division Cell is a formidable challenge, and, on occasion, errors occur Non-disjunction occurs when chromatids fail to separate, resulting in trisomy/monosomy If chromosomes become tangled or damaged and then break, this could result in loss (deletion), or rearrangement (inversion, translocation) of genomic loci. The consequence of these events could be deleterious (death, cancer) or neutral, depending on the genes involved.
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THE CELL CYCLE The cell cycle can be divided into four phases
G1 S G2 M = cell growth and preparation for DNA replication = synthesis of DNA (replication) = Growth and preparation for chromosomal segregation =Mitosis (nuclear division and cytokinesis)
Interphase (G1,S, G2) accounts for about 90% of the cell cycle in most cells.
Regulation of progression through the cell cycle insures that growth and DNA replication precede cell division.
FREQUENCY OF DIVISION VARIES WITH CELL TYPE.
Some cells divide frequently throughout life (skin cells) Some cells have the ability to divide, but don't unless they have to (liver cells) Some cells do not divide at all after maturity (nerve, muscle). Some cells stop dividing depending on conditions (THEY EXIT THE CELL CYCLE AND REMAIN IN G0).
Cycle Time for Different Eukaryotic Cells Cell Type Cell Cycle Time Bacteria 15-20 min Early frog embryos 30 min Yeast Cells 1.5-3 hours ~12 hours ~20 hours Intestinal epithelial cells Human liver cells
Mammalian fibroblasts (cultures) ~ 1 year
What controls the cell cycle?
These differences in timing and rate of cell cycle in different cell types imply several models. 1. Each event leads to the next. 2. Each event is driven by specific molecules present in cytoplasm
The following experiment first demonstrated that molecules in the cytoplasm regulate the cell cycle.
EXPERIMENT
M
G1
In each experiment, cultured cells at two different phases of the cell cycle were induced to fuse.
RESULTS
M M When a cell in the M phase was fused with a cell in G1, the G1 cell immediately began mitosis-- a spindle formed and chromatin condensed, even though the chromosomes had not been CONCLUSION duplicated. The results of fusing cells at two different phases of the cell cycle sugges cell cycle.
that molecules present in the cytoplasm control the progression through
G1 nucleus immediatedly entered S phase. If a cell in M phase was fused to any other cell in any other phase, the second nucleus entered mitosis. Conclusion from these and other experiments is that events of the cell cycle are directed by a distinct cell cycle control system, a cyclically operating set of molcules that both triggers and coordinates key events.
Similar types of fusion experiments demonstrated If one cell is in Sthat.... and the other is in G1, the
The distinct events of the cell cycle are regulated by checkpoints.
A checkpoint is the process by which an incomplete upstream event generates a signal that inhibits the initiation of downstream events. A checkpoint in the cell cycle is a critical control point where stop and go signals regulate the cycle. Three major checkpoints are found in the G1, G2, and M phases. Signals registered at checkpoints come from cellular surveillance mechanisms that indicate whether key cellular processes have been completed correctly. Checkpoints register signals from inside and from outside outside the cell.
4
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The Importance of Checkpoints
If the cell proceeds past a checkpoint, the following may occur: Insufficient cell size daughter cells will be too small to support life Incomplete chromosome replication daughter cells will have lost genetic material Incomplete attachment of chromosomes to microtubulesdaughter cells will receive incorrect number of chromosomes.
CANCER CELLS GROW OUT OF CONTROL BECAUSE THEY DON'T RESPOND TO NORMAL SIGNALS THAT REGULATE CELL CYCLE. (MOST CANCER CELLS HAVE LOST THE ABILITY TO ESTABLISH THE G1 CHECKPOINT)
Checkpoints are molecular brakes
The events of the cell cycle must occur in a particular sequence. This sequence must be preserved even if one of the steps takes a little longer than usual.
No Is DNA intact
G0 (Resting state)
For many mammalian cells, the G1 checkpoint is the most important.
If the cells receives a goahead signal, the cell proceeds to replicate its DNA If it does not receive a goahead signal, the cell exits the cycle in G1 and switches to a nondividing resting state, the G0 phase. Most human cells are in this G0 phase. Liver cells can be "called back" to the cell cycle by external cues (growth factors), but highly specialized nerve and muscle cells never divide.
G0
DNA damage Nutrition, cell size Growth factors
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Cell cycle control systems are remarkably similar in all eukaryotes.
Some of the proteins that regulate the cell cycle first appeared in organisms over a billion years ago and have been highly conserved through evolution. The human and yeast proteins are interchangeable!!! This high degree of conservation allowed the cell cycle to be studied in a simple experimental model system, which has resulted in a clear picture of the cell cycle, although there are still many unanswered questions. The significance of understanding cell cycle control mechanisms is crucial for anti-cancer therapeutics and cannot be over emphasized.
Nobel laureates S.cerevisia e Genetics of budding yeast
S. pombe Fission yeast
Xenopus (frog)
Question: What are the molecules that induce progression through the cell cycle?? Answer: cyclically activated protein kinases.
THE REACTION CATALYZED BY A PROTEIN KINASE
Protein kinases catalyze the addition of a phosphate group to a serine, threonine or tyrosine of a target protein.
Target protein Pi
Protein kinase Target protein ATP ADP Pi
Negatively charged phosphate group can change a protein's conformation (3D shape). As a result, the target protein may be activated or deactivated.
The Cell Cycle Clock: Cyclins and CyclinDependent Kinases
Two types of regulatory proteins are involved in cell cycle control: cyclins and cyclindependent kinases (Cdks) The activity of cyclins and Cdks fluctuates during the cell cycle
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Cyclins are proteins that are produced in synchrony with the cell cycle (that's how they got their name). They had no detectable enzymatic activity.
Figure 8.10 The Biology of Cancer ( Garland Science 2007)
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Substrates of cyclindependent kinases affect processes that influence progression through the cell cycle. .
P
regulatory
Cyclin
Cyclin-dependent kinase (Cdk)
catalytic
P
Rhythmic fluctuations in the abundance and activity of certain protein kinases pace the cell cycle.
It was a surprise to learn that these kinases are present in constant amounts throughout the cell cycle! To become activated, these kinases must bind a second protein, a cyclin. It is the amount of cyclins that fluctuate dramatically during the cell cycle. The complex of kinases and cyclin forms cyclin dependent kinases (Cdks).
Cyclin binding activates Cdk
Cyclin is made only at certain times of the cell cycle cyclin Cdk Cdk is always present but its active site is not always exposed
cyclin Cdk
Cyclin binding changes Cdk, exposing its active site
Protein substrate ATP
cyclin Cdk
A protein substrate and ATP bind to Cdk
A DP
cyclin Cdk
P
The phosphorylated protein regulates some of step the
Relative concentration
MPF (maturation promoting factor) was the 1st cyclin:CDK complex characterized (in frog oocytes). Its activity correlates with the concentration of cyclin protein.
M
G1
S
G2
M
G1
S
G2
M
MPF activity Cyclin
Time
Fluctuation of MPF activity (that is, kinase activity) and cyclin concentration during the cell cycle. As cyclin accumulates, it forms complexes with cdk's and activates them.
li Cyc
1
S
G
n acc umula tion
Cdk Degraded cyclin Cyclin is degraded
M
G2
G2 checkpoint MPF
Cdk Cyclin
Molecular mechanisms that help regulate the cell cycle
Fluctuation of MPF activity and cyclin concentration during the cell cycle
Mpf is Cdk-cyclin complex that acts at the G2 checkpoint as the go ahead signal for mitosis to begin. Note that concentration of cdk is constant throughout cell cycle. However the concentration of the cyclin fluctuates because of other regulatory events The net result is that the MPF complex peaks at G2/M phase then drops drastically during M.
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G2 / M checkpoint
Has all DNA been replicated?
Spindle checkpoint M
1 hr
C
Kinetochores attached to splindle? (APC, cohesin)
G2
3-4 hrs
6-8 hrs
6-12 hrs
S
G1
Is genome intact? growth factors, nutrients
G1 / S checkpoint (Start or Restriction Point)
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Replication completed DNA integrity
Active Inactive Cdk / G2 M APC C cyclin (MPF) G2 Spindle checkpoint G2 / M checkpoint G1 / S checkpoint S
Chromosomes attached at metaphase plate Active Inactive
G1
Cdk / G1 cyclin Active Inactive Growth factors Nutritional state of cell Size of cell
Activated MPF has an array of effects that trigger M phase.
Phosphorylates nuclear lamins (> nuclear envelope breakdown). Activated MPF Phosphorylates chromosomal proteins (> chromosome condensation) Phosphorylates microtubule associated proteins (> mitotic spindle activation Phosphorylates an enzyme that degrades cyclin (-> cyclin concentrations
Cyclin CdK
Cyclin dependent kinase
+
Cyclin
Internal and external cues help regulate the cell cycle
While we know that CDKs phosphorylate proteins, the identity of all the CDK target proteins is still under investigation. However, some steps in the signaling pathways that regulate the cell cycle have been well defined.
Some signals originate from inside the cell, others outside.
Example of an internal signal that regulates the cell cycle
A signal to delay anaphase originates at kinetochores that have not yet attached to spindle microtubules. This keeps the anaphase-promoting complex (APC) in an inactive state. When all kinetochores are attached, the APC is activated, triggering breakdown of cyclin and of cohesins that hold together sister chromatids. This checkpoint ensures that daughter cells do not end up with missing or extra chromosomes.
Stop and Go Signs: Internal and External Signals at the Checkpoints
An example of an internal signal is that kinetochores not attached to spindle microtubules send a molecular signal that delays anaphase Some external signals are growth factors, proteins released by certain cells that stimulate other cells to divide For example, platelet-derived growth factor (PDGF) stimulates the division of human fibroblast cells in culture
A variety of external chemical and physical factors can influence cell division. Example 1. Wound healing
Mammalian cells secrete growth factors, proteins released by one group of cells that stimulate other cells to divide. For example, plateletderived growth factor (PDGF), produced by platelet blood cells, binds to tyrosine-kinase receptors of fibroblasts, a type of connective tissue cell. In a living organism, platelets release PDGF in the vicinity of an injury. This triggers a signal-transduction pathway that leads to cell division. The resulting proliferation of fibroblasts helps heal the wound.
PDGF Mode of Action
Fibroblast PDGF platelet
PDGF receptor a tyrosine kinase receptor
Stimulation of cell division
Injury causes secretion of PDGF
The role of PDGF is easily seen in cell culture.
A MODEL FOR THE AFFECT OF GROWTH FACTORS IN PASSING THE G1 CHECKPOINT
Cyclin Cyclin Cyclin Cyclin 1. Arrival of growth factors from other cells. 2. Growth factors cause increase in cyclin concentration.
Cyclin CdK CdK Cyclin ATP ADP
Cyclin CdK Target Pprotein i
3. Cyclin activates 4. Kinases cyclindependent activate S phase kinase. proteins, leading to cell division.
Cancer cells have escaped from cell cycle controls
Cancer cells divide excessively and invade other tissues because they are free of the body's control mechanisms. Cancer cells do not stop dividing when growth factors are depleted either because they manufacture their own, have an abnormality in the signaling pathway, or have a problem in the cell cycle control system. If and when cancer cells stop dividing, they do so at random points, not at the normal checkpoints in the cell cycle.
Another example of external signals is density-dependent inhibition, in which crowded cells stop dividing Most animal cells also exhibit anchorage dependence, in which they must be attached to a substratum in order to divide
LE 12-18a
Cells anchor to dish surface and divide (anchorage dependence).
When cells have formed a complete single layer, they stop dividing (densitydependent inhibition).
If some cells are scraped away, the remaining cells divide to fill the gap and then stop (densitydependent inhibition).
Normal mammalian cells
25 m
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Cancer cells do not exhibit anchorage dependence or densitydependent inhibition.
Cancer cells
25 m
Cancer cells do not respond normally to the body's control mechanisms Cancer cells form tumors, masses of abnormal cells within otherwise normal tissue If abnormal cells remain at the original site, the lump is called a benign tumor Malignant tumors invade surrounding tissues and can metastasize, exporting cancer cells to other parts of the body, where they may form secondary
Loss of Cell Cycle Controls in Cancer Cells
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Tumor
Lymph vessel Blood vessel Cancer cell Metastatic tumor A small percentage of cancer cells may survive and establish a new tumor in another part of the body.
Glandular tissue A tumor grows from a single cancer cell. Cancer cells invade neighboring tissue.
Cancer cells spread through lymph and blood vessels to other parts of the body.
Cancer cell may divide indefinitely if they have a continual supply of nutrients.
In contrast, nearly all mammalian cells divide 20 to 50 times under culture conditions before they stop, age, and die. Cancer cells may be "immortal".
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WHY DO CANCER CELLS GROW OUT OF CONTROL?
SOME POSSIBILITIES
s
Make required growth factors themselves Have abnormal signaling pathways that fail to convey growth factor signals or that are constitutively active Have defects in Cdks or other cell cycle signaling machinery that normally insures DNA replication and mitosis occur only when conditions are favorable.
s
s
Treatments for metastasizing cancers include high-energy radiation and chemotherapy with toxic drugs. These treatments target actively dividing cells. Researchers are beginning to understand how a normal cell is transformed into a cancer cell. The causes are diverse. However, uncontrolled growth almost always involves the alteration of genes that influence the cell cycle control system.
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