StimulationofDMASynthesisinCalcium ...

[CANCER RESEARCH 40, 4541-4545, December 1980]
0008-5472/80/0040-OOOOS02.00

Stimulation of DMA Synthesis in Calcium-deprived T51B Liver Cells by

the Tumor Promoters Phénobarbital,Saccharin, and 12-0-
Tetradecanoylphorbol-13-acetate1

A. L. Boynton and J. F. Whitfield

Animal and Cell Physiology Section, Division of Biological Sciences, National Research Council of Canada, Ottawa. Canada K1A OR6

ABSTRACT MATERIALS AND METHODS

Extracellular calcium deprivation arrested the proliferative Adult T51B rat liver cells (36) were cultivated in a medium
development of T51B rat liver cells in late Gìor S phases. consisting of 90% BME (Microbiological Associates, Bethesda,
These arrested cells initiated DNA synthesis within an hr after Md.), 10% FBS (Flow Laboratories, Inc., Rockville, Md.), and
addition of calcium or a tumor promoter such as phorbol-12,13- 24 fig gentamicin (Microbiological Associates) per ml. They
didecanoate, phénobarbital, saccharin, or 12-O-tetradeca- were subcultured at weekly intervals at a 1:10 split by first
noylphorbol-13-acetate. Nonpromoting relatives of 12-O-tetra- exposing the monolayer briefly (1 min) to 5 ml of 0.1 % crystal
decanoylphorbol-13-acetate such as phorbol, 4-O-methyl-12- line trypsin solution at 37° (Sigma Chemical Co., St. Louis,
O-tetradecanoylphorbol-13-acetate, and 4a-phorbol-12,13-di- Mo.) in Ca2+- and Mg2+-free PBS, pouring off the trypsin
decanoate were unable significantly to induce the calcium-
solution, allowing the flasks to remain at room temperature for
deprived cells to initiate DNA synthesis. It is suggested that an an additional 3 min, and finally suspending the cells in 10 ml of
ability to elicit a prompt DNA-synthetic response from calcium- fresh complete medium. This cell suspension was then equally
deprived cells might be a useful in vitro indicator of tumor- distributed among 10 T-75 flasks (Lux Scientific Corp., Thou

promoting potential. sand Oaks, Calif.), each containing 10 ml BME: 10% FBS and
then incubated at 37°in an atmosphere consisting of 5% CO?:
INTRODUCTION
95% air.
In the 2-stage mechanism of carcinogenesis, a carcinogen Cells for experimental purposes were removed from stock

initiates the neoplastic process, which is then promoted by culture flasks as described above for subculturing and plated
tumor-promoting agents (3, 4). The best example of a tumor- at a density of 0.75 x 10"/sq cm on round 25-mm Thermanox
promoting agent is TPA2 from the oil of Croton tiglium (2, 3,
coverslips (Lux Scientific Corp.) in 35-mm plastic Petri dishes
28).
Although the means by which the archetypal promoter TPA (Lux Scientific Corp.) containing 2.0 ml of 1.8 rnw calcium 10%
FBS:90% BME. Twenty-four hr later, the high-calcium 10%
promotes neoplastic transformation is unknown, the agent has
been found to produce a wide variety of changes in nonneo- FBS:90% BME was removed, and the cultures were rinsed
twice with PBS and then overlaid with either 2.0 ml of complete
plastic cells which are to various extents associated with neo low-calcium FBS:BME containing only 0.02 m.Mcalcium [0.02
plastic transformation. These changes are: increased ornithine
decarboxylase activity (26, 45); release of arachidonic acid rriM calcium FBS, prepared according to the method of Borle
from plasma membrane phospholipids (24); increased prosta- and Briggs (6) using the specific calcium chelator ethylenegly-
col-bis(/?-aminoethylether)-/v',/v''-tetraacetic acid, and low-cal
glandin synthesis (24); induction of plasminogen activator (44);
loss of surface-associated fibronectin (5); decreased affinity of cium 0.02 rriM BME (Grand Island Biological Co., Grand Island,
N. Y.)] or low-calcium (0.02 DIM) and low-serum (0.1% FBS:
surface receptors for epidermal growth factor (32); reduction
or loss of proliferative anchorage dependence (17-19); in 99.9% BME) medium. Experiments began 24 to 48 hr later
when the percentage of cells in S phase had fallen to about
creased DNA synthesis (1) and cell proliferation (33, 41); and 15% for cells exposed to low-calcium FBS:BME and to about
stimulation of DNA synthesis by nonneoplastic cells blocked in 5% for cells exposed to low-calcium and low-serum FBS:BME
late G, (prereplicative) or S phases by extracellular calcium
deprivation (13, 41). from the normal 50 to 60% (10, 11, 40).
To determine the effects of calcium and/or serum on the
It is the intention of this paper to show that one of the
properties of TPA, the ability to elicit a DNA-synthetic response proportion of cells in the DNA-synthetic S phase of their growth
from calcium-deprived proliferatively quiescent nonneoplastic
cells, is shared by 3 other tumor-promoting agents (PDD, division cycle, cultures grown on coverslips were briefly (1 hr)
exposed to 5 /¿Coif [3H]thymidine per ml (specific activity, 20
phénobarbital, and saccharin) and to suggest that this could
be a general property of tumor promoters. Ci/mmol; New England Nuclear, Boston, Mass.). After expo
sure to [3H]thymidine, the coverslips were rinsed twice with
1 Issued as National Research Council of Canada No. 18736
2 The abbreviations used are: TPA, 12-O-tetradecanoylphorbol-13-acetate; PBS and then twice with a 10 mM solution of nonradioactive
PDD, phorbol-12,13-didecanoate; BME. Eagle's basal medium; FBS. fetal bovine thymidine in distilled water, mounted on slides, and finally
covered with a layer of NTB-2 nuclear track emulsion (Eastman
serum; PBS, phosphate-buffered saline [8.0 g NaCI:0.3 g KCI:O.075 g Na2HPCv Kodak, Rochester, N. Y.). Forty-eight hr later, autoradiographs
0.02 g KHzPCvl.O liter distilled water (pH 7.2)]; cyclic AMP, adenosine 3':5'-
were developed and stained, and the fraction of labeled nuclei
monophosphate. was determined. It should be noted that with this autoradi-
Received December 18, 1979; accepted September 15, 1980. ographic procedure the [3H]thymidine responsible for produc

ing the silver grains is incorporated only into cold acid-insoluble

DECEMBER 1980 4541

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A. L. Boynton and J. F. Whitfield

DNA (not the cold acid-soluble nucleotide fraction) and that the induced proliferative quiescence in T51B rat liver cells in about
silver grains (approximately 500 or more/cell) are found only 95% of the cell population at the well-known early Gìor G0
over the nuclei (10, 43). restriction point (Chart 1; Refs. 9-11,14 and 27). Replacement
of this low-serum calcium-deficient medium after 48 hr with
The known tumor promoters TPA and PDD (34) as well as
the nonpromoters 4a-phorbol-12,13-didecanoate, 4-O-methyl- one consisting of 20% FBS:80% BME containing 1.25 mM
12-O-tetradecanoylphorbol-13-acetate, and phorbol were pur calcium caused the majority of the blocked cells to initiate DNA
chased from Consolidated Midland (Brewster, N. Y.) while pure synthesis with a mean delay of 10 hr. However, if the fresh
sodium saccharin, calcium saccharin, and phénobarbital were 20% FBS:80% BME contained only 0.02 mM calcium, then
the kind gifts of Dr. D. Stoltz (Health and Welfare Canada, T51B rat liver cells did not initiate DNA synthesis. These
Ottawa, Canada). seemingly nonresponding extracellular calcium-deprived T51B

RESULTS cells must have been stimulated to advance into their G, phase
because a later addition of calcium ions [e.g., 1.25 mM at 4 hr
Lowering the serum and calcium concentrations in the me after addition of low-calcium 20% FBS:80% BME (Chart 1ß)]
dium from the usual 10% FBS:90% BME with 1.82 HIMcalcium
to 0.1 % FBS:99.9% BME with only 0.02 HIMcalcium reversibly to these cultures allowed them to initiate DNA synthesis at
exactly the same time as cells which had been stimulated with
B high-calcium 20% FBS:80% BME. Thus, extracellular calcium
ions were not required to initiate cell cycle progression of
_ 7° 70 serum-starved proliferatively quiescent T51B cells but were
needed at a much later stage of prereplicative (Gì)develop
ment. These observations also clearly showed that calcium
deprivation did not cause the cells to stop proliferative devel
opment after they had started making DNA (i.e., after entering

the S phase).

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Chart 1. A demonstration of the ability of calcium to promote the initiation of HOURS AFTER ADDITION
DMA synthesis late in the G, phase of the growth division cycle of T51B rat liver
cells. Twenty-four hr after plating, the normal serum:medium mixture was OF CALCIUM
changed to a low-calcium low-serum one consisting of 0.1% (v/v) FBS:99.9%
(v/v) BME containing only 0.02 rtiM calcium. In A, 48 hr later (Time 0 in the Chart 2. An example of the prompt initiation of DNA synthesis following the
experiments), the medium in some cultures was replaced with high-calcium (1.25 addition of calcium (to a final concentration of 1.25 mM) to cultures of T51B rat
mm) high-serum [20% (v/v)] medium (•)a, nd in some others, the medium was liver cells which had been blocked in either late G or S phases by incubation for
not changed (A). Cells treated with high-serum high-calcium medium initiated 48 hr in low-calcium (0.02 mM) medium. Points, means of the values for 4
cultures: bars. S.E.
DMA synthesis with a mean delay of 10 hr. In S. medium of the remaining cultures
was replaced at Time 0 with a low-calcium (0.02 mM) high-serum [20% (v/v)j
medium, and the calcium was either raised to 1.25 mM 4 hr later (•o) r left at
0.02 mM (O). It is evident that cultures treated at 4 hr with 1.25 mM calcium
initiated DMA synthesis at the same time as those cultures in A which received
the additional calcium at Time 0 (i.e., 4 hr earlier). Points, means of the values
from 4 cultures; bars, S.E.

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Tumor Promoters and DNA Synthesis

Further evidence suggesting, though by no means proving, (34) at concentrations between 0.007 and 0.14 ^M evoked a
a late G,-phase requirement for extracellular calcium came similar DNA-synthetic response from the calcium-deprived

from experiments in which only the extracellular calcium con T51B cells (Chart 3). By contrast, there was a much smaller
centration in 10% FBS:90% BME was lowered from the usual
1.82 to 0.02 mM. This treatment reversibly reduced the pro response to nonpromoters (33) such as phorbol, 4-O-methyl-
portion of cells making DNA in T51B liver cell cultures from the
normal 50 to 60% (10, 11, 40) to less than 20% during the 12-O-tetradecanoylphorbol-13-acetate, and 4a-phorbol-
next 24 to 48 hr. A large fraction (about 30%) of the cells in
such calcium-deprived cultures must have been arrested in 12,13-didecanoate (Chart 3).

either late Gìphase or the S phase of their growth division TPA and PDD were not the only tumor promoters to elicit a
cycle because they began making DNA within an hr after the rapid DNA-synthetic response from the calcium-deprived T51B
calcium concentration in the medium was raised from 0.02 to
1.25 mM (Chart 2). cells. Exposure to certain concentrations of the entirely differ

The classical tumor promoter TPA (at the optimally effective ent promoters phénobarbital and the calcium and sodium salts
0.02 /IM) caused T51B liver cells in low-calcium (0.02 mM)
of saccharin (22, 23, 25, 29, 38) stimulated these cells to
medium to initiate DNA synthesis as rapidly and as effectively
as raising the extracellular calcium concentration to 1.25 mM initiate DNA synthesis as rapidly and as effectively, or nearly
(Chart 3). Moreover, it did so at approximately the same con
centration at which it elicited rapid DNA-synthetic responses as effectively, as raising the extracellular calcium concentration
to 1.25 mM (Charts 4 to 6).
from BALB/3T3 mouse cells and rat thymic lymphoblasts in
low-calcium media (13, 41). The related tumor promoter PDD DISCUSSION

We conclude from the above observations that there is a
calcium-sensitive step in the middle or late d phase of the
growth division cycle of serum-stimulated T51B rat liver cells.
This conclusion is consistent with a large body of evidence
obtained with regenerating rat liver which clearly demonstrates

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Chart 3. A demonstration of the ability of various concentrations of the tumor-
promoting TPA and PDD to induce a rapid DNA-synthetic response from T51B Chart 4. An example of the different abilities of various concentrations of the
tumor-promoting phénobarbital to induce a rapid DNA-synthetic response from
rat liver cells which had been blocked in either late G, (prereplicative) or S either late-G,- or S-phase T51B rat liver cells in low-calcium (0.02 mM) medium.

phases by incubation in low-calcium (0.02-mM) medium. The tumor promoters , DNA-synthetic response to raising the calcium concentration in the
medium from 0.02 to 1.25 mM; means of the values from 4 cultures: bars, S.E.
(TPA and PDD) and their nonpromoting relatives [4a-phorbol-12,13-didecanoate

(4a-PDD), 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (4-O-Me-TPA),

and phorbol] were added to the calcium-deprived cultures at Time 0. .

DNA-synthetic response to raising the calcium concentration in the medium from

0.02 to 1.25 mM; points, means of the values from 4 cultures; bars. S.E.

DECEMBER 1980 4543

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A. L. Boynton and J. F. Whitfield LU

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Chart 6. An example of the rapid DNA-synthetic response to sodium saccharin
SACCHARIN CONCENTRATION (100 /ig/ml) of T51B rat liver cells blocked in either late G, or S phase by
incubation in low-calcium (0.02 mM) medium. Points, means of the values from
(/¿g/ml) 4 cultures; bars, S.E.

Chart 5. An example of the different abilities of various concentrations of the probably triggers the synthesis of the 4 deoxyribonucleotides
tumor-promoting sodium and calcium saccharin to elicit a rapid DNA-synthetic (10, 11, 39, 40, 42). Preventing this cyclic AMP surge with
adrenergic blockers or indomethacin, with a cyclic 3':5'-nu-
response from either late-G,- or S-phase T51B liver cells in low-calcium (0.02
cleotide phosphodiesterase activator such as imidazole, or by
mM) medium. , DNA-synthetic response to raising the calcium concentra greatly lowering the extracellular calcium concentration blocks
the initiation of DMA synthesis as effectively as calcium depri
tion in the medium from 0.02 to 1.25 mw; points, means of the values from 4 vation (10-12, 20, 39, 40, 42). Thus, the G,- or S-phase block
in calcium-deprived T51B rat liver cells can be overcome
cultures; bars. S.E.
equally effectively by adding any of the 3 components of the
that prolongedly calcium-deprived rat hepatocytes in the hy control system (;'.e., calcium, cyclic AMP, type II cyclic AMP-

pocalcémie thyroparathyroidectomized rat do not initiate DNA dependent protein kinase holoenzyme, or the catalytic subunits
synthesis when proliferatively activated by partial hepatectomy of types I and II cyclic AMP-dependent protein kinases) to the
and that a single i.p. injection of CaCI2 as late as 12 to 15 hr low-calcium medium (10, 11, 39, 40, 42). The present obser
after partial hepatectomy enables these crippled hepatocytes vations on these cells show that 4 different tumor promoters
to start making DNA at the normal time (30, 31, 37, 39, 40, overcome this block as effectively as any component of the
normal control system.
42).
Forty-eight hr of extracellular calcium deprivation of T51B Neoplastic transformation of avian and mammalian cells (of
epithelial or mesenchymal origin) by chemicals, viruses, or
rat liver cells by itself reduces the proportion of S-phase cells cultivation in vitro disconnects the initiation of DNA synthesis
from the calcium-dependent control system (7, 8, 11, 15, 16,
from the usual 50 to 60% to 15 to 20%, and the addition of 21, 39-42). Consequently, all of the 33 neoplastic cell types
calcium (1.25 mM) to the medium causes an almost immediate
resumption of DNA-synthetic activity (Chart 2). These obser so far examined can initiate DNA synthesis and proliferate
vations coupled with those on serum-stimulated cells suggest indefinitely in media which do not contain enough calcium to
that the rapidly responding calcium-deprived T51B rat liver support the proliferation of their nonneoplastic counterparts (7,
35, 39, 40). Thus, tumor promoters endow the nonneoplastic
cells are blocked late in the G, phase of their growth division T51B rat liver cells as well as BALB/3T3 mouse cells and rat
cycle, although the possibility of their being blocked in S phase
cannot be ruled out.

Although the mechanism(s) by which calcium ions participate
in the initiation of DNA synthesis are not specifically known, a
growing body of evidence suggests that extracellular calcium
ions are components of a control system which also includes
a seemingly universal middle or late prereplicative cyclic AMP
surge and its attendant burst of protein kinase activity that

4544 CANCER RESEARCH VOL. 40

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Tumor Promoters and DNA Synthesis

thymic lymphoblasts (13, 41) with the neoplastia property of cyclic AMP metabolism in proliferating lymphocytes. In: J. G. Kaplan (ed.).
being able to initiate DNA synthesis in a low-calcium medium. The Molecular Basis of Immune Cell Function, pp. 57-74. Amsterdam:

It remains to be seen how the ability of these promoters to Elsevier/North Holland, 1979.
stimulate or bypass the proliferative calcium control system of 21. Hazelton, B., Mitchell, B., and Tupper, J. Calcium, magnesium, and growth

cultivated cells is related to tumor promotion in vivo. However, control in the WI-38 human fibroblast cell. J. Cell Biol.. 83. 487-498. 1979.

even if it should be found not to be related directly to tumor 22. Hicks, R. M., Chowaniec, J., and Wakefield. J. St. J. Experimental induction
promotion, this property may prove to be useful for the rapid in of bladder tumors by a two-stage system. In: T. J. Slaga. A. Sivak. and R. K.
vitro assessment of tumor-promoting potential.
Boutwell (eds.). Mechanisms of Tumor Promotion and Cocarcinogenesis,
ACKNOWLEDGMENTS pp. 475-489. New York: Raven Press, 1978.
23. Mondai, S., Brankow, D. W., and Heidelberger. C. Enhancement of onco-
We gratefully acknowledge the technical assistance of R. J. Isaacs and R.
Tremblay and of D. J. Gillan who prepared the illustrations. genesis in C3H10T1 /2 mouse embryo cell cultures by saccharin. Science
(Wash. D. C.), 201: 1141-1143, 1978.
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Stimulation of DNA Synthesis in Calcium-deprived T51B Liver
Cells by the Tumor Promoters Phenobarbital, Saccharin, and 12-
O-Tetradecanoylphorbol-13-acetate

A. L. Boynton and J. F. Whitfield
Cancer Res 1980;40:4541-4545.

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