Hall Book Ch 4 Flashcards
Mammalian cells propagate and proliferate by mitosis. When a cell divides, two progeny cells are produced, each of which carries a chromosome complement
identical to that of the parent cell. After an interval of time has elapsed, each of the progeny may undergo a further division. The time between successive divisions is known as the ( ) or, as it is commonly called, the ( ).
mitotic cycle time, cell cycle time (Tc)
If a population of dividing cells is observed with a conventional light microscope, the only event in the entire cell cycle that can be identified and distinguished is ( ), or division itself. Just before the cell divides to form two progeny cells, the chromosomes (which are diffuse and scattered in the nucleus in the period between mitoses) condense into clearly distinguishable forms.
In addition, in monolayer cultures of cells just before mitosis, the cells round up and become loosely attached to the surface of the culture vessel. This whole process of mitosis—in preparation for which the cell rounds up, the chromosome material condenses and the cell divides into two and then stretches out again and attaches to the surface of the culture vessel—lasts only about 1 hour. The remainder of the cell cycle, the ( ), occupies all of the intermitotic period. No events of interest can be identified with a conventional microscope during this time.
mitosis, interphase
Because cell division is a cyclic phenomenon repeated in each generation of
the cells, it is usual to represent it as a circle, as shown in Figure 4.1. The
circumference of the circle represents the ( ); the period of mitosis is represented by M.
The remainder of the cell cycle can be further subdivided by using some marker of DNA synthesis. The original technique was ( ), introduced by Howard and Pelc in 1953.
full mitotic cycle time for the cells (TC), autoradiography
The basis of the technique to study cell cycle, illustrated in Figure 4.2, is to feed the cells
( ), a basic building block used for making DNA, which has been labeled
with ( ).
Cells that are actively synthesizing new DNA as part of the process of replicating their chromosome complements incorporate the ( ).
Then, the surplus ( ) is flushed from the system, the cells are fixed and stained so that they may be viewed easily, and the preparation of cells is coated with a very thin layer of nuclear (photographic) emulsion.
thymidine, radioactive tritium (3H-TdR), radioactive thymidine, radioactive thymidine
FIGURE 4.2 Cell-labeling techniques. Top panels: The principle of
( ), which may be applied to cells in culture growing as a
monolayer on a glass microscope slide or to thin sections cut from a tumor or
normal tissue.
Cells in the DNA synthetic phase (S) take up ( ).
After the cells are fixed and stained so that they are visible by light microscopy,
they are covered with a layer of ( ) and left for several weeks in a cool refrigerator.
As β-particles from the tritiated thymidine pass through the emulsion, they form latent images that appear as black grains when the emulsion is subsequently developed and fixed.
If cells are stained and autoradiographed immediately after incorporation of the tritiated thymidine, cells that are labeled are in ( ).
If staining and autoradiography are delayed for 6 to 8 hours after the pulse label, some cells may move from S to M, and labeled mitotic cells are observed (top right panel).
autoradiography, tritiated thymidine, nuclear (photographic) emulsion, S phase (top middle panel)
The lengths of the various phases of the cycle can be determined in this way.
Bottom panels: The principle of cell cycle analysis using ( )
as the DNA precursor instead of radioactively labeled thymidine. The
bromodeoxyuridine is incorporated into cells in ( ) phase.
It can be recognized by the use of a ( ) stain (which is purple) or a monoclonal antibody to ( ).
The antibody is tagged with a fluorescing dye (e.g., fluorescein), which shows up bright green under a fluorescence microscope.
If cells are stained immediately after labeling with bromodeoxyuridine, those staining darkly are in ( ) phase (bottom middle panel).
If staining is delayed for 6 to 8 hours, cells incorporating bromodeoxyuridine
may move from ( ), and a darkly staining mitotic cell is seen (bottom right
panel). (Courtesy of Dr. Charles Geard.)
5-bromodeoxyuridine, S, Giemsa, bromodeoxyuridine-substituted DNA, S, S to M
( ) from cells that have incorporated radioactive thymidine pass through the ( ) and produce a ( ) image.
When the emulsion is subsequently developed and fixed, the area through which a β-particle has passed appears as a ( ). It is then a comparatively simple matter to view the preparation of cells and to observe that some of the cells have black spots or
“grains” over them, which indicates that they were actively synthesizing DNA at
the time radioactive thymidine was made available.
Other cells do not have any grains over their nuclei; this is interpreted to mean that the cells were not actively making DNA when the radioactive label was made available to them.
Examples of labeled cells are shown in Figure 4.3. If the cells are allowed to
grow for some time after labeling with tritiated thymidine so that they move into
mitosis before being fixed, stained, and autoradiographed, then a labeled mitotic
cell may be observed (see Fig. 4.3A).
β-Particles, nuclear emulsion, latent, black spot
FIGURE 4.3 A: Autoradiograph of Chinese hamster cells in culture flash-labeled with tritiated thymidine. The black grains over some cells indicate that
they were synthesizing DNA when they were labeled. Also shown is a labeled
mitotic cell. This cell was in S phase when the culture was flash-labeled but
moved to M phase before it was stained and autoradiographed. B: Color
photomicrograph showing cells labeled and unlabeled with bromodeoxyuridine.
Cells were grown in the presence of bromodeoxyuridine and then fixed and
stained 20 hours later. Incorporated bromodeoxyuridine stains ( ).
The purple-stained interphase cell (upper right) was in S phase during the time the
bromodeoxyuridine was available. Also shown is a first-generation mitotic cell
(upper left), which had been in S phase at the time the bromodeoxyuridine was
available and had moved to M phase by the time it was fixed and stained. It can
be identified as first generation because both chromatids of each chromosome
are stained uniformly.
A second-generation mitotic cell (lower right) passed through two S phases during bromodeoxyuridine availability. One chromatid of each chromosome is ( ) because both strands of the DNA double helix have incorporated bromodeoxyuridine. One chromatid is lighter because only one strand of the DNA has incorporated bromodeoxyuridine. (Courtesy of Dr. Charles Geard.)
purple, darker
The use of tritiated thymidine to identify cells in the DNA synthetic phase
(S) has been replaced largely by the use of ( ), which differs
from thymidine only by the substitution of a bromine atom for a methyl group.
If this halogenated pyrimidine is fed to the cells, it is incorporated into DNA in place of ( ), and its presence can be detected by using an appropriate stain (see Fig. 4.3B).
5-bromodeoxyuridine, thymidine
Cells that have incorporated bromodeoxyuridine appear darkly stained a bright ( ) color. To identify cells that are in ( ) phase and have incorporated bromodeoxyuridine even more readily, one can use a ( ) antibody against (
), which fluoresces brightly under a fluorescence microscope.
purple, S, fluorochrome-tagged, bromodeoxyuridine-substituted DNA
Examples of stained and unstained cells are shown in Figure 4.3B. If time is allowed between labeling with bromodeoxyuridine and staining, then a cell may move from (
) phase, and a stained mitotic cell is observed (see Fig. 4.3B).
If the cell is in the ( ) mitosis after bromodeoxyuridine incorporation, both chromatids of each chromosome are ( ) stained, as shown in the Figure 4.3B (upper left), but by the (
) mitosis, one chromatid is stained darker than the other (lower right in Fig. 4.3B)
S to M, first, equally, second,
By using either of these techniques, it can be shown that cells synthesize
DNA only during a discrete well-defined fraction of the cycle, the S phase.
There is an interval between mitosis and DNA synthesis in which no label is
incorporated. This first “gap” in activity was named ( ) by Howard and Pelc, and
the nomenclature is used today.
After DNA synthesis has been completed, there is a second gap before mitosis, ( ).
All proliferating mammalian cells, whether in culture or growing normally in
a tissue, have a cycle of mitosis (M), followed by G1, S, and G2, after which
mitosis occurs again. The relative lengths of these various constituent parts of
the cell cycle vary according to the particular cells studied.
If cells stop progressing through the cycle (i.e., are arrested), they are said to be in ( ) (Fig. 4.4).
G1, G2, G0
The use of bromodeoxyuridine has two advantages over conventional
autoradiography using tritiated thymidine.
First, it does not involve ( ) material.
Second, it greatly ( ) the time to produce a result because if cells
are coated with emulsion to produce an autoradiograph, they must be stored in a
refrigerator for about a month to allow ( )
to produce a latent image in the emulsion.
radioactive, shortens, β-particles from the incorporated tritium
FIGURE 4.4 Update of the phases of the cell cycle, showing how they are
regulated by the periodic activation of different members of the ( ) family.
Various ( ) complexes are required to phosphorylate several protein substrates, which drive key events, including the initiation of ( ) and the onset of ( ).
cyclin-dependent kinase (Cdk), Cdk–cyclin, DNA replication, mitosis
The characteristics of two cell lines commonly used for in vitro culture are
summarized in Table 4.1. HeLa cells have a total cell cycle time of about ( )
hours, which is more than double that of the Chinese hamster cell, which has a
cell cycle time of about 11 hours.
Mitosis lasts only a relatively short time, about ( ), and is not very different for those two cell lines or for most others. The S phase is 8 hours for HeLa cells and 6 hours for hamster cells; in all cell lines studied in culture or growing in vivo, the S phase never exceeds about ( ) hours.
The ( ) period is very similar in HeLa and hamster cells; in fact, the difference in
the total cell cycle time between these two cell lines is accounted for almost
entirely by the difference in the length of the G1 period.
24, 1 hour, 15, G2
This is an important point: The difference among mammalian cell cycle
times in different circumstances, varying from about ( ) hours for a hamster cell
grown in culture to ( ) of hours for stem cells in some self-renewal tissues,
is the result of a dramatic variation in the length of the ( ) period. The remaining
components of the cell cycle (M, S, and G2) vary comparatively little among
different cells in different circumstances.
10, hundreds, G1
The description of the principal phases of the cell cycle (M, G1, S, and G2)
dates from Howard and Pelc in 1953, as previously discussed. During a complete
cell cycle, the cell must accurately replicate the DNA once during S phase and
distribute an identical set of chromosomes equally to two progeny cells during M
phase. In recent years, we have learned much more about the mechanisms by
which the cycle is regulated in eukaryotic cells.
Regulation occurs by the periodic activation of different members of the ( ) family.
In its active form, each Cdk is complexed with a particular ( ).
cyclin-dependent kinase (Cdk), cyclin
Different Cdk–cyclin complexes are required to phosphorylate several protein
substrates that drive such cell cycle events as the initiation of DNA replication or
the onset of mitosis.
Cdk–cyclin complexes are also vital in preventing the initiation of a cell cycle event at the wrong time.
Extensive regulation of Cdk–cyclin activity by several transcriptional and
posttranscriptional mechanisms ensures perfect timing and coordination of cell
cycle events.
The Cdk catalytic subunit by itself is inactive, requiring association
with a cyclin subunit and ( ) residue to become fully active.
phosphorylation of a key threonine
The Cdk–cyclin complex is reversibly ( ) either by
phosphorylation on a ( ) residue located in the ( ) domain or by association with Cdk inhibitory proteins.
After the completion of the cell cycle transition, the complex is inactivated irreversibly by ( ) subunit.
inactivated, tyrosine, adenosine triphosphate–binding, ubiquitin-mediated degradation of the cyclin
Entry into ( ) phase is controlled by Cdks that are sequentially regulated by cyclins ( ).
( )-type cyclins act as growth factor sensors, with their expression depending more on the extracellular cues than on the cell’s position in the cycle.
S, D, E, and A, D
( ) stimulation governs both their synthesis and complex formation with ( ) and ( ), and catalytic activity of the assembled complexes persists through the cycle as long as mitogenic stimulation continues.
Mitogenic, Cdk4, Cdk6
Cyclin ( ) expression in proliferating cells is normally periodic and maximal at
the ( ) transition, and throughout this interval, it enters into active complexes
with its catalytic partner, ( ). Figure 4.4 illustrates this view of the cell cycle
and its regulation. This is, in essence, an update of Figure 4.1 and is discussed in
more detail in Chapter 18.
E, G1/S, Cdk2
In the discussion of survival curves in Chapter 3, the assumption was implicit
that the population of irradiated cells was ( ); that is, it consisted of
cells distributed throughout ( ) phases of the cell cycle.
Study of the variation of radiosensitivity with the position or age of the cell in the cell cycle was made possible only by the development of techniques to produce ( ) dividing cell cultures—populations of cells in which all of the cells occupy the ( ) phase of the cell cycle at a given time.
asynchronous, all, synchronously, same
There are essentially two techniques that have been used to produce a
synchronously dividing cell population.
The first is the ( ) technique, first described by Terasima and Tolmach. This technique can be used only for cultures that grow in ( ) attached to the surface of the growth vessel.
It exploits the fact that if such cells are close to mitosis, they round up
and become loosely attached to the surface. If at this stage the growth medium
over the cells is subjected to gentle motion (by shaking), the mitotic cells
become detached from the surface and float in the medium.
If this medium is then removed from the culture vessel and plated out into new petri dishes, the population consists almost entirely of ( ) cells.
Incubation of these cell cultures at 37° C then causes the cells to move together ( ) in step through their mitotic cycles.
By delivering a dose of radiation at various times after the initial harvesting of mitotic cells, one can irradiate cells at various phases of the cell cycle.
mitotic harvest, monolayers, mitotic, synchronously
An alternative method for synchronizing cells, which is applicable to cells in
a tissue as well as cells grown in culture, involves the use of a ( ).
Several different substances may be used. One of the most widely applicable drugs is
( ). If this drug is added to a population of dividing cells, it has two
effects on the cell population.
First, all cells that are synthesizing DNA (S phase) take up the drug and are ( ).
Second, the drug imposes a block at the end of the ( ) period; cells that occupy the G2, M, and G1 compartments when the drug is added progress through the cell cycle and accumulate at this block.
drug, hydroxyurea, killed, G1
The dynamics of the action of hydroxyurea are illustrated in Figure 4.5. The
drug is left in position for a period equal to the combined lengths of ( ) for that particular cell line.
By the end of the treatment period, all of the viable cells left in the population are situated in a narrow ( ), poised and ready to enter ( ) phase.
If the drug is then removed from the system, this synchronized cohort of cells proceeds through the cell cycle.
For example, in hamster cells, 5 hours after the removal of the drug, the cohort of synchronized cells occupies a position late in the ( ) phase. Some 9 hours after the removal of the drug, the cohort of cells is at or close to mitosis.
G2, M, and G1
“window” at the end of G1
S
S
FIGURE 4.5 Mode of action of hydroxyurea as an agent to induce ( ).
A: This drug kills cells in ( ) phase and imposes a “block” at the end of the ( )
phase.
B: Cells in ( ) accumulate at this block when the drug is added.
C: If the block is removed, the synchronized cohort of cells moves on through the cycle.
synchrony, S, G1
G2, M, and G1
Techniques involving one or another of a wide range of drugs have been
used to produce ( ) dividing cell populations in culture, in organized
tissues (in a limited number of cases), and even in the whole animal.
Figure 4.6 is a photomicrograph of a squash preparation of the root tip of a ( ) seedling 11 hours after synchrony was induced with ( ). A very large
proportion of the cells are in mitosis.
synchronously, Vicia faba plant, hydroxyurea
