Journal of Gertmtaloiiv
1979 Vol. 34, No. 3, 323-327
DNA Synthesis in the Human Diploid
Cell Strain WI-38 During in Vitro
Aging: An Autoradiography Study1
The percent of cells in a WI-38 cell population which did not incorporate tritiated thymidine (3H-TdR)
was determined by autoradiography from the measurement of percent nonlabeled nuclei and the ratio
of total cell numbers at the initiation and at the termination of exposure to 3H-TdR. This percentage was
minimally affected by factors influencing the proliferation of labeled cells, but was dependent on the
population doubling level- (PDL). The results suggest the presence, in a proliferating WI-38 population,
of subpopulation(s) with an extremely slow rate of S phase entrance. The parameter was useful in estimating, empirically, the doubling potential of a cell population.
HE limited proliferative capacity of human
T
diploid cells (HDC) has been interpreted
to be aging at the cell level (Hayflick, 1965a;
Hayflick, 1977; Hayflick & Moorhead, 1961).
Studies of mass populations of HDC as they
age in vitro reveal, among other decrements,
a progressive decline in proliferation rate,
cell density, and in average cell size (Hayflick,
1965a; Macieira-Coelho etal., 1966; Schneider
& Fowlkes, 1976). It has been observed that:
(1) cells plated at low density contain nondividing cells, the proportion of which increases with the population doubling level
(PDL) of a culture (Absher et al., 1974; Martin
et al., 1974; Merz & Ross, 1969; Smith &
Hayflick, 1974); (2) cloned cells varied in
their doubling potential (Martin et al., 1974;
Smith & Hayflick, 1974; Smith et al., 1977);
(3) the percent of labeled cells (labeling index) in an autoradiography study reached a
plateau after a 24 to 30-hour exposure to
tritiated thymidine (3H-TdR); and (4) the
labeling index decreased as a function of PDL
(Cristofalo & Sharf, 1973). These observations
show that individual cells in a HDC population
vary in proliferation rate and metabolic activity. The labeling index has been used as
an empirical parameter to estimate the doubling potential of a cell population (Cristofalo
& Sharf, 1973; Nichols et al., 1977; Vincent
et al., 1976). However, because this index is
a complex function of PDL, 3H-TdR concentration, time of exposure to 3H-TdR, and
the rate of cell proliferation during exposure
(Cristofalo, 1976; Good, 1974; MacieiraCoelho, 1974), it is difficult to investigate
quantitatively the heterogeneity of a HDC
population by use of this index.
It is the purpose of this study to develop
a method of autoradiographic analysis for the
determination of that proportion of cells which
do not enter S phase during exposure to 3HTdR in a HDC population. Evaluation of this
method for the measurement of doubling
potential as well as for the analyses of population heterogeneity is described.
MATERIALS AND METHODS
'This study was carried out at Stanford Univ. School of Medicine and
was supported, in pan. by NIH Grants CA-184S6 and AG-00428, and by
the Ministry of Education, Science and Culture, Japan. The authors wish
to acknowledge the assistance of Mr. Stephen Swan berg and Miss Noriko
Munezawa, and discussions with Drs. James R. Smith, and Gerald Masover.
2
Dept. of Cancer Cell Research, Inst. of Medical Science, Univ. of
Tokyo, Shirokanedai, Minato-ku, Tokyo 108.
}
Dept. of Dermatology, Stanford Univ. School of Medicine, Stanford,
CA 94305.
'Children's Hospital Medical Ctr. of Northern California, Bruce Lyon
Memorial Research Laboratory, 51st and Grove Streets, Oakland, CA 94609.
Monolayer cell cultures of the human diploid
cell strain WI-38 were serially passaged in
T-75 flasks as previously described (Hayflick,
1965a; Hayflick & Moorhead, 1961). The culture medium used (30 ml/T-75 flask) was
Eagle's Basal Medium (90% v/v of the culture
medium) (Flow Laboratories, Rockville, MD),
323
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Toshiharu Matsumura, PhD, 2 Eva A. Pfendt, MA, 3
and Leonard Hayflick, PhD 4
324
MATSUMURA, PFENDT AND HAYFLICK
N(t)
2 Rn(t)
10-* 1
2
3
INCUBATION TIME
Fig. 1 (A & B). Diagrammatic representation of the
derivation (B) of a nonlabeled cell ratio corrected for
cell proliferation —^-L
— from the three measurable
parameters (A) [i.e., cell density on incubation time 0
(No), cell density on incubation time t (N(t)), and the
labeling index on day t (1-Y(t)/N(t))]. Incubation time
is given in an arbitrary unit. The nonlabeled cell ratio
(Rn(t)) is equal to Y(t)/N(t). Open circles diagrammatically indicate those cells which do not incorporate 3HTdR and are not labeled. Solid circles are those cells
which have incorporated 3H-TdR and are labeled. Some
of these cells may proliferate by repeating cell division.
X indicates a hypothetical population of cells which
possess no capacity to incorporate 3H-TdR during the
incubation period and are presumed to be present in the
total population of No when the incorporation experiment
was initiated. It should be noted that the corrected ratio
given does not represent the fraction of nonlabeled cells
to total cells at the time of harvest, but to total cells at
the initiation of exposure.
cent nonlabeled nuclei corrected for cell
proliferation is described in the legend for
Fig. 1. Cell density on the day of initiation
of exposure was determined by averaging
cell numbers of duplicate windows in a replicate culture. This determination caused some
errors common in any replicate culture experiment, which we presume to be minor in evaluating the corrected values.
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28 mM 4-(2-hydroxymethyl)-l-piperazineethane-sulfonic acid (HEPES, Calbiochem., La
Jolla, CA), and 50 /Ag/ml aureomycin (Lederle
Lab., Pearl River, NY), and fetal bovine serum
(10% v/v of the culture medium) obtained
from either Microbiological Associates, Walkersville, MD, or International Scientific Ind.,
Cary, IL). The PDL of a culture was determined as the cumulative sum of the Iog2 (split
ratio). Cultures were routinely monitored for
mycoplasma contamination and found to be
negative (Hayflick, 1965b).
For tritiated thymidine (3H-TdR) incorporation and autoradiography, the cells were
trypsinized, and a 0.4 ml aliquot of suspension
of known cell density was inoculated into
each of eight chambers of a Lab-Tek chamber
slide (Lab-Tek Products #4808, Naperville,
IL). Each chamber has a 1 cm2 surface area
on the slide glass, which will be referred to as
a window. To start an experiment, three to
seven chamber slides were prepared. The
method of 3H-TdR incorporation and preparation of an autogram was modified from that
previously described (Cristofalo & Sharf,
1973). Briefly, after 24 hours of incubation (on
the day of initiation of exposure), a chamber
slide was removed from the incubator, rinsed,
fixed in methanol, dried and stained with
Giemsa solution. The rest of the chamber
slides received 3H-TdR (New England Nuclear, Boston, MA, 2 Ci/mM and 24 Ci/mM) at
a final concentration of 0.05 /uCi/ml. Three
days after supplementation with 3H-TdR, the
medium was renewed with 3H-TdR. During a
period of six days with 3H-TdR, chamber slides
were removed from the incubator, rinsed, fixed
in Carnoy's solution, dehydrated, dried,
coated with emulsion (Kodak NTB-2), kept in
a cold dark room, and then developed, fixed
and stained with Harris hematoxylin, and
mounted. An autogram was analyzed microscopically by the use of a square frame inserted in the eyepiece. The frame indicated
a field of 0.1276 mm2 on an autogram. By
increasing the number of fields which were
chosen on a random basis, at least 400 cells,
with or without labeled nuclei (five silver
grains or more), were counted unless otherwise described. From such counts, the cell
density (N/cm2 = the density of cells with
labeled and nonlabeled nuclei), and the percent nonlabeled nuclei, were derived for
each of the windows. Determination of per-
DNA SYNTHESIS OF HUMAN FIBROBLASTS
RESULTS
new cells entering DNA synthesis, but also
to the second or greater round of DNA synthesis during the exposure. Inasmuch as 0.05
/xCi/ml of 3H-TdR did not entirely stop cell
proliferation, a dilution of nonlabeled cells
by an increasing number of labeled cells may
have occurred. Thus, the labeling index was
not expected to remain constant even after
24 to 48 hours of exposure to 3H-TdR. In the
next experiment, the cells were incubated with
0.05 /uCi/ml 3H-TdR (2 Ci/mM), and the
dark exposure was for eight days.
The parameter, nonlabeled cell ratio corrected for cell proliferation, was introduced
as described in Fig. 1 to determine the fraction
of nonlabeled cells without the interference
of the isotope effect. This parameter was the
product of the nonlabeled cell ratio, and the
ratio of total cell number at the initiation and
that at the termination of exposure to 3H-TdR.
As shown diagrammatic ally in Fig. 1, this
parameter is closely related to the fraction
in the initial cell population of cells that remain without incorporation of 3H-TdR during
exposure to 3H-TdR. In a hypothetical case
where the isotope does not affect the survival
rate of nonincorporating cells this parameter
is, by definition, independent of the rate of
proliferation and survival of those cells which
have incorporated 3H-TdR. In the following
experiments, percent nonlabeled nuclei were
used to approximate the percent nonlabeled
cells, because there are few multinucleated
cells in a proliferating population of WI-38.
The percent of nonlabeled nuclei, and that
corrected for cell proliferation, for a culture
at PDL 32 are shown as a function of incubation time in Fig. 2. The percent decreased over
four days and then no longer decreased. On
the other hand, the corrected percent decreased during the first 24 hours of incubation,
then the rapid decrease stopped until the end of
the six-day incubation. The corrected percent was not significantly influenced by the
cell inoculum. It was observed, in repeated
experiments, that the corrected percent after
one day may not remain at a plateau, but may
decrease very slowly (Matsumura et al., 1979).
The corrected percent was determined after
exposure to 3H-TdR for six days for cultures
at PDL ranging from 18 to 55. The parameter
was determined for cells inoculated at several
varying densities. For relatively early passage cultures, inoculation densities were low,
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In a preliminary experiment cultures at
PDLs 22, 26, 40, 52, and 58 were used to investigate the effect of TdR concentration on
the labeling index. The cells were exposed
for 48 hours to 3H-TdR (2 Ci/mM and 24
Ci/mM) at final concentrations of, respectively, 5 x 10"8 and 4 x 10"9 M, which gave a
concentration of 0.1 /xCi/ml, in the incubation medium. They were exposed for four days
in the dark. No significant difference was
found at any PDL tested between the two
groups of labeling indices, respectively, for
the two different 3H-TdR concentration (data
not presented). In all subsequent experiments,
2 Ci/mM 3H-TdR was added to the medium at
a final concentration of 0.05 to 0.1 /u,Ci/ml,
or 2.5 to 5 x 10-8 M.
The isotope effect of 3H-TdR, and the time
of exposure in the dark are variables thought
to influence the labeling index. Using a culture
at PDL 32 we compared several combinations
of those conditions. When the cells were exposed to 3H-TdR for 24 hours neither reduction of 3H-TdR concentration from 0.1 /tCi/ml
to 0.05 /uCi/ml nor the time of exposure in the
dark (from four days to eight days) had a significant effect on the labeling index (data not
presented). These results confirm those previously presented (Cristofalo, 1976; Cristofalo & Sharf, 1973) in that the labeling index
remains constant with 3H-TdR concentration
and the time of dark exposure respectively
within adequate ranges. When the cells were
exposed to 3H-TdR for longer than 24 hours,
however, the isotope effect became evident
as described below. We started the incubation of a culture at PDL 40 in the absence of
3
H-TdR with 0.65 and 1.3 x 104 cells/cm2 as
the inoculum density. An approximately logarithmic proliferation continued for five and
four days respectively, and yielded the cell
densities of 2.0 and 1.8 x lOVcm2 on day
5 and day 4 respectively. Subsequently, the
rate of proliferation decreased. In the presence
of 0.05 fiCUml 3H-TdR, however, the cells
proliferated but not exponentially after 24
hours of incubation. After six days with
3
H-TdR the densities were 2.9 x 104 and
4.8 x 104 cells/cm2, respectively, for the cells
inoculated at 0.65 and 1.3 x 104 cells/cm2. Cell
number increased approximately four times
during the exposure, indicating that the increase of labeled cells was due, not only to the
325
MATSUMURA, PFENDT AND HAYFLICK
326
|100
• O 6.7 x 10 3 /cm 2
D • 3.3 x 10 3 /cm 2
< 50 -
It
1
1
1
/
/a
10h
z =
,
V
Q-
_J O
I
/
• 3.3 x 10 3 /cm 2
D1.7x 10 3 /cm 2
i
i
• 13.3x 103/cm2
O6.7x 103/cm2
5 -
O 0.5
10
20
30
40
50
60
POPULATION DOUBLING LEVEL
Fig. 3. Percent nonlabeled nuclei corrected for cell
proliferation, which were obtained after six days incubation with 3H-TdR, as a function of PDL. The values
for the four PDLs were obtained hot from a single series
of WI-38 cultures, but from, respectively, four independent mother cultures.
1 L
in the following discussion, that the effect of
3
H-TdR on nonlabeled cells is much less than
that on labeled cells. As shown earlier, the
Fig. 2. Percent nonlabeled WI-38 nuclei and those cor- corrected percent for middle PDL cells derected for cell proliferation, which were obtained from a creased rapidly during the first day of incubaculture at PDL 32, as a function of incubation time tion, and from then on, extremely slowly. The
with 3H-TdR. The numerical values indicated in the
percent without correction did not show this
figure show the inoculation densities.
rapid change. At day 1 of incubation, the
cell density was considered to be low enough
while for relatively late passage cultures, they to permit exponential cell proliferation. One
were elevated. The reason for the choice of of the simplest conclusions to be reached from
relatively high densities in the latter was be- the preceding considerations is that WI-38 at
cause cell proliferation was less rapid. As middle PDLs consists of at least two subshown in Fig. 3, it was found that a semiloga- populations, one with a high rate of entrance
rithmic plot of the corrected percent was ap- into S phase, and the other with a very slow
proximately a linear function of PDL, and was rate of entrance into S phase. The present
not significantly influenced by the cell density. result supports the presence of subpopulations
in accordance with what was observed in
sparse cultures by Merz and Ross (1969), Smith
and Hayflick (1974), Absher et al. (1974), and
DISCUSSION
As described in Results, the parameter, per- Grove and Cristofalo (1976), considering the
cent nonlabeled cells corrected for cell pro- possibility that nondividing cells they observed
liferation, provides an estimation of the pro- might, in part, have the capacity to synthesize
portion of cells in a population which do not DNA. In an observation in progress, a small
enter S phase during exposure to 3H-TdR. proportion of Phase II cells show similar morWhen the cells are exposed to 3H-TdR, no phological progression as described for cells
matter how it influences the labeled cells, that in a senescent culture (Matsumura et al, 1979).
parameter remains constant so long as it does This observation suggests that the slowly
not influence nonlabeled cells. We presume, cycling cells in a Phase II population are
o
1
2
3
4
5
DAYS OF INCUBATION
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1 -
DNA SYNTHESIS OF HUMAN FIBROBLASTS
SUMMARY
Heterogeneity in the rate of entrance into
S phase of proliferating WI-38 human diploid
cells was investigated by autoradiography. The
percent of cells in the initial cell population
which did not incorporate 3H-TdR was determined from measurements of percent nonlabeled nuclei and the ratio of total cell
numbers at the initiation and at the termination of exposure to tritiated thymidine (3HTdR). This percentage decreased rapidly
during the first 24 hours of exposure, and
from then on, extremely slowly. The fraction
of cells with an extremely slow entrance
into S phase increased in parallel with the increasing population doubling level of the
cell population.
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Pendergrass, W. R., Clonal selection, attenuation
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related to the whole population of a senescent culture (details to be described elsewhere).
In regard to the practical use of parameters
to estimate in vitro age of a cell population,
the percent nonlabeled nuclei corrected for cell
proliferation may be an empirical parameter as
valuable as the labeling index. Perhaps the
most desirable way to determine a value of percent nonlabeled nuclei, corrected for cell proliferation, is to extrapolate the linear portion
of the time course to day 0. However, values
determined at day 6 seem to be useful as an
empirical parameter. The correlation coefficient between PDL and the correct percent
determined at day 6 was larger thaji the coefficient between PDL and the percent determined after 24 to 30-hour exposure without
correction (Cristofalo & Sharf, 1973), but comparable to that obtained by Merz and Ross
(1969) between passage number and the percent of nondividing cells.
327