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Cancer Letters, 15 (1982) 271-279 Elsevier/North-Holland Scientific Publishers Ltd.
271
CALCIUM DEPENDENCE OF CHEMICAL CARCINOGEN INDUCED MORPHOLOGICAL TRANSFORMATION OF SYRIAN HAMSTER EMBRYO CELLS
CHARLES H. EVANS and ALTON L. BOYNTONa
Tumor Biology Section, Laboratory of Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205 (U.S.A.) and aAnimal and Cell Physiology Group, Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario KlAOR6 (Canada)
(Received 2 October 1981) (Revised version received 14 December 1981) (Accepted 14 December 1981)
SUMMARY
The effect of extracellular calcium upon carcinogen induced morphologi- cal transformation was evaluated in Syrian hamster embryo cells. Reduction in [Ca”] from 1.8 mM to 0.2 mM throughout the 6 days between exposure of the cells to 2.5 pg benzo[a]pyrene (BP)/ml and examination of the cells for transformation inhibited both cell proliferation and transformation as measured by the frequencies of colony formation and morphological trans- formation. The transformation frequency among surviving cells, i.e. fre- quency/cell colony, however, was nearly equivalent indicating that the inhibition of transformation largely resulted from inhibition of cell colony formation. Proliferation and transformation were unaffected when [Ca2’] was reduced to 0.2 mM on days 3-6. Reduction to 0.01 mM Ca2+ during the same period, however, completely abolished transformation by BP, UV-irradiation or n-acetoxyacetylaminofluorene (AcAAF) without reducing cell proliferation. Thus, the expression of morphological transformation in newly transformed hamster cells is dependent upon extracellular [ Ca”] to a greater degree than is cell proliferation.
Abbreviations: AcAAF, n-acetoxyacetylaminofluorene; BP, benzo[a]pyrene ; decal FBS, EGTA treated FBS with Ca’+ reduced to approximately 0.02 mM; DMEM, Dulbecco’s modification of minimal essential medium; EGTA, [ethylenebis(oxyethylenenitrilo)]- tetraacetic acid; FBS, fetal bovine serum containing 2.3 mM ionic Ca”; MCA, 3-methyl- cholanthrene; MEM, minima! essential medium; Nocal DMEM, DMEM prepared without calcium salts and containing 0.01 mM Ca”; UV, 254 nm ultraviolet irradiation.
0304-3835/82/0000-0000/$02.75 0 1982 Elsevier/North-Holland Scientific Publishers Ltd.
272
INTRODUCTION
In a previous investigation it was observed that there were different extracellular calcium requirements for proliferation of non-neoplastic, preneoplastic, and neoplastic mouse cells [ 71. Neoplastic fibrosarcoma forming MCA-C3H/lOT% type III mouse fetal fibroblasts synthesized DNA and multiplied indefinitely in the presence of extremely low con- centrations of extracellular free calcium (0.001 -0.005 mM). DNA synthesis and proliferation were inhibited in preneoplastic C3H/lOT% and MCA- C3H/lOT% type I mouse fetal fibroblasts at calcium levels of 0.01 mM or less. Primary passage non-tumorigenic C3H mouse epidermal cells ceased DNA synthesis and proliferation when the extracellular free calcium level was reduced to 0.05-0.1 mM. Those data suggested that the control of proliferation of mouse fibroblasts by extracellular calcium is lost in at least 2 stages as the cell and its decendents advance toward the neoplastic state. Many tumor cells require substantially less calcium to proliferate compared to non-tumorigenic cells [2,4--6,9,17,20,21,23,24] including Syrian hamster cells which acquire a lower requirement for extracellular calcium in association with the development of chemical carcinogen induced neoplastic transformation [ 251. In the present investigation the levels of extracellular calcium in the cell culture medium were reduced after carcino- gen treatment of Syrian hamster embryo cells and cell proliferation and the frequency of carcinogen induced morphological transformation subsequently examined 6 days after carcinogen treatment to further evaluate the role of extracellular calcium during the early stages of carcinogenesis in mam- malian cells.
METHODS
The effect of extracellular calcium was studied in the Syrian hamster embryo fibroblast in vitro model of carcinogenesis in which the frequency of carcinogen-induced morphological transformation is quantifiable and indicative of the cells’ neoplastic potential 1141. In each transformation experiment, secondary cultures of Syrian hamster embryo cells, obtained from 12-day gestation fetuses of a single hamster and stored in liquid nitro- gen were cultured and treated with a carcinogen as previously described [ 131. Tissue culture dishes (100 mm diameter) were seeded with 10’ cells. The cells were grown in Dulbecco’s MEM, containing 1.8 mM calcium and supplemented with 3 X 10d6 M thymidine, 3 X lo-’ M hypoxanthine and 10% fetal bovine serum (FBS) without antibiotics, at 37°C in a 10% CO, in a water saturated atmosphere of PHARM-AIR (Pall Trincor Corp., Cortland, NY) purified compressed air [ 161. After 3 days, the cells were trypsinized and 100 mm dishes were seeded with 2.5 X lo6 cells in 10 ml of medium with 10% serum. Cells were trypsinized 48 h later, and 300 cells together with 6 X lo4 X-irradiated sister culture cells were seeded/
273
60 mm dish in 4 ml medium with 10% FBS. After additional 24 h another 4 ml of medium containing a chemical carcinogen was added or the medium was removed, the cells irradiated with UV irradiation and the dishes re-fed with 8 ml medium. Twelve dishes were treated with each concentration or dose of carcinogen. In some experiments, calcium-free Dulbecco’s MEM and/or decalcified serum were substituted for the normal calcium-containing medium or serum. The ionic (physiologically available) calcium content of the serum was reduced to about 0.020 mM with EGTA and measured by the fluorometric method of Borle and Briggs [3] as previously described [7]. The medium over the carcinogen treated cells was removed and re- placed with various combinations of medium and 10% serum with reduced extracellular calcium simultaneously with or 1, 3,4 or 5 days after carcino- gen treatment as described in individual treatments. Six days after carcino- gen treatment, unless stated otherwise, the dishes were stained and cell colonies greater than 2 mm in diameter examined for morphological trans- formation, a random piled up layering of spindle shaped fibroblasts not observed in non-carcinogen treated cultures and correlating with the ability of the cells to grow as tumors in vivo.
RESULTS
Reduction in the extracellular ionic calcium from 1.8 mM to 0.2 mM throughout the 6 days between carcinogen exposure and evaluation of morphological transformation results in decreased cell proliferation and a decrease in the frequency of transformation as shown in Table 1. The reduction in transformation may result predominantly from decreased cell proliferation since the transformation frequency/colony formed in 0.2 mM calcium is similar to that in the presence of 1.8 mM calcium. Both cell proliferation and transformation are also decreased if the reduction from 1.8 to 0.2 mM calcium is instituted 1 day after carcinogen treatment (Table 1). The results in the latter experiment, however, suggest that there is a greater reduction in the frequency of transformation than there is in cell proliferation. If, however, the calcium level in the medium is reduced to 0.2 mM commencing 3 days after carcinogen exposure (Table 2) cell pro- liferation and transformation are unaffected unless the -calcium is further reduced to 0.01 mM. When that is done, there is little effect upon cell proliferation as judged by colony formation, but transformation is com- pletely inhibited. The inhibition is due to the reduction in the calcium and not to the presence of the chelator EGTA in the decalcified serum as inclusion of the decalcified serum in the regular Dulbecco’s medium sup-
ports transformation by BP, UV irradiation or AcAAF (Table 3). Morpho- logical transformation, furthermore, is sensitive to reduction in extracellular calcium to 0.01 mM as late as 5 days after carcinogen treatment as illustrated in Table 3.
P
TA
BL
E
1
EF
FE
CT
O
F
RE
DU
CE
D
EX
TR
AC
EL
LU
LA
R
CA
LC
IUM
U
PO
N
CE
LL
P
RO
LIF
ER
AT
ION
A
ND
M
OR
PH
OL
OG
ICA
L
TR
AN
S-
FO
RM
AT
ION
Tre
atm
ent
[Cal
+]
mM
C
olon
y fo
rmat
ion
(%
Y
No.
of
tran
sfor
med
co
lon
ies
per
dish
a pe
r to
tal
colo
nie
s (%
)*
DM
EM
+ 1
0% F
BS
D
ME
M +
10%
FB
S +
BP
5 w
g/m
l N
ocal
DM
EM
+ 1
0% F
BS
N
ocal
DM
EM
+ 1
0% F
BS
t
BP
5 g
g/m
l D
ME
M +
10%
FB
S a
nd
re-f
ed o
n d
ay 1
wit
h D
ME
M +
10%
FB
Sb
DM
EM
+ 1
0% F
BS
an
d re
-fed
on
day
1
wit
h D
ME
M +
BP
5 r
g/m
l D
ME
M +
10%
FB
S a
nd
re-f
ed o
n d
ay 1
wit
h N
ocai
D
ME
M
+ 1
0% F
BS
b D
ME
M +
10%
FB
S a
nd
re-f
ed o
n d
ay 1
w
ith
Noc
al D
ME
M
+ B
P 5
pg/
ml
1.8
1.8
0.2
0.2
1.8
1.8
1.8
- 1.
8 1.
8 0.
2
1.8
- 0.
2 14
* 0
.4
28 *
0.4
0
0 26
f
0.5
3.9
f 0.
5 4.
7 f
0.6
10 f
0.
3 0
0 5
f 0.
4 0.
5 f
0.2
3.3
f 0.
4
28 f
0.
4 0
23 f
0.
6 3.
2 *
0.5
21 f
0.
4 0 1.
0 f
0.3
2.4
f 0.
3
0 4.7
* 0.
7
0
aMea
n f
S
.E.
bDay
1
= t
he
day
foll
owin
g ca
rcin
ogen
ex
posu
re.
TA
BL
E
2
SE
NS
ITIV
ITY
O
F
MO
RP
HO
LO
GIC
AL
T
RA
NS
FO
RM
AT
ION
T
O
RE
DU
CT
ION
IN
E
XT
RA
CE
LL
UL
AR
C
AL
CIU
M
Tre
atm
ent
in
DM
EM
+
10
%
FB
S
(Ca’
+
1.8
mM
)
Con
trol
B
P
1 fi
g/m
l B
P
2.5
pg/m
l B
P 5
pg/
ml
Re-
fed
3 da
ys
afte
r C
olon
y N
o.
of
tran
sfor
med
co
lon
ies
carc
inog
en
wit
h
form
atio
n
(%)”
pe
r di
sha
per
tota
l co
lon
ies
( %)a
DM
EM
+
10
%
FB
S
(Ca2
+ 1
.8
mM
) 26
f
0.6
0 0
DM
EM
+
10
%
FB
S
(Ca’
+
1.8
mM
) 20
f
0.6
1.5
f 0.
4 2.
4 *
0.7
DM
EM
+
10
%
FB
S
(Ca’
+
1.8
mM
) 20
f
0.7
2.3
* 0.
5 3.
9 f
0.8
DM
EM
+
10
%
FB
S
(Ca*
+
1.8
mM
) 19
*
0.8
3.2
f 0.
2 5.
3 f
0.5
Con
trol
B
P 1
r g
/ml
BP
2.
5 rg
/ml
BP
5
rg/m
l
Con
trol
B
P 1
rg/
ml
BP
2.
5 fi
g/m
l B
P
5 rg
/ml
*Mea
n
* S
.E.
Noc
al
DM
EM
+
10
%
FB
S
(Ca’
+
0.2
mM
) 20
f
0.6
0 0
Noc
al
DM
EM
+
10
%
FB
S
(Cal
+
0.2
mM
) 17
f
0.6
0.7
f 0.
2 1.
3 f
0.3
Noc
al
DM
EM
+
10
%
FB
S
(Ca’
+
0.2
mM
) 18
*
0.8
2.0
f 0.
3 2.
7 f
0
Noc
al
DM
EM
+
10
%
FB
S
(Ca”
0.
2 m
M)
17
f 0.
6 3.
3 f
0.3
6.6
f 0.
3
Noc
al
DM
EM
+
10
%
deca
l F
BS
(C
a’+
0.
01
mM
) 19
f
0.3
0 0
Noc
al
DM
EM
+
10
%
deca
l F
BS
(C
al+
0.
01
mM
) 16
f
0.6
0 0
Noc
al
DM
EM
+
10
%
deca
l F
BS
(C
a’+
0.
01
mM
) 14
f
0.5
0 0
Noc
al
DM
EM
+
10
%
deca
l F
BS
(C
al+
0.
01
mM
) 15
f
1.0
0 0
TA
BL
E
3
RE
VE
RS
ION
O
F M
OR
PH
OL
OG
ICA
L
TR
AN
SF
OR
MA
TIO
N
DU
E T
O A
DE
CR
EA
SE
IN
EX
TR
AC
EL
LU
LA
R
CA
LC
IUM
Tre
atm
ent
in
Re-
fed
3,4
or
DM
EM
+ 1
0% d
ecal
FB
S
5 da
ys a
fter
(C
a*+
1.6
mM
) ca
rcin
ogen
w
ith
Non
e D
ME
M
+ 1
0% d
ecal
FB
S (
Ca’
+ 1
.6 m
M)
19
0 15
0
15
0 B
P 1
pg/
ml
DM
EM
t 10%
deca
l F
BS
(C
a*+
1.6
mM
) 13
1.
3 12
1.
8 12
1.
5 U
V 6
J/m
’ D
ME
M t
10
% d
ecal
FB
S (
Ca*
+ 1
.6 m
M)
5 1.
0 7
1.0
6 0.
3 A
cAA
F
1 fi
g/m
l D
ME
M +
10%
dec
al F
BS
(C
a’+
1.6
mM
) 14
0.
7 12
1.
0 8
1.3
Re-
fed
afte
r ca
rcin
ogen
on
Day
3
Day
4
Day
5
stai
ned
day
6
stai
ned
day
7
stai
ned
day
8
CF
a T
/Db
CF
T
/D
CF
T
/D
Non
e N
ocal
D
ME
M +
10%
dec
al F
BS
(C
a’*
0.01
m
M)
15
0 16
0
15
0 B
P 1
Mg/
ml
Noc
al
DM
EM
+ 1
0% d
ecal
FB
S (
Ca’
+
0.01
m
M)
10
0.3
11
0 11
0
UV
6 J
/m’
Noc
al
DM
EM
+ 1
0% d
ecal
FB
S (
Ca’
+
0.01
m
M)
4 0
6 0
5 0
AcA
AF
1
pg/m
l N
ocal
D
ME
M
t 10
% d
ecal
FB
S (
Ca*
+ 0
.01
mM
) 10
0.
2 11
0
11
0
Yol
ony
form
atio
n,
%.
bNo.
of
tra
nsf
orm
ed
colo
nie
s/di
sh.
277
DISCUSSION
The present observations clearly demonstrate that morphological trans- formation of secondary cultures of Syrian hamster embryo cells by BP, UV irradiation or AcAAF in a 7 day cell colony assay is dependent on the ionic extracellular calcium concentration. The induction of morphological transformation requires cell proliferation and thus is dependent upon extra- cellular calcium. The induction of morphologic transformation is not, however, intrinsically calcium dependent since the transformation fre- quency per cell colony is unaffected by reduction in extracellular calcium to 0.2 mM simultaneously with, or 1, or 3 days after carcinogen treatment. The expression of morphological transformation, however, is intrinsically calcium dependent. Reduction of the extracellular calcium concentration in the medium serum mixture from 1.6 mM to 0.01 mM after morpho- logical transformation has been expressed (i.e. day 5; Table 3) causes re- version to the ‘normal’ non-transformed phenotype. The ability of 0.01 mM calcium to suppress the expression of morphological transformation of hamster cells is reminiscent of the change from monolayer to focally strat- ified growth of spontaneously transformed mouse epidermal cells induced by a shift from 0.07 mM to 1.2 mM calcium in the culture medium [26].
The calcium-dependent expression of morphological transformation in hamster cells is not due to reduced cell proliferation of morphologically transformed cells because by 3 days after carcinogen exposure the low- calcium medium-serum mixture (0.01 mM) does not appreciably affect cell proliferation as determined by colony formation (Tables 1 and 3). However, if the cultures are treated with a medium-serum mixture con- taining only 0.2 mM calcium from the onset of the experiment, cell pro- liferation is reduced by 65-80s (Table 1) thus confirming the proliferative sensitivity of primary and secondary non-neoplastic cells in culture to extracellular calcium ions [ 71. Colony formation in this model of carcino- genesis is a good measure of cell proliferation as the developing cell colonies at 3 days are less than 2 mm in diameter and either cell detachment, dis- solution, or inhibition of proliferation due to reduction in extracellular calcium at 3 days would be reflected by a reduced colony formation at 6 days. Since this did not occur the expression of morphological trans- formation is more dependent upon extracellular calcium than is cell pro- liferation in newly transformed cells.
Calcium ions, calmodulin (the ubiquitous calcium-binding protein and mediator of calcium action), cyclic AMP and cyclic AMP-dependent protein kinase activity are needed briefly in the later Gl phase of the cell cycle for the completion of prereplicative development and the initiation of DNA synthesis in non-neoplastic cells [ 5,7-11,23,24]. On the other hand, neoplastic cells both of mesenchymal and epithelial origin are able to initiate DNA synthesis and proliferate indefinitely in very low extracellular calcium concentrations [2,4-9,17,20,21,23,24]. The available evidence suggests that the diminished dependency of malignant cells upon extracellular cal-
273
cium may result from a persistent increase in intracellular levels of cal- modulin as reported for some virus transformed cells [12,X428] and found in a wide variety of virus, chemical or spontaneously transformed human and rodent tumor cells (Boynton, unpublished observations) or perhaps from alteration of the late prereplicative calcium--cyclic AMP protein kinase system [11,19,22,27]. In either case, the effect would be to reduce the extracellular calcium requirement for neoplastic cells by either sen- sitizing, altering or bypassing the late Gl calcium-calmodulin cyclic AMP protein kinase regulatory system.
It is not known whether the calcium dependency of carcinogenesis in its earlier stages as described in this investigation is mediated through calcium binding proteins, protein kinases, and/or whether carcinogen induced changes in membrane permeability [l] are involved. Future investigations to examine these possibilities are important, for as with corticosteroid [16] and lymphotoxin [15] prevention of transformation, the ability of calcium to inhibit morphological transformation provides another example of physiological mechanisms that in the absence of inhibiting cell prolifera- tion can regulate the expression of properties accompanying the develop- ment of carcinogenesis.
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