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PHASES&OF&MITOSIS&

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!!

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o Important&step&to&ensure&that&each&new&cell&ends&up&with&the&

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o Replace&dead&cells&and&repair&damaged&tissue&o Growth&

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How Cancer Affects the Cell Cycle Sep&05,&2010 | By Dr. Shavon Jackson-Michel

A cancer can be described as a group of rapidly growing cells that lose the ability

to be controlled by the command center of the cell. The command center of the

cell is the nucleus. DNA, a component of the nucleus, provides the cell with the

directives to grow and divide and also the inhibitory signals to stop growth and

even self-destruct, if things go awry. The cell cycle, according to Emory

University's Cancer Quest, is the natural and orderly progression that cells take

to undergo proper division.

The Cell Cycle

The cell cycle is a series of four steps. The four steps are known as G1, S, G2 and M,

according to a National Cancer Institute transcript. The steps conclude in the creation of a

new cell. The cell cycle stages represent the activity of the nucleus and other cellular

components. The G1 phase prepares the cell for copying, by increasing its growth. The

instruction provided by the nucleus in the S phase, also known as the synthesis phase,

directs the copying of the DNA. Once the DNA is replicated, the Institute notes that G2,

otherwise known as gap 2, leads to further cell growth and reorganization in preparation

for division. The M phase is the last stage of the cell cycle. Also known as mitosis phase,

M represents the stage where the single mother cell divides into two daughter cells.

Division Deterrents!

The relay of information that allows one level to proceed to the next is monitored by a

system of checks and balances, according to a 2007 "Proceedings of the National

Academy of Sciences" article. The checkpoint system ensures that each phase is

thoroughly completed and no errors have occurred, before permitting movement into the

next phase. The article also notes that cancer influences the cell cycle by reducing the

sensitivity of the cell to its innate checkpoint system. Alterations in this system will allow

damaged cells to proceed with growth that would have otherwise been signaled to stop

growing and await better conditions, or simply die. The deterrents to optimal cell cycle

progression are damaged DNA, incomplete replication of DNA during the S-phase, and

insufficient nutrients to support growth.

Growth Without Stimulation!

Only a small percentage of cells are going through the cell cycle at any one time. Emory

University notes that the cells that are replicating are responding to external stimuli.

Cancer cells appear to be able to grow without the presence of external stimuli. Under

normal conditions, cells will grow or regenerate after injury, pre-programmed cell death

or enticing environmental conditions. The usual chemical stimulants are hormones,

proteins or other specialized growth factors. The stimulating substances bind to the

receptors on the cell and signal the nucleus to turn on the genes that start the cell cycle.

Cancer cells are uniquely able to signal cell cycle progression without the binding of

external stimuli. Emory University likens this action to driving a car without first

pressing the gas pedal.

Ignore Stop Signals!

The cell cycle utilizes the checkpoint system to immediately halt the process of cell

division if a problem is encountered. The system has inborn stop signals, according to the

Emory University Cancer Quest Center. A primary deterrent to cell division is damaged

DNA. A normal cell will not continue to advance through the G1, M and G2 phases if its

DNA became damaged. Prevention of this action will stop stop the damaged DNA from

being passed down to the next generation of cells. A cancer cell, however, can ignore the

stop signal, complete cell division and continue to proliferate and propagate the faulty

DNA further along. Emory describes this faulty DNA as mutations and notes that

mutated genes cause the cells to act inappropriately and encourage increased tumor

growth.

Evades Warnings!

Neighboring cells also help dividing cells monitor their need. The neighboring cells

maintain a supply-demand relationship between the cells undergoing replication. The

National Cancer Institute notes that chemicals are sent between neighbors to help keep

the level of new cell growth measure in close proximity to the rate of cell death. Emory

notes that crowding of cells, trigger warning signals that should slow down or stop the

rate of growth in an area that is becoming overpopulated. Cancer cells also evade these

warnings. They continue to grow within a limited space and create a pile-up of cells,

often described as a tumor or cancer mass.

Cell Cycle in Cancer The cell cycle, the process by which cells progress and divide, lies at the heart of cancer. In normal cells, the cell cycle is controlled by a complex series of signaling pathways by which a cell grows, replicates its DNA and divides. This process also includes mechanisms to ensure errors are corrected, and if not, the cells commit suicide (apoptosis). In cancer, as a result of genetic mutations, this regulatory process malfunctions, resulting in uncontrolled cell proliferation.

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The cell cycle involves a complex series of molecular and biochemical signaling pathways. As illustrated in the diagram above the cell cycle has four phases:

the G1, or gap, phase, in which the cell grows and prepares to synthesize DNA; the S, or synthesis, phase, in which the cell synthesizes DNA; the G2, or second gap, phase, in which the cell prepares to divide; and the M, or mitosis, phase, in which cell division occurs.

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As a cell approaches the end of the G1 phase it is controlled at a vital checkpoint, called G1/S, where the cell determines whether or not to replicate its DNA. At this checkpoint the cell is checked for DNA damage to ensure that it has all the necessary cellular machinery to allow for successful cell division. As a result of this check, which involves the interactions of various proteins, a "molecular switch" is toggled on or off. Cells with intact DNA continue to S phase; cells with damaged DNA that cannot be repaired are arrested and "commit suicide" through apoptosis, or programmed cell death. A second such checkpoint occurs at the G2 phase following the synthesis of DNA in S phase but before cell division in M phase. Cells use a complex set of enzymes called kinases to control various steps in the cell cycle. Cyclin Dependent Kinases, or CDKs, are a specific enzyme family that use signals to switch on cell cycle mechanisms. CDKs themselves are activated by forming complexes with cyclins, another group of regulatory proteins only present for short periods in the cell cycle. When functioning properly, cell cycle regulatory proteins act as the body's own tumor suppressors by controlling cell growth and inducing the death of damaged cells. Genetic mutations causing the malfunction or absence of one or more of the regulatory proteins at cell cycle checkpoints can result in the "molecular switch" being turned permanently on, permitting uncontrolled multiplication of the cell, leading to carcinogenesis, or tumor development.

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Cancerous Cells: How is a normal cell transformed into a cancerous cell? The proteins involved in cell division events no longer appropriately drive

progression from one cell cycle stage to the next.

By: Amy Y. Chow, Ph.D. (Division of Tumor Cell Biology, Beckman Research Institute, City of Hope) © 2010 Nature Education

How is a normal cell transformed into a cancerous cell? The proteins involved in regulating cell division events no longer appropriately drive progression from one cell cycle stage to the next. Rather than lacking function, cancer cells reproduce at a rate far beyond the normally tightly regulated boundaries of the cell cycle. Cancer can be distinguished from many other human diseases because its root cause is not a lack of, or reduction in, cell function. For example, individuals with diabetes may lack insulin production or the ability to respond to insulin. With coronary heart disease, poor blood supply to the heart can cause the organ to eventually fail. In the case of acquired immune deficiency syndrome (AIDS), the immune system loses the cells it needs to fend off infection. And with many infectious diseases, foreign microorganisms wreak havoc on the host they have invaded, causing a loss of function within cells, tissues or entire organ systems. Cancers, however, occur due to an alteration of a normal biological process — cell division. Cells that progress through the cell cycle unchecked may eventually form malignant tumors, where masses of cells grow and divide uncontrollably, then develop the ability to spread and migrate throughout the body. Fortunately, cancer prevention usually occurs through the strict regulation of the cell cycle by groups of proteins that interact with each other in a very specific sequence of events. It is these events that determine whether the cell cycle will go forward or remain stalled between stages.

Figure 1: Cells growing in a tissue culture petri dish, adhered to dish bottom and immersed in liquid medium (pink) Given that cancer is fundamentally a disorder at the cellular level, the technological achievement of cultivating cells in vitro, or outside theorganism as a whole, has allowed investigators to determine what events lead to tumor formation and to identify the proteins involved in the process. Typically called cell culture or tissue culture, researchers now regularly grow cells immersed in nutrient-rich liquid also known as media (Figure 1). Normal, non-cancerous, cells will grow in a single, uncrowded layer attached to plastic dishes. These cells generally undergo a limited number division cycles, depending on space and nutrient availability, among other constraints. What, then, can go wrong with this orderly cell growth system? And what can be learned when things go awry? The answers to these questions serve as the basis for fundamental discoveries made by researchers in tumor cell biology. Abnormal or cancerous cells, grown in vitro have been transformed from their normal phenotype due to genetic changes affecting proteins involved in cell cycle control. Historically,

these transformation assays have led to the identification of the genes and proteins important for driving the cell cycle forward. As a class, these genes have been named oncogenes. More recently, creative scientists have used advances in methods of genetic manipulation in combination with the transformation assay to identify the genes and proteins important for restricting cell cycle progression. As a class, these genes are called tumor suppressors. Here we focus on the transformation assay, which scientists first used to identify oncogenes and tumor suppressors and study their affect on cell cycle progression. Even more important will be understanding the specific sequence of events in which multiple oncogenes and tumor suppressors must act in combination to promote cancerous cell growths (Kinzler 1996; Hahn 2002).

Early Work in Tumor Biology Led to the Discovery of a Standard Technique: The Transformation Assay &As early as 1911, Peyton Rous demonstrated through his studies of tumorous growths in chickens that the potential for tumor generation could be transferred from animal to animal in cell-free extracts. These extracts were eventually shown to contain viruses, whose ability to promote abnormally increased cell division in their hosts served to enhance their own replication. Thus the same processes stimulated by viral replication could lead to tumor production. This field of tumor virology was instrumental in developing the "cellular transformation assay" still used today to assess tumor growth.

Figure 2: Transformation assay for mouse fibroblasts As pictured in (c), transformed cells will form a focus (plural: foci), or colony, of cells growing at a very high density. Here, transformation occurs in the absence of Kruppel-like factor 4 (KLF4), a transcription factor with tumor suppressive activity in colorectal cancer. Normal, non-transformed cells, do not progress past a four cell stage as depicted in panels (a) and (b). How do transformed cells grow? First, they no longer require contact with the surface of a culture dish. The transformed cells are, instead, capable of replicating in agar or in suspension (Figure 2). This ability reflects a cancer cell's enhanced mobility, its ability to break down substances around it in order to create more space to grow and divide, and a reduction in the contact inhibition that normally prevents the cells from becoming too crowded. Second, transformed cells will grow in more than one layer, producing abnormally abundant layers of cells. While untransformed cells grow parallel in orientation to one another in a single layer, transformed cells will pile up in chaotic fashion (Figure 1). This feature is reminiscent of cancer cells that have reduced contact inhibition of growth. A third characteristic of transformed cells is their requirement for fewer nutrients in the media. This reflects tumor cells' ability to grow and divide even in the absence of growth factors. Finally, transformed cells overcome the restriction of limited rounds of replication seen in normal cells and essentially become immortal (Varmus 1983). Researchers made use of these results from early virology studies with cellular assays. In these experiments, they infected the cultured cells with various viruses and then looked for "transformations" to occur (Todaro 1966). Since viral genomes are relatively small, researchers could more easily determine the genetic components responsible for transformation. In fact, the first oncogenes they identified were derived from viruses and called viral oncogenes. Remarkably, researchers soon also

realized that the source of these viral oncogenes came from cellular counterparts that had been transferred by viruses from one cell to another (Varmus 1983).

The Accelerator Gets Stuck: Activated Oncogenes Drive Uncontrolled Cell Cycling

Figure 3: Cell cycle control by tumor suppressors and oncogenes Checkpoints are depicted as thick red bars. The stages of the cell cycle (G1: Gap 1, S: DNA synthesis, G2: Gap 2, and M: mitosis) are indicated. Tumor suppressors act to maintain checkpoints (arrows) whereas oncogenes allow for checkpoints to be overcome (stop lines) (Adapted from Kopnin 2000). What, then, are oncogene products? These are the proteins involved in cell cycle regulation that operate by stimulating cellular growth and division (Figure 3). A common analogy equates oncogenes to an automobile's gas pedal stuck in the acceleration mode. Though the driver does not have his foot on the pedal, the car continues to speed up. Likewise, oncogenes code for proteins that function to drive the cell cycle forward, typically causing cells to proceed from one of the G (gap) phases to either chromosome replication (S phase) or chromosome segregation (mitosis). Examples include receptors at the cell surface that bind to growth factors, proteins that interact withDNA to initiate replication, and signaling molecules that link the receptors to the replication initiators through various pathways. In their normal state, genes that code for the normal proteins controlling these critical processes are called proto-oncogenes. However, once they are altered (see below) to become oncogenes, their abnormal protein products exhibit increased activity that contributes to tumor growth. Therefore, instead of stopping within a G phase as it normally should, a tumor cell continues to progress through subsequent phases of the cell cycle, leading to uncontrolled cell division. In addition, oncogenes can also rescue cells from programmed cell death.

How does a proto-oncogene become converted to an oncogene? (Figure 4) Occasionally, mutations will permanently activate proteins that normally interchange between active or inactive states. For example, Ras proteins function as molecular switches that are turned on and off depending on the form of nucleotide (di-phosphate or tri-phosphate) to which it is bound. In an "on" state, the products of

these proto-oncogenes relay proliferation-stimulating signals. Problems arise, however, when mutations convert the proto-oncogene to an oncogene, rendering Ras permanently active regardless of the signals the cell receives.

Figure 4: Schematic representation of three major types of genetic alterations leading to oncogene activation (bottom) The proto-oncogene (top) is depicted as a regulatory sequence (RS) followed by the coding region (gene). In the first example, a star indicates the location of the nucleotide substitution on the transcribed portion of the gene. In the case of the translocation example, a different regulatory sequence becomes responsible for stimulating transcription of a resultant fusion protein. For the amplification example, the presence of multiple copies of the gene results in excessive expression (Adapted Kufe et al. 2003). A second type of genetic alteration that converts a proto-oncogene to an oncogene is a chromosomal translocation. This occurs when the pieces of broken chromosomes reattach haphazardly, leading either to the formation of a fusion protein containing the N-terminus of one protein and the C-terminus of another, or leading to altered regulation of protein expression (Figure 4). One example of an oncogene generated by this type of chromosomal translocation is BCR/ABL, whose protein product consists of the N-terminus of Bcr (breakpoint cluster region) and the C-terminus of Abl, a tyrosine kinase that relays proliferative signals. The fused chromosome is known as the Philadelphia chromosome, and it is widely present in patients with chronic myelogenous leukemia, a blood cell cancer. Formation of thefusion protein renders Abl permanently active, leading to unregulated cell cycling. Yet another means of generating oncogenes does not change the proto-oncogene directly at all. Instead, the proto-oncogene may exist in multiple copies in the cell, resulting in amplified expression. This is the case for c-MYC, for which 8 to 30 copies are present in each HL60 cell, a promyelocytic

leukemia cell line. As myc is a transcription factor, its increased expression will, in turn, lead to the increased expression of its transcriptional targets, many of which function to drive the cell cycle forward. Since all of these genetic alterations result in a gain of function, only one affected chromosome is needed to induce the transformed cell phenotype (Vogelstein 2002). The Broken Brake: Defective Tumor Suppressors Fail to Restrict Cell Cycling &How do tumor suppressors differ? In contrast to the cellular proliferation-stimulating function of proto-oncogenes and oncogenes that drive the cell cycle forward, tumor suppressor genes code for proteins that normally operate to restrict cellular growth and division or even promote programmed cell death (apoptosis). To continue the automobile analogy, tumor suppressors are like the brakes on a car. Examples include inhibitors of cell cycle progression, factors involved in maintenance of cell cycle checkpoints, and proteins required for apoptosis induction. One of the best-studied factors of this type is a protein known as retinoblastoma protein (pRb) and its corresponding gene, RB1, the firsttumor suppressor gene to be identified. Since pRb activity stops the expression of genes required for progression into S phase of the cell cycle, its inactivation allows for uncontrolled cell division (Figure 3). In fact, this principle applies to all tumor suppressors: genetic alterations in the gene leading to tumorigenesis prevent the regulatory protein from inhibiting cell proliferation. In other words, when the brakes on a car don't work, the car cannot stop. The types of genetic alterations leading to pRb inactivity most often involve frameshifts or deletions in the RB1 gene causing premature introduction of a stop codon and defective protein expression. In some instances, expression of pRb may be normal, but the pathway in which it functions is defective due to inactivity of other pathway components. By definition, then, these other components would also be considered tumor suppressors (Burkhart, 2008). Yet another example of a tumor suppressor, and the most commonly mutated gene in human tumors, is the p53 gene (Vogelstein, 2004). As a transcription factor that activates expression of proliferation-inhibiting and apoptosis-promoting proteins in response to DNA damage, p53 plays a critical role in maintaining the G1 to S cell cycle checkpoint (Figure 3). Genetic alterations that inactivate p53 will inhibit the DNA damage response that prevents cell cycle progression. When this occurs, a cell continues to divide even in the presence of DNA damage. Since inactivation of tumor suppressors results in a loss of function, both maternal and paternal copies of a gene coding for a tumor suppressor must usually be altered for tumorigenesis to occur — one good copy of the gene may provide sufficient activity for the cell to maintain proper growth and division. Summary &Two classes of genes, oncogenes and tumor suppressor genes, link cell cycle control to tumor formation and development. Oncogenes in their proto-oncogene state drive the cell cycle forward, allowing cells to proceed from one cell cycle stage to the next. This highly regulated process becomes dysregulated due to activating genetic alterations that lead to cellular transformation. Tumor suppressor genes, on the other hand, restrict cell cycle progression. Their control over cell division is lost with genetic alterations leading to their inactivation. The role that both types of genes play in tumor formation can be experimentally determined using in vitro transformation assays or more complex in vivo animal models. These sorts of experiments will lead to a more thorough understanding of the genetic basis for cancer, more effective therapeutics, and a deeper appreciation of the intricacies of cell cycle regulation. &

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