The Discrete Phases of the Cell Cycle: Autoradiographic, Physical, and Chemical Evidences
Claudio Nicolini
1
2,3
The life of a cell cycle includes a series of metabolic
events that are important in the overall process of cell
division. The idealized model of this cycle, i.e., from midpoint of mitosis to midpoint of a successive mitosis, has
been described as a series of metabolic progressions
through four distinct phases (1, 2). These are generally
assumed, at least operationally, to be discrete and identified in the literature as: a) G 1 (pre-DNA-synthesis phase),
during which cells synthesize RNA and proteins; b) S
(DNA-synthesis phase), during which the amount of
DNA is duplicated while RNA and protein syntheses continue; c) G 2 (post-DNA-synthesis phase), characterized by
no net synthesis of DNA and continued RNA and protein synthesis; d) M (mistosis), extending from the beginning of prophase to completion of telophase.
Since mitosis is the only visually distinguishable phase
in the nuclear cycle, until recently most of our knowledge
of the cell cycle was obtained by indirect means such as
the use of a radioactive label combined with high resolution autoradiographic techniques (3-7). The most commonly used precursor is tritium-labeled thymidine (3H_
TDR), a deoxyribonucleoside which is incorporated into
DNA (8). Ordinarily in such studies, cells whose grain
counts exceed the background counting threshold are
considered to have been in a state of active DNA synthesis (S phase) at the time of 3H-TDR exposure, whereas
cells whose grain counts fall below the threshold are considered to have been at a different phase of the cycle,
completely devoid of DNA synthesis.
This all-or-none phenomenon of DNA synthesis has
been recently (9, 10) put in doubt, and a series of percent
labeled mitoses (PLM) curves and related radioautographic data have repeatedly supported the idea of DNA
synthesis occurring continuously during the entire cycle
of a cell, at different rates (9-12). Analogous variations
on PLM curves as functions of counting threshold and
emulsion exposure duration were published in five
papers (9-13) as the main, if not only, experimental evidence for such a drastic conclusion. Usually repetition is
effective for making a point, but in this instance, the
more we reread the description of a continuous cell cycle
model (with a given DNA synthetic rate function), the
more we became suspicious of the equivalence of the
continuous model (11, 12) with the discrete phase model
under steady-state conditions in terms of the kinetics of
a cell population as a whole. However, the implication
of DNA synthesis as a continuum on cell biology could
be profound. The search for events causally related, at
the membrane and nuclear level, to DNA synthesis and
cell proliferation, could become less significant if the
synthesis of DNA should really occur continuously in
a given cell during the entire cycle.
For this reason, in this communication we critically
analyze the autoradiographic data obtained with PLM,
in terms of the technique's limitations and inconveniences that have long been known to investigators. Some
attention will -.lso be paid to the usefulness of a so-called
"radioautogr<'~dic transfer function" (13), which seems
to be an arbitrary mathematical expression of old autoradiographic observations. Recently, the introduction of
laser microfluorometry and microphotometry (14) allows
a faster, more reliable, and quantitative analysis of cell
kinetics, so that most of the limiting and misleading observations of traditional autoradiography are bypassed.
A brief review will also be presented of recent evidences (by physical, chemical, and computer image analysis) of the existence of discrete phases in the cell cycle;
such data rule out the hypothesis of a continuous cell
cycle model with varying DNA synthesis rate function.
AUTORADIOGRAPHY
In autoradiography, the emulsion registers charged
particles emitted by disintegrating radioactive atoms.
When a radioactive atom decays, it emits one or more
kinds of ionizing radiation that produce in photographic
emulsion (i.e., silver halide crystals) an invisible image
(the latent image), which is transformed into a visible
image when the film is developed. To obtain high-resolution autoradiography, the tissue sample containing a
radioactive compound is usually in direct contact, within
a few microns, with the sensitized emulsion. Since the
spectrum of energies of the beta particles emitted isotropically by tritium (the most commonly used tracer)
ranges between 0 and 18 kiloelectron volts (keV), with an
average of 5.5 keY, less than 1% of the radiation reaches
the emulsion if the source is 2 p. away (on the assumption
that the density for the tissue section is :::::= 1.3); this fact
alone would recommend that for any quantitative study
the section should be 1 or 2 p. thick (5).
Background
Even if the normal pathway for the synthesis of the
TMP is methylation of dUMP, the phosphorylation of
Received March 17, 1975; accepted June 9,1975.
Department of Biophysics and Physiology, Committee of Biophysics and Bioengineering, Temple University Health Sciences
Center, Philadelphia, Pa. 19140.
3 I thank Drs. R. Baserga, T. Borun, C. Desaive, and F. Kendall,
whose comments and suggestions were most helpful in the preparation of this manuscript.
1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 55, NO.4, OCTOBER 1975
821
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SUMMARY-Recently the discrete model of the cell cycle, described as a series of metabolic progressions through four
distinct phases, has been challenged by a few curves of percent
labeled mitoses (PLM) and related autoradiographic data, which
have been questionably interpreted in terms of a "continuous"
cell cycle model. This conclusion is based in part on a few
questionable observations which we summarize, along with our
objections and explanations. We present a critical analysis of
the autoradiographic data obtained with the PLM, in terms of the
limitations and inconveniences of the technique itself. Some
attention is paid to the usefulness of the "radioautographic transfer function," which seems an arbitrary mathematical expression of old autoradiographic observations. Finally, a review is
presented of the old (by autoradiography) and new (by physical, chemical, and computer image analysis) evidences of
the existence of discrete phases in the cell cycle, which in turn
support the idea of an all-or-none phenomenon for DNA synthesis, and which rule out the hypothesis of a continuous cell
cycle model with a varying DNA synthesis rate function.-J
Natl Cancer Inst 55: 821-826, 1975.
822
NICOLINI
I.-Background grain counts from mitoses of crypt cells of the
smaU intestine, SD-S5 minutes after Be inoculation of mice with
IH-TDR
TABLE
IH-TDR/~ body
('" i)
0.5
1.0
1.0
wt Emulsion e~08ure Number of grains/mitosis
duration ( ays)
Mean
Range
10
10
28
0.06
0.10
0.34
0-1
0-3
0-6
DNA Synthesis Rate
Investigators have equated (9-13,26) grain counts over
cells labeled with 3H-TDR with rate of DNA synthesis.
To determine the rate of DNA synthesis, one needs to
know the specific activity of the precursor pool, the size
of the endogenous thymidine pool, activity of thymidine
kinase, the route of injection (when an animal is used),
the activity of catabolic enzymes, the competition from
other cell populations, and the activity of other synthetic
pathways. These factors greatly modify the incorporation
of 3H-TDR into DNA, even when the rate of DNA synthesis remains unchanged. Many biologists have difficulty
accepting this concept. The incorporation data do not
give even relative measurements of the rate of synthesis.
Awareness of it will avoid misleading conclusions. The
dTTP pool size can be estimated by the use of a mathematical model that describes the labeling of DNA by
3H-TDR in a culture of exponentially growing L cells
(27). The rate at which the DNA becomes labeled is a
function of both the rate of DNA synthesis and the specific activity of the precursor pool; i.e., during S phase
the rate at which extracellular labeled thymidine is converted into intracellular dTTP competes with the synthesis of cold dTTP from endogenous de novo pathways.
In the most ideal situation, the grain counts, after the
proper background subtraction, measure- only 3H-TDR
incorporation into DNA.
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on May 12, 2016
3H-TDR is efficiently used in mammalian cells. Thus
when cells are exposed to 3H-TDR, they can incorporate
it into DNA, which then becomes radioactive. In addition, thymidine is incorporated in the intact animal
within the first 30-40 minutes (2,5,15,16), or it is rapidly
catabolized to non utilizable products, which (as well as
thymidine itself and its phosphorylated derivatives) are
easily soluble in fixatives. Thus by fixation of the tissue,
most radioactivity should be removed, except that
incorporated in DNA.
However, some factors contribute to the overall background: 1) 3H atoms from broken-down products of injected 3H-TDR; these may find their way into precursors
of proteins or carbohydrates (5). 2) Degradation of thymidine; frequently reports have appeared in the literature on the labeling of amino acids, glycogen, RNA, and
proteins after the administration of 3H-TDR. The lipids
and proteins obtained from rat liver cytoplasmic fraction were heavily labeled when thymidine was given to
animals (17). 3) Reutilization of 3H-TDR from dying
cells (18). 4) Trapping, i.e., coprecipitation, especially
when tissues are not extensively washed before fixation
(5). The blackening of the emulsion can also be due to
causes other than radiation from the experimental source.
5) Chemical and mechanical fogging (19, 20). 6) Cosmic
rays, present in any ordinary environment, which integrate over the time, increasing proportionally with the
exposure.
At least five of these six background contributions are
functions of the time exposure and/or the magnitude of
the 3H-TDR pulse. Therefore, it is surprising that background count distribution shows no increase over a range
of exposure times from 2 to 16 days (9, 10, 13). The compensatory effect due to latent image fading (21) (i.e., the
number of disintegrations required to activate silver
grains increases as exposure time increases) is not sufficient to explain such invariance of background with respect to exposure duration. Table 1 shows how, with the
use of crypt cells of the intestinal tracts of mice, background count does depend both on exposure duration
and amount of 3H-TDR injected. Confusingly enough,
Shackney (13) contradicts his previous results (9, 10) by
admitting a background dependence from time exposure.
Frequently, background corrections are applied by the
arbitrary setting of a threshold grain count below which
a cell is treated as nonlabeled. Evidently this setting is
artificial and may be misleading, but various methods for
background correction have been devised: a) by the use
of a standard from cells not exposed to radioisotopes (22);
b) by plots of the grain count distribution, so that a first
peak of "unlabeled" population is separated by a break
from a broader peak of "labeled" cells; c) when time between exposure of cells to thymidine and fixing is of
short duration (40 min or less), use of the number of
grains per mitosis as a background value; and d) for
mammary tissue, parallel preparations of tissue from animals ovariectomized 3-4 days previously and given injections at the same time of radioactive precursors. Ovariectomy stops DNA synthesis and the remaining grains
are supposed to be background grains (16).
The advantage of these last three methods is that possible nonspecific incorporation of thymidine can be
checked, since background data are obtained from the
autoradiography of cells actually exposed to 3H-TDR.
Shackney et al. (9-12) found no significant increase in
background grain counts with increasing emulsion exposure duration. One possible explanation is that, by the
evaluation of background counts over three areas (of
comparable size) adjacent to the cell, the chemical fogging and aspecific incorporation of thymidine into the
cell were underestimated. Furthermore, the introduction of his so-called radioautographic transfer function
(13) sounds as arbitrary as the setting of an a priori
threshold which he criticizes, with good reason. Only a
qualitative justification was given of both the use of a
cylindrical iluclear model and an upper boundary for the
grain counting window. Even with the assumption that
his model is sound, in view of the many idealized assumptions introduced [that ignore the effect of "variation in
size, shape, orientation of nuclei, partial nuclear sectioning, local variation in thickness, presence and variation
of a tritium-free absorbing layer" (13)], it is difficult to
understand how the cylindrical model can be an improvement over the spherical model (23,24), or how the
radioautographic transfer function is an advancement
with respect to the computer program developed for
automatic background subtraction (25). When the simple
software written for an IBM 1130 "small" computer is
used (25), no idealized assumptions are made and the
true labeling index is estimated even when background
(grains per mitosis after 3H-TDR) and experimental
grain distributions are incompatible.
823
CELL CYCLE DISCRETE PHASES
PLM
The existence of discrete phases of the cell cycle (1, 2, 5)
has been established previously on the basis of the following observations from the PLM curve, found at various
intervals after a singleSH-TDR pulse, of a homogeneous
He La cell population in vitro:
CONTINUOUS MODEL FOR THE CELL CYCLE
Analyzing their PLM curves, some authors (9-13) have
suggested the existence of continuous DNA synthesis during the entire cycle. This conclusion is based on a few
observations which we will summarize along with possible objections or explanations.
1) "Mitosis unlabeled or very lightly labeled at short
emulsion exposure duration appears progressively more
labeled as emulsion exposure duration is increased" (13).
This does not appear to be true for the PLM curve of
sarcoma 180 after 2 days of population growth (9, 13),
when it goes from 2 to 4 days' emulsion exposure: The
cells are consistently and drastically less labeled at longer
exposure, at every threshold. This indicates either a tremendous biologic variability or some improper condition
when autoradiography was performed. Both suggest prudence in trying to force conclusions from such PLM data.
2) The "increase in height and labeling intensity of the
first trough as emulsion exposure duration increases" (13)
(ignoring the 4 days' exposure PLM curves), could be due
to: a) background radioactivity from reutilization of
radioactive thymidine from dying cells (18); chemical
fogging (19, 20); 3H-TDR finding its way into precursors
of proteins, lipids, or carbohydrates (5, 17); or mitochondrial DNA synthesis.
b) Effect of desynchronization due to the stochastic
progression of the cells during the cycle (like many natural phenomena) and to a log-normal distribution of the
residence times. We have performed, with a Monte Carlo
method (28-30), several least chi-square fits of PLM
curves from homogeneous cell populations (crypts of
small intestines of mice, He La cells), and the chi-square
values have consistently high confidence levels, even after
the cells were followed for two or more cycles. 4 The loss
of synchrony can be proved by an analysis of the data
obtained with a population of HeLa cells synchronized
by selective detachment. The standard deviations of both
the labeling index from autoradiography and the thymidine incorporation by chemical assays increase considerably with time after mitosis (31).
2.-Optimized total cell cycle times of epithelial cells from the
small intestine and esophagus of Fels A mice a
Organ
Mean (hr)
Small intestine _____ 14.5 (12.9-16.4)
E6ophagus ________ 44.6 (39.1-50.6)
Percentage of cells in the
peak of time distribution
16.3
9.3
• Optimization was done by a least-squares fit between the experimental labeled
mitosis and the value predicted by a Monte Carlo method, which takes into account
also a G. absorptive phase. Numb.r. in par.nth•••• represent lower and upper
bounds of 1 log-normal SD of the total cell cycle time distributions.
TABLE
3.-Grain count distributions from mitoses of epithelial cells of
the gastrointestinal tract of F els A mice a
Organ
Total cell cycle
time (hr)
Mean (grains/mitosis)
Small intestine ____ _
Large intestine ___ _
~ophagus _______ _
14.5
29.7
44.6
5.8 (3.6-9.0)
5.2 (3.4-8.3)
7.1 (4.9-12.0)
• Six hours after a single sc injection of 'H-TDR (0.5I'Ci/g body wt), the labeling
index of all three cell populations had the same value, corresponding to S phase.
The emulsion exposure duration was 10 days. Numb.r. in parenthe.e. represent
lower and upper bounds of 1 log-normal 8D.
3) The "flattening of the second wave" (13) is consistent with a deterioration of signal/background ratio,
and is due also in part to the dilution of labeled DNA
during successive division.
4) "Cell cycle-related DNA synthesis pattern is suggested by PLM curves at different threshold values" (13)_
First, a general objection can be made about the equation between grain counts and rate of DNA synthesis
(see previous comments). The "spreading and decreasing
of maximal thymidine incorporation with the age of a
given cell" (13) could be explained in terms of an increasing standard deviation of residence time with age.
This increase seems to occur, in effect, if we compare the
optimized total cell cycle time distribution of epithelial
cells from the esophagus with those from the small intestine (table 2). However, we found that the means and
standard deviations of grain count distributions from
mitoses of different cell populations do not change proportionately with the total length of the cycle. They remain invariant, going from the large intestine (29.7 hr)
to the small intestine (14.6 hr), when emulsion exposure
and thymidine pulse remain constant (table 3). The maximal thymidine incorporation in the esophagus seems
even to increase with the age of the cell population
(table 3): exactly the opposite of the expectation of the
model (9-12) for a cycle-age-related DNA synthesis rate.
Furthermore, a cycle-age-related DNA synthesis rate
pattern contradicts earlier reports (32-34), which have
shown that the lengths of the S periods are independent
of the total generation times; i.e., variations in generation times are mainly due to variations in the lengths of
the G 1 periods.
In the literature, several reports of PLM curve analysis,
with homogeneous cell populations, proper technical
handling, background subtraction, and computer analysis, strongly suggest the existence of discrete phases of the
cell cycle. All PLM data obtained by Shackney et al.
(9-13) can be explained within the framework of the stochastic growth of the cells during the cycle, a log-normal
4
Nicolini C, Kendall F: Manuscript in preparation.
Downloaded from http://jnci.oxfordjournals.org/ at Pennsylvania State University on May 12, 2016
1) No mitoses are found labeled up to 2 hours after thymidine
removal; since mitosis lasts approximately half an hour, there
must be a period before mitosis during which cells do not
synthesize DNA (~).
2) No mitoses are labeled immediately after removal of 3H-TDR,
but approximately 30% (HeLa cells) of interphase cells are
labeled. This indicates that DNA was synthesized during
interphase and not mitosis.
3) As the time interval between removal of 3H-TDR and fixation of cells increases, the PLM increases rapidly to 100%.
These are the labeled mitoses in DNA synthesis when 3HTDR was added. Originally, on empirical bases, the two 50%
points on the ascending and descending limbs of the PLM
curve have been used as an indication of the duration of the
phase during which DNA is synthesized (S phase). Analogous
results on a more quantitative basis can be obtained with a
Monte Carlo simulation program (28-30) or various algorithms.
4) After a low point is reached (very rarely zero, due to the
variability of residence times), the PLM increases again to a
second maximum. The interval between two corresponding
points on two successive waves gives the total cell cycle time.
By the difference (with a duration for the mitosis assumed
to be 0.5-1 hour), it is possible to evaluate the length of the
G 1 period.
TABLE
824
NICOLINI
distribution of the residence times, and the pitfalls of
radioautographic techniques. Moreover, any definitive
and quantitative implication about the cell cycle (existence of discrete phases, DNA synthesis rate) based only
on PLM data does not take into consideration all the
recent technical and theoretical advancements. A few
authors (30) have also drawn conclusions on the cell
cycle (i.e., significance of Go phase) only from PLM data
on human tumors, ignoring the heterogeneity of such cell
populations and the abnormality of their growth. These
attitudes sound to me as if the events occurring at the
subnuclear level were being studied with a hammer and
anvil and a Polaroid camera.
PHYSICAL AND CHEMICAL EVIDENCE OF A
DISCRETE CELL CYCLE
Quantitative Cytochemical Analysis
Automated Image Analysis
On the basis of visual observation (light microscopy),
it is difficult to judge if the morphology of the interphase
nucleus changes during the cycle. Objective analysis, by
means of a computer digitizing the image projected from
a microscope onto the face plate of a television scanner
(41,42), allowed the determination of the optical density
(OD) of each picture point, the area (at a selected OD
Circular Dichroism and Dye Binding Studies
Differences in the chromatin of M, GlJ S, and G~ cells
have been reported with respect to the amount of actinomycin D bound per unit DNA (44) and the sensitivity of
chromatin to digestion by DNase (45). These differences
in the "chromosome cycle," which are visualized by light
microscopy only during mitosis, are resolved by a more
sensitive probe during interphase as well. More recently
(31), a more direct proof of the structural alteration
occurring in the chromatin during the He La cell cycle
has been done by circular dichroism sepctra and by spectropolarimetric binding studies of ethidium bromide.
When chromatin was extracted at different intervals after
mitosis from a synchronized population of He La S3 cells,
both the molar ellipticity (at 272 nm) and the primary
binding sites changed abruptly between M and G I , remained constant during the 6- to 7-hour period of G I ,
increased constantly during S phase (corresponding to
the increase of DNA synthesis detected by thymidine incorporation), then decreased during G:2 (31). The synchrony of the HeLa cell is progressively reduced due to
the variation in the length of the G 1 period, as previously
reported also by other authors (35, 38-40). All these conformation studies are compatible with the existence of
a well-defined physical state of the chromatin for every
phase of the cell cycle, during which the DNA can be
characterized in terms of a different degree of supercoil,
i.e., the number of super helices per unit DNA (as suggested by ethidium bromide-binding studies at low concentrations and by the existence of a pre melting in the
chromatin)5. Analogous evidence has recently been accumulated (46) about the conformation of chromatin from
quiescent WI-38 cells (Go). Interestingly enough, the circular dichroism spectra and the number of primary binding sites of the chromatin from WI-38 cells stimulated to
proliferate (G 1 ) are identical to those of a synchronized
HeLa population 1-6 hours after mitosis (G 1).
Biochemical Analysis
Additional independent evidence of the discreteness
of the G C S-G2 transitions is given by the biochemical
analysis of the fraction-l histone phosphorylation and its
correlation with DNA synthesis during the cycle of synchronized populations of HeLa (47) and HTC cells (48).
During the first 6-7 hours after mitosis (G1 ), fraction-l
his tones are dephosphorylated, become heavily phosphorylated as the cells enter S phase, and then reveal
new sites of phosphorylation during (;2 and mitosis (47).
Laser Microfluorometry
The development of flow microfluorometric techniques
(14, 49) for the measurement of DNA content of single
cells at a high rate (>20,000/min) has permitted a statistical precision and sensitivity not obtainable previously.
5
Nicolini C: Manuscript in preparation.
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Many examples exist in the literature of the validity
of the discrete phase model, but one of the most clear,
in my judgment, is given by the combined use of cytochemistry and time-lapse cinematography of individual
cells in vitro (35). It is worthwhile to notice how only the
development of new, rapid-scanning instruments (36, 37)
makes possible this investigation by the analysis of many
cells. Advancements in biology are bound, more than
ever before, to the development of physical techniques,
both old and new.
Mouse fibroblast cells in the logarithmic growth were
cultured in Eagle's minimum essential medium supplemented with 10% calf serum. By the analysis of a series
of photographs, taken at 30-minute intervals for 30 hours
in an incubator room, of the same field of each slide, the
age of an individual fixed cell was the time interval between its preceding division (anaphase) and its fixation
(35). Each individual cell, aged, was then analyzed for
protein, DNA, and RNA content (36) by the combined
use of a rapid scanning interferometer and microphotometer. By plotting the measured cell DNA, RNA, and
dry mass of the individual cells against their experimentally determined age [see figs. 1-3 in (35)], the authors
show that, whereas RNA and protein are synthesized
during the whole interphase, DNA is synthesized at
constant rate only during a discrete part of interphase
(S period). For 8 hours (G 1 ) preceding DNA synthesis,
the amount of DNA is rigorously constant, then increases at a constant slope for 6 hours (S) until it reaches
an amount greater by about a factor of 2, then remains
constant for 5 hours (G2 ) until the next anaphase.
Analogous results were obtained by several other investigators with different cell lines (38-40). These results
show, furthermore, that there was a significant intracellular variation in the length of the G 1 period and that the
dry mass variation among early S cells was significantly
less than the initial mass variation, which suggests that
DNA synthesis is initiated at a critical mass level (additional evidence against the hypothesis of a "continuous"
model).
threshold), and the integrated OD of a given cell component. A recent report (43) has shown that the total
nuclear area, the areas of chromatin at different OD
thresholds, and the mean OD of chromatin significantly
vary between G I , middle S, and G 2 nuclei of WI-38
human fibroblasts stained with the Feulgen method. It
is interesting that morphologic changes of interphase
nuclei during the cell cycle are the reflection of structural
changes in the chromatin.
825
CELL CYCLE DISCRETE PHASES
TABLE 4.-Comparisan of normal and log-normal statistics from a
typical fluorescence histogram produced by a homogeneous populatian
of confluent WI-38 cells dyed with ethidium bromide being fed through
a laser microfluorometer a
Statistics
Mean
Median
(predicted)
Mode
(observed peak)
Correlation
coefficien t
Linear normal_
Log-normal. _ _
44
36
38
34
34
34
0.882
0.965
G
Number8 refer to the channels of the multianalyzer in a given scale expansion.
CONCLUSIONS
The goal of a more quantitative and rigorous study of
the cell cycle, its phases, and its kinetics, cannot be
achieved by the rephrasing, in one's own terms, of various autoradiographic observations that have long been
known to investigators. Such circular logic can only further deepen the pitfalls of autoradiography.
The concept of the discrete phase through which the
cells proceed stochastically with certain log-normal distributions, besides being supported originally by PLM
analyses, is well supported by physical and chemical evidence obtained by the use of the most varied and sophisticated techniques.
The final objective of this review is to stress that only
through the development of new techniques and the
characterization of additional independent physicalchemical parameters of the cell will it be possible to
further elucidate the mechanism of growth during the
cell cycle.
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This has opened a new approach to cell characterization
in general, and to the study of the cell cycle in particular.
Computer-based mathematical techniques have allowed a
further independent identification of G v S, and G,2-M
phase cells and the analysis of the fluorometric spectra of
DNA distribution (50). Ordinarily in these flow cytofluorographs, the optical interaction with a single cell of
a laser beam includes scatter and fluorescence. Our frequency histograms of fluorescence and distribution in
size (light scattered of a homogeneous population of Go
cells (confluent WI-38 human flbroblasts) in suspension
with ethidium bromide are best fitted by a log-normal
distribution. Of many distributions tested, the log-normal
regression provided the best fit to the data, as indicated
by the largest correlation coefficient (>0.96) and the
closest correspondence betwen the median channel predicted by the regression, the log-normal mean, and the
observed value of the peak channel (table 4). This lognormal distribution in size (light scattered) and flourescence of a cell in a given phase of the cycle is compatible
with a log-normal hypothesis of distribution for residence
times (28, 29). A given distribution could also be due to
machine artifact. Furthermore, the variations in the size
of a Go cell is compatible with the large variation in the
duration of G 1 phase (29,30,31,35,43) if the cell has to
reach a critical mass for the initiation of DNA synthesis
as proposed in (35). We believe that it would be premature to claim that these data support the critical mass
hypothesis.
826
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