Cancer, the excessive proliferation of a body’s own cells, strikes one in two men and one in three women at least once in their lifetime [DePinho, 2000], and is the second leading cause of death in the United States [Minino and Smith, 2000]. These cancerous cells can remain in one location in the body and form tumors. They can also metastasize, by entering the blood stream or lymph vessels, migrating, and then replacing normal tissue in parts of the body other than where the cancer originated.Cancer begins when a normal cell undergoes mutations that allow the cell to proliferate (divide) more than it should. The number of normal cells that can survive in a tissue is determined by two factors: the blood supply to the tissue, which provides nutrients and oxygen to cells, and the presence of chemicals, called survival factors, that are released by surrounding cells. In normal cells, the process of cell division (mitosis), which also usually requires these survival factors, halts when the cell detects damage to its own DNA. To form a tumor, the cancer cells must be able to survive and divide in tissues where a normal cell would stop mitosis, or die. A normal cell that is in an environment deficient in nutrients or survival factors (if there are too many cells occupying the same tissue) will first stop dividing, and then activate a suicide program known as apoptosis. Cancer occurs when a cell accumulates enough mutations (changes in the genetic information contained in DNA) that it can to escape these natural growth-control mechanisms. This process is known as carcinogenesis.
Cells Kill Themselves
Cell division is easily observed in the development of an embryo and in the growth of a child. Cell death, on the other hand, occurs quietly, frequently going unnoticed. The two types of cell death are necrosis, which results from injury or exposure to toxins, and apoptosis, or programmed cell-suicide. (See Figure 1.) Necrosis is easily observed, as it causes cells to explode: the membrane ruptures, and the contents of the inside of the cell leak into the surrounding tissue. This triggers an inflammatory response. Apoptosis is much more subtle: the cell shrinks and the membrane splits into small pieces containing parts of the cell. These membrane-bound portions of the dying cell are then absorbed by the surrounding living cells, without inflammation or other observable effects [Alberts, et. al., 1994].
Both mitosis and apoptosis are required for proper development and tissue maintenance throughout an organism’s life. During embryonic development, it is apoptosis that removes the web-like skin found between an embryo’s fingers, and removes a tadpole’s tail as it becomes a frog. In the adult, apoptosis kills potentially dangerous immune cells and controls cell numbers by eliminating excess cells generated by mitosis [Raff, 1998]. Apoptosis also removes cells with damaged DNA, and cells that have migrated inaccurately. But when this “clean-up” procedure goes wrong, these damaged cells do not die, and cancer can develop.
Treatment of Cancer
Current cancer therapies are frequently painful and many times ineffective. Chemotherapy works by inducing apoptosis in cancer cells, rather than through interruption of the cell cycle, as it is frequently portrayed [Green and Evan, 2002]. The good news is that during carcinogenesis, cells that make up a tumor are more susceptible to the induction of apoptosis than the surrounding normal cells. The bad news is that chemotherapy causes both cancerous and normal cells to undergo this induced apoptosis, and this leads to many of the side effects of chemotherapeutic treatment, such as extreme exhaustion and hair loss. In the early stages of a cancer, chemotherapy causes the death of many cancer cells and few normal ones, presumably because the cancer cells are still in an environment that is deficient in nutrients and survival factors. In later-stage cancers, after the tumor has caused the growth of new blood vessels and the cells of the tumor have escaped the need for survival factors, such chemotherapy causes the death of many normal cells and only a few cancerous ones, because these cancer cells have now effectively blocked the apoptotic pathway (the biological assembly line) that leads to cell death. (This death-machinery remains in most cancer cells, but it has been disabled.)The specific portion of the pathway that is disrupted varies in different cancers. This suggests that the ultimate cancer treatment would involve finding a way around the blockage to trigger apoptosis only in cancer cells, without activating the pathway in normal cells, thus killing the cancer without side effects [Green and Evan, 2002].
Triggering Cell death
When cells either stop receiving survival signals or receive death signals, they die, and are said to have committed suicide. They may have lost the competition with their neighbors for limited survival factors, and die through activation of the stress (mitochondrial) pathway. Alternatively, they may have received death signals from other cells, and die following activation of the death receptor pathway. Both are considered “suicides” because cell death is caused by factors within the cell that are activated by other factors within the same cell. There are many different triggers of apoptosis: For example, detection (by the cell) of DNA damage at a cell-cycle checkpoint activates a protein within the cell, p53, that prevents that cell from dividing until other proteins have repaired that DNA damage. However, if the damage is too extensive to be easily repaired, p53 instead triggers death via apoptosis [Evan and Littlewood, 1998]. Apoptotic pathways are complicated by the many checks and balances that prevent accidental cell death and yet ensure that death occurs when necessary. (See Figure 2.) There are two different but interacting basic pathways by which apoptosis can occur (See Figure 3): The death-receptor pathway (more like cell-cell murder, in which one cell responds to a death-signal released by another cell), and the mitochondrial (stress) pathway (the mitochondria -- part of the cell, separated by a membrane, responsible for energy production -- release their contents into the cell, causing death.) Proteins responsible for causing apoptotic cell death are called caspases. Caspases break specific protein bonds, either breaking off a portion of the protein, thus making it functional, or breaking apart the functional part of the protein, thus destroying the protein. These processes are analogous to shaving off part of a dull pencil to allow its use or breaking it into pieces that are too short to be functional. Most caspases are activated when bonds are broken by other caspases. The first caspase in the cascade (caspase-9 in the mitochondrial pathway, caspase-8 in the death-receptor pathway) is activated by the trigger of apoptosis. The later caspases are activated when bonds are broken by already-active caspases. Activated caspases cause cell death by destroying many of the necessary cellular proteins. In one important event, caspases inactivate a protein that blocks a nuclease. This activates the nuclease, which then breaks the cell’s DNA apart, thereby ensuring death. Caspases also break many of the proteins that provide cellular structure, thereby causing the cell to lose its shape and break apart, whereupon it is digested by neighboring cells [Hengartner, 2000].
Cell-Death Mechanisms
Activating apoptosis via the death-receptor pathway begins when a death-inducing ligand (specific molecule) binds to a death receptor located on the membrane of the cell. (See Figure 4.) Each death receptor matches to a specific ligand, as a specific lock will match to a specific key. This ligand can be a soluble molecule in the extracellular space that has been released by another cell, or it can be a molecule that is attached to a membrane protein of a neighboring cell. There are several different such ligand-receptor pairs, and the basic mechanism by which each of these works is the same. Cellular membranes are fluid, and proteins can move through them. Receptor proteins such as death receptors span the membrane, so that the protein is effectively divided into three parts: The portion outside of the cell, the portion through the membrane, and the portion in the cytoplasm of the cell on the inside of the membrane. A ligand binds to the extracellular part of the receptor, and when it does so, it causes three receptor proteins to join, forming a trimeric unit. A fourth protein binds to the intracellular portion of these three receptor proteins, and it is this protein that is responsible for activating the first caspase in the caspase cascade. In this way, the receptor proteins can transduce a signal from the outside of the cell (where the ligand is) to the inside of the cell (where apoptosis can be activated). Several different caspases participate in the death of the cell, each destroying specific other proteins in the cell, leading to death [Kaufmann and Gores, 2000].
The mitochondrial pathway also leads to caspase activation, but it uses a different mechanism. (See Figure 5.) This pathway is activated by cellular stress, such as insufficient nutrients or survival factors, UV radiation-induced DNA damage, and the cell-cycle control protein p53, if activated. These stress triggers cause the protein Bid to bind to proteins located on the mitochondrial membrane called the Bcl-2 family proteins [Evan and Littlewood, 1998]. This family includes proteins that activate apoptosis and proteins that inhibit apoptosis. The end result is that majority rules. Bax and Bak work together to cause cell death, while Bcl-2 and Bcl-XL, other mitochondrial membrane proteins in that same family, work together to fend off death. The balance of these factors determines the cell’s fate. (See Figure 6.) When the Bax and Bak proteins outnumber the inhibitors (Bcl-2 and Bcl-XL), they bind to each other. This action forms a pore in the membrane, and allows cytochrome C to enter the cytoplasm. Cytochrome C, which plays an important role in energy production, is toxic when released into the cytoplasm because it binds to and activates a protein, Apaf-1, which, in turn, activates caspase-9, which in turn activates caspases 3 and 7. [Kaufmann and Gores, 2000] This is the same caspase cascade as occurs in the death-receptor pathway, and the caspase-caused destruction of cellular proteins and DNA finally kills the cell.
Cancer Cell Survival
By evolving a way around the apoptotic pathway, cancer cells survive in situations that would kill normal cells. Each time a cell divides, it must replicate its DNA, and mistakes (mutations) can occur during this process. Rapidly dividing cancerous cells have much higher mutation rates than do normal cells because the cancerous cells lost the checks on their growth during carcinogenesis. When a cancerous cell acquires a mutation that allows it to survive better than its neighbors, that mutation will become present in more cells within the tumor. Eventually, cells with that mutation will predominate -- natural selection on a cellular level.Although DNA is the genetic information of a cell, it is proteins that perform the actions in the cell. A mutation in the DNA will result in a mutated protein, as the DNA sequence for a protein is directly correlated to that protein’s structure and function. (See Figure 7.) But a mutant protein can also arise without a mutation in the DNA if it is damaged later in the process of formation. A DNA-based mutation is analogous to a damaged candy mold that cannot correctly shape candy, and a non-mutant-DNA protein mutation is analogous to an incomplete (and thus misshapen) piece of candy coming from a good mold. Mutant proteins generated from correct DNA degrade within one generation of the cell, and thus do not contribute to the changes leading to cancer. Only DNA-based mutations can do this. Loss-of-function mutations in proteins that promote apoptosis, and gain-of-function mutations in proteins that inhibit apoptosis are two different types of mutations that promote the survival of cancer cells. (See Figure 8.)
Loss -of-function mutations generate proteins that are incapable of performing their usual function, usually because the mutation has caused the deletion of part or all of the gene for the protein, or the exchange of one amino acid (the structural unit for a protein) for another in the protein. A common class of mutations that increase death resistance in cancer cells are those that affect p53, which normally triggers cell death when irreparable DNA damage is detected. Mutations that affect the pro-death Bcl-2 family members (including Bax and Bak), preventing them from forming the pore in the in the mitochondria, also block death, thereby promoting cell survival.
Gain-of-function mutations generate more active copies of a protein than are present in a normal cell in a similar environment. These mutations can arise in several different ways, including the deregulation of a protein’s expression, a loss-of-function mutation in an inhibitor (for a protein that is normally present in the cell, but not active), and chromosomal translocations, in which the gene (A) for a protein gets moved next to a gene (B) for a protein that is normally present at much higher levels than the first protein, and that causes the translocated gene (A) to produce protein at that same high level. As an example, by over-expressing the anti-death members of the Bcl-2 family (Bcl-2, Bcl-XL, Mcl-1), so that they outnumber the pro-death proteins, a cancer cell can avoid death. Another such mutation results in the over-expression of decoy death receptors. A decoy death receptor looks like a functional death receptor from the outside of the cell (all that the ligand can “see”), but does not have a portion inside of the cell to transduce the signal. These receptors mimic functional death-receptors in ligand binding, but do not transmit the signal to the rest of the cell. (See Figure 9.)
Cancerous cells contain these mutations. Approximately fifty percent of all cancers have a loss-of-function mutation in the p53 gene. Without the p53 protein, which can trigger death, a cell with severe DNA damage can survive. Fifty percent of lung and colon tumor cells show increased numbers of a decoy-death receptor, DR3, and do not respond well to extracellular death-inducing signals. Some cancer cells also have an increased amount of Bcl-2, which fends off death. Because each of these mutations (and others) cause cells to block the initial proteins in the pathway, but not the caspases that actually kill the cell, it may be possible to activate any still-functional apoptotic machinery that is located “downstream” from these blocked proteins.
Killing Just the Villains
Chemotherapy causes high levels of DNA damage. This leads to cellular stress, which activates the p53 death pathway, causing apoptosis. Cancer cells are more sensitive to apoptosis than are normal cells, because they compete with each other for limited nutrients and survival factors. In its early stages, a tumor is still receiving the standard amount of blood for the tissue it develops in, and nutrients are scarce. Additionally, healthy cells only release sufficient survival factors for their normal number of surrounding cells. A developing tumor creates many more cells, all of which are competing for that standard issue of survival factors. (Each cancer cell is not receiving its recommended daily value of vitamins!)Chemotherapy is most effective in killing early cancers, before they can generate new blood vessel growth and can deactivate apoptotic pathways. It is also more effective at killing cancer cells that still have a functional p53 gene, because when chemotherapy-induced stress causes expression of p53, this can trigger apoptosis [Wu, et. al., 1999]. Statistically, fifty percent of cancer diagnoses have a functional p53 gene, so chemotherapy is effective for only half of all cancer patients. Chemotherapy is also less effective when cancer cells have increased levels of Bcl-2, which blocks p53-induced (mitochondrial) apoptosis [Bold, et. al., 1997].
Chemotherapy also causes damage to currently dividing healthy cells, but successful chemotherapy kills more cancer cells than it does healthy cells. It is the death of healthy cells, such as white-blood cells and hair cells that causes the commonly known side effects of the therapy. The ideal cancer therapy would activate apoptosis only in cancer cells, and not damage any healthy cells. This will require being able to target the differences between cancerous and healthy cells. One such difference is that some cancer cells express specific death receptors (DR4 and DR5) not expressed in most normal cells. These death-receptors bind a ligand (TRAIL) that does not induce death via any other death-receptor. Therapy targeted at activating just these receptors kills tumor cells while not affecting most normal cells [Kaufmann and Gores, 2000].
A different approach to therapy would be to restore the function of the apoptotic pathway in the cancer cell, and then use it to kill the cell. Recent research, focused on different ways to do this, uses cells that have different mutations in this pathway. Gene therapy, currently in phase III trials, aims at decreasing the Bcl-2 function in tumor cells, thus making them more susceptible to apoptosis. This genetic therapy involves injecting cells with small pieces of DNA that bind to the Bcl-2 gene DNA that is present, and that block the gene from being made into a protein by preventing transcription (the DNA‡RNA step; see Figure 10.) Proteins degrade over time and must be replenished, so when formation of new Bcl-2 is blocked, the decreasing level of Bcl-2 in the cell reverses the apoptosis-resistance previously gained by the tumor cell. The previously chemo-resistant cells should then respond to chemotherapy.
Most cancers that are non-responsive to chemotherapy lack p53 function; but it is proving much easier to block gene transcription than to fix a gene that has been mutated [Bullock and Fersht, 2002].
Hope For the Future
A fourth of all deaths in the United States are caused by cancers that did not respond to treatment. In 2003, 1.3 million people will be diagnosed with cancer [Minino and Smith, 2000; Cancer Registries, 2002], and these numbers drive the research to find new cancer therapies. By understanding the details of apoptotic pathways, and identifying the different specific mutations in cancer cells, researchers are taking the first step in designing therapies that will counter the effects of these mutations, whether by finding a drug that blocks the action of a death-inhibitor protein, restoring the natural function of a cell-death protein, decreasing the amount of a death-inhibitor protein produced in a cell, or by triggering apoptosis without employing a damaged cell-death protein. Curing cancer may never be easy, but the direct activation of apoptotic pathways offers hope.
References:
Alberts, et al. 1994. Molecular Biology of the Cell. New York: Garland Publishing. p. 1173-1175.
An Introduction to Clinical Trials. 2003. <http://www.clinicaltrials.gov/ct/gui/info/whatis> Accessed 13 March 2003.
Apoptosis. <http://www.student.livjm.ac.uk/bmsbhosk/apoptosis review.doc> Accessed 29 November 2002.
Bold, Richard; Termuhlen, Paula, and McConkey, David. 1997. Apoptosis, Cancer, and Cancer Therapy. Surgical Oncology. Volume6(3):133-142.
Bullock, Alex, and Fersht, Alan. Rescuing the Function of Mutant p53. Nature Reviews: Cancer. Volume 1: 68-74. <www.nature.com/reviews/cancer> Accessed 29 November 2002.
Cancer Registries: The Foundation for Cancer Prevention and Control. <http://www.cdc.gov/cancer/npcr/register.htm> Accessed 1 December 2002.
Cappello, Paola et al. 2001. Death Receptor Ligands in Tumors. Journal of Immunology. Volume 25 (1):1-15.
Cory, Suzanne, and Adams, Jerry. 2002. The Bcl2 Family: Regulators of the Cellular Life-or-Death Switch. Nature Reviews: Cancer. Volume 2:647-656. <www.nature.com/reviews/cancer> Accessed 29 November 2002.
DePinho, Ronald A. 2000. The Age of Cancer. Nature. Volume 408: 248-253.
Evan, Gerard, and Littlewood, Trevor. 1998. A Matter of Life and Cell Death. Science. Volume 281:1317-1321.
Evan, Gerard, and Vousden, Karen. 2001. Proliferation, Cell Cycle, and Apoptosis in Cancer. Nature. Volume 411:342-347.
Focosi, Daniele. Death Receptor Signaling. <http://digilander.libero.it/danielefocosi/deathmap.html> Accessed 29 November 2002.
Green, Douglas, and Evan, Gerard. 2002. A Matter of Life and Death. Cancer Cell: Volume 1: 19-28.
Hengartner, Michael. 2000. The Biochemistry of Apoptosis. Nature: Volume 407: 770-775.
Igney, Frederk, and Krammer, Peter. Death and Anti-Death: Tumour Resistance to Apoptosis. Nature Reviews: Cancer. Volume 2:277-286. <www.nature.com/reviews/cancer> Accessed 29 November 2001.
Kaufmann, Scott, and Gores, Gregory. 2000. Apoptosis in Cancer: Cause and Cure. BioEssays. Volume 22: 1007-1017.
Minino, Arialdi, and Smith, Betty. 2000. Deaths: Preliminary Data for 2000. National Vital Statistics Report. Vol. 49(12). <http://www.cdc.gov/nchs/data/nvsr/nvsr49/nvsr49_12.pdf> Accessed 2 November 2002.
Packham, Graham. Regulation of Apoptosis in Normal and Malignant Cell. <http://www.som.soton.ac.uk/research/cancersciences/
members/packham/gpackham.htm> Accessed 29 November 2002.
Raff, Martin. 1998. Cell Suicide For Beginners. Nature. Volume 396: 119-122.
Timmer, Tineke; de Vries, Elisabeth; and de Jong, Steven. 2002. Fas Receptor-mediated Apoptosis: A Clinical Application? Journal of Pathology. Volume 196:125-134.
Wu, Min; Ding, Han-Fei; and Fisher, David. 1999. Apoptosis: Molecular Mechanisms. Encyclopedia of Life Sciences. <http://www.els.net> Accessed 23 October 2002.
Vaux, David, and Korsmeyer, Stanley. 1999. Cell Death in Development. Cell. Volume 96:245-254.
Zörnig, Martin; Hueber, Anne-Odile; Baum, Wiebke; and Evan, Gerard. 2001. Apoptosis Regulators and Their Role in Tumorigenesis. Biochimica Et Biophysica Acta. Volume 1551: F1-F37.
