TGFβ-Smad signaling pathway are frequently inactivated in human cancers. For example, loss-of-function mutations in the TGFβ receptor are common in colon and stomach cancers, and occur in some cancers of the breast, ovary, and pancreas as well.
Loss-of-function mutations in Smad proteins are likewise observed in a variety of cancers, including 50% of all pancreatic cancers and about 30% of colon cancers. Such evidence indicates that the genes coding for TGFβ receptors and Smads both qualify as tumor suppressors.
vi. One Gene Produces Two Tumor Suppressor Proteins: p16 and ARF:
Thus far, this article has described the relationship between tumor suppressor genes and several signaling pathways for inhibiting cell proliferation or promoting cell death. The next tumor suppressor to be covered, known as the CDKN2A gene, exhibits the rather unusual property of coding for two different proteins that act independently on two of these pathways, the Rb pathway and the p53 pathway.
How does the CDKN2A gene produce two entirely different tumor suppressor proteins? Because the genetic code is read three bases at a time, changing the start point by one or two nucleotides will completely change the message contained in a base sequence.
For example, the sequence AAAGGGCCC can be read in three different reading frames starting from the first, second, or third base—that is, starting as AAA-GGG . . ., AAG- GGC …, or AGG-GCC …, respectively. A shift in the normal reading frame usually creates a garbled message that does not code for a functional protein. In the ease of the CDKN2A gene, however, a shift in the reading frame leads to the production of an alternative protein that is fully functional.
The first of the two proteins produced by the CDKN2A gene is the pl6 protein (also called INK4a), a Cdk inhibitor that suppresses the activity of the Cdk- cyclin complex that normally phosphorylates the Rb protein. Loss-of-function mutations affecting p16 lead to excessive Cdk-cyclin activity and inappropriate Rb phosphorylation. Since the phosphorylated form of Rb cannot restrain the cell cycle at the restriction point, the net result is a loss of cell cycle control.
The second protein produced by the CDKN2A gene is called the ARF (for Alternative Reading Frame) protein. Although they are produced by the same gene, p16 and ARF are completely different proteins exhibiting no sequence similarity. Whereas p16 is a Cdk inhibitor, ARF binds to and promotes the degradation of Mdm2, the ubiquitin ligase that normally targets p53 for destruction by tagging it with ubiquitin (see Figures 5 and 6).
By promoting the degradation of Mdm2, ARF facilitates the stabilization and accumulation of p53. Conversely, loss- of-function mutations affecting ARF interfere with the ability of p53 to accumulate and perform its function in triggering cell cycle arrest and cell death.
The CDKN2A gene therefore influences cell proliferation and survival through two independent proteins: the p16 protein, which is required for proper Rb signaling, and the ARF protein, which is required for proper p53 signaling (Figure 11).
Loss-of-function mutations in CDKN2A have been observed in numerous human cancers, including 15% to 30% of all cancers originating in the breast, lung, pancreas, and bladder. Deletion of both copies of the CDKN2A gene, which leads to complete absence of both the p16 and ARF proteins, is common in such cases.
2. Essay on Tumor Suppressor Genes: (Around 2500 Words)
Roles in DNA Repair and Genetic Stability:
Although they are involved in a variety of different signaling pathways, the tumor suppressor genes discussed thus far share a fundamental feature in common. They produce proteins whose normal function is to inhibit cell proliferation and survival. Loss-of-function mutations in such genes therefore have the opposite effect, namely increased cell proliferation and survival.
A second group of tumor suppressors act through their effects on DNA repair and the maintenance of chromosome integrity. Unlike genes that exert direct effects on cell proliferation and whose inactivation can lead directly to tumor formation, the inactivation of genes involved in DNA maintenance and repair acts indirectly by permitting an increased mutation rate for all genes. This increased mutation rate in turn increases the likelihood that alterations will arise in other genes that directly affect cell proliferation.
The terms gatekeepers and caretakers are used to distinguish between these two classes of tumor suppressor genes. The tumor suppressors described in the first part of this article, which exert direct effects on cell proliferation and survival, are considered to be “gatekeepers” because the loss of such genes directly opens the gates to tumor formation.
Tumor suppressors involved in DNA maintenance and repair, on the other hand, are “caretakers” that preserve the integrity of the genome and whose inactivation leads to mutations in other genes (including gatekeepers) that actually trigger the development of cancer. We will examine the functions of some of these caretaker genes.
i. Genes Involved in Excision and Mismatch Repair Help Prevent the Accumulation of Localized DNA Errors:
Cancer cells accumulate mutations at rates that can be hundreds or even thousands of times higher than normal. This condition, called genetic instability, does not by itself disrupt the normal controls on cell proliferation.
In fact, most of the mutations that arise in genetically unstable cells are likely to be harmful mutations that hinder cell survival. But elevated mutation rates also increase the probability that occasional mutations will arise that allows cells to escape from the normal constraints on cell proliferation and survival.
Cells that randomly incur such mutations will tend to outgrow their neighbors, an important first step in the development of cancer. Increased mutation rates also facilitate tumor progression in which cells acquire additional traits—for example, faster growth rate, increased invasiveness, ability to survive in the bloodstream, resistance to immune attack, ability to grow in other organs, resistance to drugs, and evasion of death-triggering mechanisms—that allow cancers to become increasingly more aggressive.
Genetic instability occurs in several different forms that differ in their underlying mechanisms. The simplest type is caused by defects in the DNA repair mechanisms that cells use for correcting localized errors involving one or a few nucleotides. These localized errors typically arise either from exposure to DNA- damaging agents or from base-pairing mistakes that take place during DNA replication.
There are two types of repair mechanisms employed for correcting such errors. Excision repair, is capable of repairing abnormal bases created by exposure to DNA-damaging agents, and mismatch repair, is used for correcting inappropriately paired bases that arise spontaneously during DNA replication.
Individuals who inherit loss-of-function mutations involving genes required for either of these repair mechanisms exhibit an increased cancer risk. For example, inherited mutations in excision repair genes cause xeroderma pigmentosum, a hereditary cancer syndrome involving an extremely high risk for skin cancer.
In a similar fashion, inherited mutations in genes coding for proteins involved in mismatch repair are responsible for hereditary nonpolyposis colon cancer (HNPCC), a hereditary syndrome associated with a high risk for colon cancer.
Although both of these hereditary syndromes involve a striking increase in cancer risk, xeroderma pigmentosum exhibits a recessive pattern of inheritance and HNPCC exhibits a dominant pattern of inheritance. In other words, inheriting an elevated cancer risk requires two defective copies of an excision repair gene but only one defective copy of a mismatch repair gene.
The reason for this difference appears to be related to how many steps are required to create genetic instability in the two cases (Figure 12). In a person who inherits a single defective mismatch repair gene all that is required to start accumulating DNA errors at a high rate is for the second copy of the gene to undergo mutation. This second “hit” will immediately permit uncorrected errors to accumulate during normal DNA replication because of the absence of mismatch repair.
In contrast, if a person were to inherit a single defective excision repair gene, subsequent mutation of the second copy of the gene would debilitate excision repair but would not immediately lead to the accumulation of mutations.
A third step, namely exposure to a DNA-damaging agent such as ultraviolet light, is needed to actually create the mutations. Thus more steps are needed to create genetic instability involving excision repair than is the case for mismatch repair.
Inherited mutations in genes required for excision or mismatch repair create a dramatic increase in the risk for certain hereditary cancers, but mutations in these two classes of genes are less important for most nonhereditary forms of cancer.
Nonetheless, mutations in excision or mismatch repair have been detected in about 15% of colon cancers and in several other kinds of cancer as well, suggesting that deficiencies in DNA repair occasionally contribute to the genetic instabilities observed in non- hereditary cancers.
Proteins Produced by the BRCA1 and BRCA2 Genes Assist in the Repair of Double-Strand DNA Breaks:
Another type of genetic instability exhibited by cancer cells involves their tendency to acquire gross abnormalities in chromosome structure and number. Such chromosomal instabilities can be caused by defects in a variety of different tumor suppressors, including the BRCA1 and BRCA2 genes.
Women who inherit a mutation in one of the BRCA genes typically exhibit a lifetime cancer risk of 40% to 80% for breast cancer and 15% to 65% for ovarian cancer. The BRCA1 and BRCA2 genes were initially thought to exert their effects directly on cell proliferation, but later studies revealed that they produce proteins involved in pathways for sensing DNA damage and performing the necessary repairs.
The two BRCA tumor suppressor genes code for large nuclear proteins that bear little resemblance to one another. An early clue regarding their cellular role came from the observation that cells deficient in either of the BRCA proteins exhibit large numbers of chromosomal abnormalities, including broken chromosomes and chromosomal translocations.
The apparent reason for these abnormalities is that the two BRCA proteins are involved in the process by which cells repair double-strand breaks in DNA. Double-strand breaks are more difficult to repair than single-strand breaks because with single-strand breaks, the remaining strand of the DNA double helix remains intact and can serve as a template for aligning and repairing the defective strand.
In contrast, double-strand breaks completely cleave the DNA double helix into two separate fragments and the repair machinery is therefore confronted with the problem of identifying the correct two fragments and rejoining their broken ends without losing any nucleotides.
The two main ways of repairing double-strand breaks are nonhomologous end-joining and homologous recombination. Of the two mechanisms, homologous recombination is less prone to error because it uses the DNA present in the unbroken homologous chromosome to serve as a template for guiding the repair of the DNA from the broken chromosome.
Repairing double-strand breaks by homologous recombination is a complex process that requires the participation of a large number of different proteins, including BRCA1 and BRCA2. The pathway is activated by the same ATM kinase whose role in detecting and responding to DNA damage was introduced earlier in this article (see Figure 5).
We have already seen that in response to DNA damage, the ATM kinase catalyzes the phosphorylation of the p53 protein, which then halts the cell cycle to permit time for repair to occur. The ATM kinase also phosphorylates and activates more than a dozen additional proteins involved in cell cycle control and DNA repair, including BRCA1 and other molecules required for repairing double-strand breaks.
Figure 13 shows that the mechanism for repairing double-strand breaks by homologous recombination involves two main phases. First, a group of proteins called the Rad50 exonuclease complex removes nucleotides from one strand of the broken end of a DNA double helix to expose a single-stranded segment on the opposite strand.
In the second phase, a multi-protein assembly called the Rad51 repair complex carries out a “strand invasion” reaction in which the exposed single-stranded DNA segment at the end of the broken DNA molecule displaces one of the two strands of the intact DNA molecule being used as a template.
In this step, Rad51 first coats the single-stranded DNA; the coated strand then invades and moves along the target DNA double helix until it reaches a complementary sequence. Once it has been located, the complementary sequence is used as a template for guiding repair of the broken DNA.
Although their roles are not completely understood, the BRCA1 and BRCA2 proteins are both required for efficient repair of double-strand breaks. BRCA2 binds tightly to and controls the activity of Rad51, the central protein responsible for carrying out strand invasion during repair by homologous recombination.
BRCA1 is associated with both the Rad50 exonuclease complex and the Rad51 repair complex. Moreover, it is known that ATM phosphorylates BRCA1 in response to DNA damage, suggesting that BRCA1 plays an early role in activating the pathway for repairing double-strand breaks.
Cells deficient in either BRCA1 or BRCA2 are extremely sensitive to carcinogenic agents that produce double-strand DNA breaks. In such cells, double-strand breaks can only be repaired by error-prone mechanisms, such as non-homologous end-joining, that lead to broken, rearranged, and translocated chromosomes. The resulting chromosomal instability is thought to play a large role in the cancer risks exhibited by women who inherit BRCA1 or BRCA2 mutations.
Mutations in Genes that Influence Mitotic Spindle Behavior can Lead to Chromosomal Instabilities:
We have just seen how broken and translocated chromosomes arise in cancer cells as a result of mutations that disrupt tumor suppressor genes needed for repairing double-strand DNA breaks. Another chromosomal abnormality frequently observed in cancer cells is the tendency for whole chromosomes to be lost or gained, thereby leading to aneuploid cells that possess an abnormal number of chromosomes (Figure 14).
The various mechanisms that underlie the development of aneuploidy are just beginning to be unraveled, but evidence already points to the existence of tumor suppressor genes whose loss contributes to this type of chromosomal instability.
To explain how these tumor suppressors work, we first need to review the normal mechanisms used by cells for sorting and parceling out chromosomes during cell division. In a normal cell cycle, chromosomal DNA is first replicated during S phase to create duplicate copies of each chromosome, and the duplicate copies are then separated into the two new cells formed by the subsequent mitotic cell division.
Accurate separation of the duplicated chromosomes is accomplished by attaching the chromosomes to the mitotic spindle, which separates and moves the chromosomes in a way that ensures that each new cell receives a complete set of chromosomes (Figure 15).
A critical moment occurs at the end of metaphase, when the chromosomes line up at the center of the mitotic spindle just before being parceled out to the two new cells. If chromosome movement toward opposite spindle poles were to begin before the chromosomes is all attached to the spindle, a newly forming cell might receive extra copies of some chromosomes and no copies of others.
To protect against this possible danger, cells possess a control mechanism called the spindle checkpoint that monitors chromosome attachment to the spindle and prevents chromosome movement from beginning until all chromosomes are properly attached. In the absence of such a mechanism, there would be no guarantee that each newly forming cell would receive a complete set of chromosomes (see Figure 15, bottom right).
The key to the spindle checkpoint is the anaphase- promoting complex, a multiprotein complex that triggers the onset of anaphase—the stage of mitosis when the chromosomes move toward opposite poles of the mitotic spindle.
As shown in Figure 16a, the anaphase-promoting complex initiate’s chromosome movement by activating separase, an enzyme that breaks down proteins called cohesins that hold the duplicated chromosomes together. As long as they are joined together by cohesins, the duplicated chromosomes cannot separate from each other and move toward opposite spindle poles.
To prevent premature separation, chromosomes that are not yet attached to the mitotic spindle send a “wait” signal that inhibits the anaphase-promoting complex, thereby blocking the activation of separase. The “wait” signal is transmitted by proteins that are members of the Mad and Bub families.
The Mad and Bub proteins bind to chromosomes that are unattached to the mitotic spindle and are converted into a Mad-Bub multiprotein complex, which inhibits the anaphase-promoting complex by blocking the action of one of its essential activators, the Cdc20 protein (see Figure 16b).
After the chromosomes have all become attached to the spindle, the Mad and Bub proteins are no longer converted into this inhibitory complex and the anaphase-promoting complex is free to initiate the onset of anaphase.
Mutations that cause the loss or inactivation of Mad or Bub proteins have been linked to certain types of cancer, which indicates that genes coding for some of the Mad and Bub proteins behave as tumor suppressor genes. A lack of Mad or Bub proteins caused by loss-of-function mutations in these tumor suppressor genes disrupts the “wait” mechanism and impedes the ability of the spindle checkpoint to operate properly.
Under such conditions, chromosome movement toward the spindle poles begins before all the chromosomes are properly attached to the mitotic spindle. The result is a state of chromosomal instability in which cell division creates aneuploid cells lacking some chromosomes and possessing extra copies of others.
Another route to chromosomal instability involves the mechanism responsible for assembling the mitotic spindle. Formation of a mitotic spindle requires two small structures called centrosomes, one located at each end of the spindle (see Figure 15). Centrosomes promote the assembly of the spindle microtubules, which form in the space between the two centrosomes. Cancer cells often possess extra centrosomes and therefore produce aberrant mitotic spindles.
In Figure 17, we see a cancer cell with three centrosomes that have assembled a spindle with three poles. Multipolar spindles containing three or more poles, which are rare in normal tissues but common in cancer cells, contribute to the development of aneuploidy because they cannot sort the two sets of chromosomes accurately. Cells produced by mitosis involving an abnormal spindle will often be missing certain chromosomes and thus will lack any tumor suppressor genes that the missing chromosomes would normally possess.
3. Essay on Tumor Suppressor Genes: (Around 2000 Words)
Role of Mutation and Non-Mutation in Converting Normal Cells into Cancer Cells:
Mutations in cancer-related genes, and the genetic instability that facilitates the accumulation of such mutations, are centrally involved in the mechanisms by which cancers arise.
Yet one cannot explain the behavior of a malignant tumor by pointing solely to gene mutations. The final part of this article will provide a broad overview of the role played not just by mutations but also by non-mutational changes in converting normal cells into cancer cells.
i. Cancers Vary in their Gene Expression Profiles:
Mutations that create oncogenes or disrupt the function of tumor suppressor genes are central to the development of cancer, but they do not explain all the cellular changes that accompany the conversion of normal cells into cancer cells.
Many of the properties exhibited by cancer cells are triggered not by gene mutations, but by switching on (or off) the expression of normal genes, thereby leading to increases (or decreases) in the production of hundreds of different proteins. The term epigenetic change is employed when referring to such alterations that are based on changing the expression of a gene rather than mutating it.
Measuring epigenetic changes requires techniques that can monitor the expression of thousands of genes simultaneously. One very powerful tool is the DNA microarray, a fingernail-sized, thin chip of glass or plastic that has been spotted at fixed locations with thousands of DNA fragments corresponding to various genes of interest.
A single microarray may contain 10,000 or more spots, each representing a different gene. To determine which genes are being expressed in any given cell population, one begins by extracting molecules of messenger RNA (mRNA), which represent the products of gene transcription. The mRNA is then copied with reverse transcriptase, an enzyme that makes single-stranded DNA copies that are complementary in sequence to each mRNA.
The resulting single-stranded DNA (called cDNA for complementary DNA) is then attached to a fluorescent dye. When the microarray is bathed with the fluorescent cDNA, each cDNA molecule binds or hybridizes by complementary base-pairing to the spot containing the specific gene to which it corresponds.
Figure 18 illustrates how DNA microarrays can be used to create a gene expression profile that compares the patterns of gene expression in cancer cells and a corresponding population of normal cells. In this particular example, two fluorescent dyes are used: a red dye to label cDNAs derived from cancer cells and a green dye to label cDNAs derived from the corresponding normal cells.
When the red and green cDNAs are mixed together and placed on a DNA microarray, the red cDNAs bind to genes expressed in cancer cells and the green cDNAs bind to genes expressed in normal cells.
Red spots therefore represent higher expression of a gene in cancer cells, green spots represent higher expression of a gene in normal cells, yellow spots (caused by a mixture of red and green fluorescence) represent genes whose expression is roughly the same, and black spots (absence of fluorescence) represent genes expressed in neither cell type.
Thus the relative expression of thousands of genes in cancer and normal cells can be compared by measuring the intensity and color of the fluorescence of each spot. Such analyses have revealed that the expression of hundreds of different genes is typically altered in cancer cells compared with normal cells of the same tissue. Moreover, significant variations in gene expression are often detected when the same type of cancer is examined in different patients.
The changes in gene expression commonly exhibited by cancer cells arise in several difference ways. One well- documented mechanism involves epigenetic silencing by DNA methylation, a process in which methyl groups are attached to the base C in DNA at sites where it is located adjacent to the base G.
In vertebrate DNA, these -CG- sequences are preferentially located near the beginning of genes (about half of all human genes are associated with -CG- sites). When -CG- sequences undergo methylation, the transcription of adjacent genes is inhibited or “silenced.”
Most -CG- sites are un-methylated in normal cells, but extensive methylation is often seen in cancer cells, where it leads to the inappropriate silencing of a variety of different genes. Tumor suppressor genes are frequently among the genes to be silenced by this mechanism.
In fact, the tumor suppressor genes of cancer cells are inactivated by epigenetic silencing at least as often as they are inactivated by DNA mutation. Loss of gene function through inappropriate methylation may therefore be as important to cancer cells as mutation induced loss of function.
ii. Colon Cancer Illustrates How a Stepwise Series of Mutations can Lead to Malignancy:
Cancer arises via a multistep process in which cellular properties gradually change over time as mutations confer new traits that impart selective advantages to the cells in which they arise.
Now that we have described the main classes of cancer-related genes and the molecular pathways in which they participate, it is appropriate to return to the concept of multistep carcinogenesis to see how a specific sequence of gene mutations can lead to cancer.
Current estimates indicate that there are more than 100 different oncogenes and several dozen tumor suppressor genes. For cancer to arise, it is rarely sufficient to have a defect in just one of these genes, nor is it necessary for a large number to be involved.
Instead, each type of cancer tends to be characterized by a small handful of mutations involving the inactivation of tumor suppressor genes as well as the conversion of proto-oncogenes into oncogenes. In other words, creating a cancer cell usually requires that the brakes on cell growth (tumor suppressor genes) be released and the accelerators for cell growth (oncogenes) be activated.
This principle is nicely illustrated by the stepwise progression toward malignancy observed in colon cancer. Scientists have isolated DNA from a large number of colon cancer patients and examined it for the presence of mutations. The most common pattern to be detected is the presence of a KRAS oncogene (a member of the RAS gene family) accompanied by loss-of-function mutations in the tumor suppressor genes APC, p53, and SMAD4.
Rapidly growing colon cancers tend to exhibit all four genetic alterations, whereas benign tumors have only one or two, suggesting that mutations in the four genes occur in a stepwise fashion that correlates with increasingly aggressive behavior.
As shown in Figure 19, the earliest mutation to be routinely detected is loss of function of the APC gene, which frequently occurs in small polyps before cancer has even arisen. Mutations in KRAS tend to be seen when the polyps get larger, and mutations in SMAD4 and p53 usually appear as cancer finally begins to develop.
These mutations, however, do not always occur in the same sequence or with the same exact set of genes. For example, APC mutations are found in about two-thirds of all colon cancers, which means that the APC gene is normal in one out of every three cases.
Analysis of tumors containing normal APC genes has revealed that many of them possess oncogenes that produce an abnormal, hyperactive form of β-catenin, a protein that—like the APC protein—is involved in Wnt signaling (see Figure 8).
Because APC inhibits the Wnt pathway and β-catenin stimulates it, mutations leading to the loss of APC and mutations that create hyperactive forms of β-catenin have the same basic effect- Both enhance cell proliferation by increasing the activity of the Wnt pathway.
Another pathway frequently disrupted in colon cancer is the TGFβ-Smad pathway, which inhibits rather than stimulates epithelial cell proliferation. Loss-of-function mutations in genes coding for components of this pathway, such as the TGFβ receptor or Smad4, are commonly detected in colon cancers. Such mutations disrupt the growth-inhibiting activity of the TGFβ-Smad pathway and thereby contribute to enhanced cell proliferation.
Overall, the general principle illustrated by the various colon cancer mutations is that different tumor suppressor genes and oncogenes can affect the same pathway, and it is the disruption of particular signaling pathways that is important in cancer cells rather than the particular gene mutations through which the disruption is achieved (Table 2).
iii. The Various Causes of Cancer can be brought Together into a Single Model:
Colon cancer illustrates how normal cells can be converted into cancer cells by a small number of genetic changes, each affecting a particular pathway and conferring some type of selective advantage. Of course, colon cancer is just one among dozens of different human cancers, and the few genes commonly mutated in colon cancer are only a tiny fraction of the more than 100 different oncogenes and tumor suppressor genes.
When various kinds of tumors are compared, it is found that different combinations of gene mutations can lead to cancer and that each type of cancer tends to exhibit its own characteristic mutation patterns.
Despite this variability, a number of shared principles are apparent in the various routes to cancer. An overview is provided by the model illustrated in Figure 20, which begins with the four main causes of cancer: chemicals, radiation, infectious agents, and heredity.
Each of these four factors contributes to the development of malignancy. While the details may differ, the bottom line is that one way or another, each of the four causes of cancer leads to DNA alterations.
In the case of either viruses that introduce specific oncogenes into cells or cancer syndromes that arise from inherited gene defects, the DNA alterations involve a specific gene. Most of the DNA mutations induced by carcinogens, on the other hand, are random. The higher the dose and potency of the carcinogen, the greater the DNA damage and therefore the greater the probability that a random mutation will disrupt a critical gene.
But critical genes (proto-oncogenes and tumor suppressor genes) represent only a tiny fraction of the chromosomal DNA, so the random nature of mutation means that luck plays a significant role; if two people are exposed to the same dose of a carcinogen, one may develop cancer while the other does not simply because random mutations happen to damage a critical proto-oncogene or tumor suppressor gene in the unlucky individual.
The random nature of mutation contributes to the long period of time that is usually required for cancer to develop. Moreover, when DNA repair mechanisms and DNA damage checkpoints are operating properly, many mutations are either repaired or the cells containing them are destroyed by apoptosis. Taken together, such considerations may help explain why cancer is largely a disease of older age.
For cancer to develop, cells need to gradually accumulate a stepwise series of appropriate mutations involving the inactivation of tumor suppressor genes as well as the conversion of proto-oncogenes into oncogenes.