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Cell Cycle and Cell Division
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Cell Cycle Control by Oncogenes and Tumor Suppressors: Driving the
Transformation of Normal Cells into Cancerous Cells
By: Am y Y. Chow , Ph.D. (Division of Tumor Cell Biology, Beckman Research Institute, City of Hope) © 2010 Nature Education
Citation: Chow , A. Y. (2010) Cell Cycle Control by Oncogenes and Tum or Suppressors: Driving the
Transform ation of Norm al Cells into Cancerous Cells. Nature Education 3(9):7
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.
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 w ith 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 w ith many infectious diseases, foreign microorganisms w reak havoc on the host they have invaded, causing a loss of function
w ithin cells, tissues or entire organ systems. Cancers, how ever, 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, w here 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 w ith each other in a very specific sequence of events. It is these events that determine w hether the cell cycle w ill go
forw ard or remain stalled betw een stages.
Given that cancer is fundamentally a disorder at the cellular level, the
technological achievement of cultivating cells in vitro, or outside the organism
as a w hole, has allow ed investigators to determine w hat events lead to tumor
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 Cell Cycle and Cell Division
formation and to identify the proteins involved in the process. Typically called
Scientific
cell culture or tissue culture, researchers now regularly grow cells immersed
Com m unication
in nutrient­rich liquid also know n as media (Figure 1). Normal, non­cancerous,
Career Planning
cells w ill grow in a single, uncrow ded 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 w rong
w ith this orderly cell grow th system? And w hat can be learned w hen things
Figure 1: Cells grow ing in a tissue culture petri dish,
go aw ry?
adhered to dish bottom and im m ersed in liquid m edium
The answ ers to these questions serve as the basis for fundamental (pink)
discoveries made by researchers in tumor cell biology. Abnormal or © 2010 Nature Education All rights reserved.
cancerous cells, grow n in vitro have been transform ed 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 forw ard. As a
class, these genes have been named oncogenes. More recently, creative scientists have used advances in methods of genetic manipulation in
combination w ith the transformation assay to identify the genes and proteins important for restricting cell cycle progression. As a class, these genes are
called tumor suppressors.

People Groups Here w e focus on the transformation assay, w hich scientists first used to identify oncogenes and tumor suppressors and study their affect on cell cycle
progression. Even more important w ill be understanding the specific sequence of events in w hich multiple oncogenes and tumor suppressors must act in
combination to promote cancerous cell grow ths (Kinzler 1996; Hahn 2002).
Nature
Education
Early Work in Tumor Biology Led to the Discovery of a Standard Technique: The Transformation Assay
Nick As early as 1911, Peyton Rous demonstrated through his studies of tumorous grow ths in chickens that the potential for tumor generation could be
Morris transferred from animal to animal in cell­free extracts. These extracts w ere eventually show n to contain viruses, w hose ability to promote abnormally
increased cell division in their hosts served to enhance their ow n replication. Thus the same processes stimulated by viral replication could lead to tumor
Ras
production. This field of tumor virology w as instrumental in developing the "cellular transformation assay" still used today to assess tumor grow th.
Kw eku
How do transformed cells grow ? First, they no longer require contact w ith the
Colin
surface of a culture dish. The transformed cells are, instead, capable of
Brow ne
replicating in agar or in suspension (Figure 2). This ability reflects a cancer
Anayansi cell's enhanced mobility, its ability to break dow n substances around it in order
Sierralta to create more space to grow and divide, and a reduction in the contact
inhibition that normally prevents the cells from becoming too crow ded.
Vincent Second, transformed cells w ill grow in more than one layer, producing Figure 2: Transform ation assay for m ouse fibroblasts
Shyu abnormally abundant layers of cells. While untransformed cells grow parallel
in orientation to one another in a single layer, transformed cells w ill pile up in As pictured in (c), transformed cells w ill form a focus (plural: foci),
Deb
chaotic fashion (Figure 1). This feature is reminiscent of cancer cells that or colony, of cells grow ing at a very high density. Here,
Majszak
have reduced contact inhibition of grow th. A third characteristic of transformation occurs in the absence of Kruppel­like factor 4
Jennifer transformed cells is their requirement for few er nutrients in the media. This (KLF4), a transcription factor w ith tumor suppressive activity in
Schisa reflects tumor cells' ability to grow and divide even in the absence of grow th colorectal cancer. Normal, non­transformed cells, do not progress
factors. Finally, transformed cells overcome the restriction of limited rounds of past a four cell stage as depicted in panels (a) and (b).
next replication seen in normal cells and essentially become immortal (Varmus © 2009 Nature Publishing Group Hagos, E. G., A. M. Ghaleb, et
1983). al. Mouse em bryonic fibroblasts null for the Krüppel-like
factor 4 gene are genetically unstable. Oncogene 28, 1197-
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 Blogs factor 4 gene are genetically unstable. Oncogene 28, 1197-
Researchers made use of these results from early virology studies w ith 1205 (2009). All rights reserved.
cellular assays. In these experiments, they infected the cultured cells w ith
Student Voices
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
Creature Cast
oncogenes they identified w ere derived from viruses and called viral oncogenes. Remarkably, researchers soon also realized that the source of these
NatureEdCast viral oncogenes came from cellular counterparts that had been transferred by viruses from one cell to another (Varmus 1983).

Sim ply Science The Accelerator Gets Stuck: Activated Oncogenes Drive Uncontrolled Cell Cycling
What, then, are oncogene products? These are the proteins involved in cell
cycle regulation that operate by stimulating cellular grow th and division (Figure
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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. Likew ise, oncogenes code for proteins
that function to drive the cell cycle forw ard, 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 grow th factors, proteins that interact w ith DNA to initiate
replication, and signaling molecules that link the receptors to the replication
initiators through various pathw ays.

In their normal state, genes that code for the normal proteins controlling these
critical processes are called proto­oncogenes. How ever, once they are
altered (see below ) to become oncogenes, their abnormal protein products
exhibit increased activity that contributes to tumor grow th. Therefore, instead
of stopping w ithin a G phase as it normally should, a tumor cell continues to
progress through subsequent phases of the cell cycle, leading to uncontrolled Figure 3: Cell cycle control by tum or suppressors and
cell division. In addition, oncogenes can also rescue cells from programmed oncogenes
cell death. 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
How does a proto­oncogene become converted to an oncogene? (Figure 4) indicated. Tumor suppressors act to maintain checkpoints (arrow s)
Occasionally, mutations w ill permanently activate proteins that normally w hereas oncogenes allow for checkpoints to be overcome (stop
interchange betw een active or inactive states. For example, Ras proteins lines) (Adapted from Kopnin 2000).
function as molecular sw itches that are turned on and off depending on the
© 2010 Nature Education All rights reserved.
form of nucleotide (di­phosphate or tri­phosphate) to w hich it is bound. In an
"on" state, the products of these proto­oncogenes relay proliferation­
stimulating signals. Problems arise, how ever, w hen mutations convert the proto­oncogene to an oncogene, rendering Ras permanently active regardless
of the signals the cell receives.

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 Figure 4: Schem atic representation of three m ajor types of genetic alterations leading to oncogene activation (bottom )

The proto­oncogene (top) is depicted as a regulatory sequence (RS) follow ed 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).
© 2010 Nature Education All rights reserved.

A second type of genetic alteration that converts a proto­oncogene to an oncogene is a chromosomal translocation. This occurs w hen the pieces of
broken chromosomes reattach haphazardly, leading either to the formation of a fusion protein containing the N­terminus of one protein and the C­terminus
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, w hose protein product consists of the N­terminus of Bcr (breakpoint cluster region) and the C­terminus of Abl, a tyrosine
kinase that relays proliferative signals. The fused chromosome is know n as the Philadelphia chromosome, and it is w idely present in patients w ith chronic
myelogenous leukemia, a blood cell cancer. Formation of the fusion protein renders Abl permanently active, leading to unregulated cell cycling.

Yet another means of generating oncogenes does not change the proto­oncogene directly at all. Instead, the proto­oncogene may exist in multiple copies
in the cell, resulting in amplified expression. This is the case for c­MYC, for w hich 8 to 30 copies are present in each HL60 cell, a promyelocytic leukemia
cell line. As myc is a transcription factor, its increased expression w ill, in turn, lead to the increased expression of its transcriptional targets, many of
w hich function to drive the cell cycle forw ard. 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).

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 The Broken Brake: Defective Tumor Suppressors Fail to Restrict Cell Cycling
How do tumor suppressors differ? In contrast to the cellular proliferation­stimulating function of proto­oncogenes and oncogenes that drive the cell cycle
forw ard, tumor suppressor genes code for proteins that normally operate to restrict cellular grow th 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 best­studied factors of this type is a protein know n as retinoblastoma protein (pRb) and its corresponding gene, RB1, the first tumor
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
allow s 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 w ords, w hen the brakes on a car don't w ork, 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 pathw ay in w hich it functions is defective
due to inactivity of other pathw ay components. By definition, then, these other components w ould 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 proliferation­inhibiting and apoptosis­promoting 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 w ill 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 grow th and division.

Modern Strategies for Understanding the Molecular Basis for Cancer

How do scientists currently use the transformation assay to identify oncogenes and tumor suppressors? These days, manipulating gene expression in
cultured cells is easily done in the lab. This makes the transformation assay extremely useful for dissecting the genetics responsible for cellular change.
Insertion of oncogenes into normal cells in vitro, follow ed by assessment of the transformation criteria, allow s for fairly straightforw ard identification of
the genetic changes leading to tumorigenesis. While researchers use this strategy more commonly for oncogene assessment, they are now also
applying it to tumor suppressor identification w here shRNA expression knocks dow n tumor suppressor protein expression and induces a transformed
cell phenotype (Bric 2009; Sung 2009). Since determining loss­of­function effects is inherently more difficult than evaluating the acquired characteristics
of transformed cells, how ever, identification of tumor suppressor genes has long been more challenging than identification of oncogenes. Nevertheless,
many of the modern strategies described below have been instrumental in making strides tow ards understanding both types of genetic changes.

In contrast to the first oncogenes that w ere identified through investigations in tumor virology, the first tumor suppressors (e.g., retinoblastoma gene­RB)
w ere described follow ing analysis of pedigrees of children afflicted w ith familial retinal tumors. In line w ith the idea that multiple genetic alterations are
often necessary for full­blow n tumor progression, these individuals also exhibited defects in other pathw ays affecting cell grow th and division. And
although it has since been established that having tw o defective copies of RB is the most critical feature that initiates tumor development in many
different types of cancer, the fact that other genetic events must occur highlights the need for further understanding the tumorigenesis process
(Burkhart 2008).

With the fully sequenced human genome and a multitude of strategies for genetic analysis of individuals (e.g., single nucleotide polymorphism analysis,
microarray analysis, linkage analysis, sequencing individual genomes), discovery of new genes influencing tumor initiation continues at a rapid pace.
Delineating the pathw ays affected by these genes, and the role they play in controlling the cell cycle, may prove more challenging (Vogelstein 2004).

The cellular transformation assay described above w as a start, but scientists must also demonstrate that particular genetic alterations are both

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 necessary and sufficient for the spontaneous development and spread of primary tumors w ithin a human body. Transplantation of transformed cells into
"humanized" animal models, such as mice w hose hematopoietic systems have been reconstituted w ith human stem cells, has elucidated genes and
pathw ays involved in the development of w hite blood cell tumors (leukemias and lymphomas) (Yu 2008). Indeed, a w ide variety of mouse models in
w hich inactivating mutations of tumor suppressors leads to spontaneous tumor development have already highlighted pathw ays important for
tumorigenesis (Vogelstein 2004). Just as critical as identifying oncogenes and tumor suppressors, understanding the w ays in w hich they interact to
bring about uncontrolled cell cycle progression continues to challenge scientists seeking not only to treat and prevent cancer but also to understand how
the cell cycle is fundamentally regulated.

Summary
Tw o 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 forw ard, allow ing 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 w ith 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 w ill
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.

References and Recommended Reading

Bric, A., Miething, C. et al. Functional identification of tumor­suppressor genes through an in vivo RNA interference screen in a mouse lymphoma model.
Cancer Cell 16, 324–335 (2009).

Burkhart, D. L. & Sage, J. Cellular mechanisms of tumour suppression by the retinoblastoma gene. Nature Reviews Cancer 8, 671–682 (2008).

Hahn, W. C. & Weinberg, R. A. Rules for making human tumor cells. New England Journal of Medicine 347, 1593–1603.

Kinzler, K. W. & Vogelstein, B. Lessons from hereditary colorectal cancer. Cell 87, 159–170 (1996).

Kopnin, B. P.Targets of oncogenes and tumor suppressors: key for understanding basic mechanisms of carcinogenesis. Biokhimiya 65, 2–27 (2000).

Kufe, D. W., Pollock, R. E., et al. eds. Holland­Frei Cancer Medicine. Hamilton, Ontario: BC Decker, 2003.

Sung, Y.M., Xu, X., et al. Tumor suppressor function of syk in human MCF10A in vitro and normal mouse mammary epithelium in vivo. PLoS ONE 4,
e7445.

Todaro, G. J. & Green, H. High frequency of SV40 transformation of mouse cell line 3T3. Virology 28, 756–759 (1966).

Varmus, H. & Levine, A. J. eds. Readings in Tumor Virology. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1983.

Vogelstein, B. & Kinzler, K. W. Cancer genes and the pathw ays they control. Nature Medicine 10, 789–799 (2004).

Vogelstein, B. & Kinzler, K. W. The Genetic Basis of Human Cancer. 2nd ed. New York, NY: McGraw ­Hill Medical Publishing Division, 2002.

Yu, C. I., Gallegos, M., et al. Broad influenza­specific CD8+ T­cell responses in humanized mice vaccinated w ith influenza virus vaccines. Blood 112,
3671–3678 (2008).

Outline | Keyw ords | Add Content to Group

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 Explore This Topic
BASIC INTERMEDIATE
Eukaryotes and Cell Cycle Aging and Cell Division
CDK Cell Cycle Control by Oncogenes and Tumor
Mitosis Suppressors: Driving the Transformation of Normal Cells
into Cancerous Cells
Cell Differentiation and Tissue
Collaboration of Mitotic Kinases in Cell Cycle Control
Cell Division and Cancer
DNA Replication and Checkpoint Control in S Phase

ADVANCED Germ Cells and Epigenetics

Coordination of Cell Cycle Events by Ran GTPase
Cytokinesis Mechanisms in Yeast
Maternal mRNA and the PolyA Tail in Oocytes
Recovering a Stalled Replication Fork
Replication Fork Stalling and the Fork Protection Complex
p53 : The Most Frequently Altered Gene in Human
Cancers
Regulation of mRNA Splicing by Signal Transduction
Repairing Double­Strand DNA Breaks
The DNA Replication Checkpoint and Preserving Genomic
Integrity During DNA Synthesis
The Domino and Clock Models of Cell Cycle Regulation
The Formation of Heterochromatin and RNA interference
Turning Somatic Cells into Pluripotent Stem Cells

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