167
Cancer Letters, 63 (1992) 167 - 170 Elsevier Scientific Publishers Ireland Ltd.
The role of protein kinase c in migration of rat glioma cells from spheroid cultures Przemyslaw Kowalczyk Deportment (Received (Accepted
Janik, Bozena Szaniawska,
of Tumor Biology,
Cancer
Center
Joanna
Institute of Oncology,
The role of protein kinase C in migration of tumor cells from spheroid cultures was inuestigated using parental rat glioma cells and their TPA resistant counterparts. These two lines differed in their PKC content as judged by the histone phosphorylation method. Also 4 days of treatment with IRA led to PKC downregulation. Cells hauing a drastically decreased PKC level migrated better then those having a normal PKC content.
Keywords: glioma
protein
kinase C; cell migration;
Introduction Protein kinase C (PKC) plays an important role in the transduction of extra cellular signals for cell proliferation and function [8]. It is well recognized that cells treated with promoting phorbol ester (TPA) change their shape and become more elongated [3]. Since TPA specifically binds and stimulates protein kinase C [2] it is reasonable to expect that the enzyme Correspondence
Warsaw,
Wawelska
Ewa Pietruszewska,
I&00-973
Dariusz.
Warsaw (Poland)
27 January 1992) 10 February 1992)
Summary
Cancer
Miloszewska,
Center
to: P. Janik, Department of Tumor Biology, Institute of Oncology, Wawelska 15.00-973
Poland.
0304.3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific in Ireland
Publishers
may have an impact on migration of cells. Prolonged treatment with TPA results in down regulation of PKC [ 111. These two opposite effects of TPA treatment allows us to analyse cells that have the same origin but differing protein kinase C content. In the experiments reported here we have examined the role of protein kinase C on tumor cell migration from spheroid cultures. This has been done by using normal glioma cells treated with TPA as well as their counterparts which have been exposed to TPA for a prolonged period. Materials
and Methods
Cells The BT5C rat glioma cell line obtained from fetal brain cells which underwent transformation after in vitro exposure to ethylnitrosourea [6] was used in the experiments. BT5C-R (PKC depleted) cell line was obtained after prolonged cultivation with 200 ng/ml medium of TPA (more then 20 passages). Plating efficiency Two hundred per dish of BT5C and BT5CR cells were plated at the presence or absence of TPA in culture medium. Cells were allowed to grow for 7 days and colonies were scored after fixing and staining. Ireland Ltd
168
Migration assay Multicellular tumor spheroids were initiated by using the liquid over-lay culture [13]. A 3 x lo6 quantity of cells were seeded into a 10 cm* Petri dish coated with 0.75% agar dissolved in medium. In order to study the directional migration the 7-day-old spheroids were transferred by Pasteur pipette to multiwell dishes not coated with agar and covered with medium with or without TPA. After 4 days cultures were fixed and stained in situ using crystal violet. The area of migration was calculated from two orthogonal diameters as described previously [ 121. PKC actioity PKC was assayed by the method of histone phosphorylation, as described earlier [5,7]. Protein concentrations was determined by the method of Bradford [l]. Results Plating efficiency Table 1 shows that 6 days of culture in the presence of 200 ng/ml TPA inhibits plating efficiency of BT5C cells, whereas BT5C-R cells chronically propagated with TPA are resistant to the inhibitory effect of TPA. PKC study Table 2 shows results of PKC determinations in glioma cells. The original BT5C cell line displayed PKC activity in membranous and cytosolic fractions. When these cells were treated for 4 days with TPA they show very
Table 2. PKC activity (pmol 32P/min per mg protein) in cytosol and membrane fractions of BT5C cells and their TPA resistant clone-BT5C-R.
BT5C BTSC zt TPA BT5C-R
Membranes
Cytosol
375.2 zt 31.9 8.3 zt 4.1 1.9 l 0.9
415.0 34.2 27.0
f f f
19.8 17.1 13.0
Mean from 5 experiments =t S.D. Cells were treated for 4 days with TPA (200 ng/ml medium).
of
low levels of PKC activity in their membranes and cytosol. The membrane fraction of the TPA resistant line (BT5C-R) had barely detectable PKC activity. These results confirm that resistance to TPA treatment is associated with depletion of PKC from the membrane fraction. Furthermore, the resistant BT5C-R cell line can grow independently of protein kinase C. Migration study Table 3 shows the results of 4 days of TPA treatment on the migration of BT5C cells and their TPA resistant line. The most interesting finding was that cells, depleted of PKC by either prolonged cultivation with TPA or by 4 days treatment with TPA migrated better then control untreated parental BT5C cells. As could be expected, TPA treatment of the resistant BT5C-R line had no effect on migration. Discussion
Table 1. Effect of TPA treatment on plating efficiency of BT5C cells and TPA resistant clone BT5C-R. TPA ng/ml of medium
BT5C BT5C-R
0
100
200
46.3 zt 2.7’ 47.8 f 3.3
40.4 f 5.2 49.1 f 2.9
27.4 f 6.3’ 51.5 jz 5.1
Mean of 10 dishes (Student’s t-test).
*
S.D.
‘Statistically
significant
It has been presented before that promoting phorbol esters like TPA change the shape of cells [3] and stimulate migration of brain endothelial cells [9]. Also staurosporin, a protein kinase C inhibitor reduced the rate of migration of bladder carcinoma cells [lo]. However, staurosporin is also an inhibitor of various kinases including CAMP and cGMP. TPA on one hand is a stimulator of PKC but on the other hand it also induces the down-regulation
169
Table 3.
Effect of TPA treatment
on directional
Area of migration TPA (ng/ml)
0 200
migration
BT5C and BT5C-R cells from spheroid.
(Fm*) BT5CR
BT5C Day 0
Day 4
Day 0
Day 4
9.2 n = 8.9 n =
653.6 f 10.3 n = 134 835.2 zt 22.4’ n = 36
9.4 f 3.1 n = 95 9.6 z+z2.6 n = 42
772.0
f
3.0 180 f 2.2 40
Mean f S.E.; n, number of spheroids used. *Statistically significant from TPA 0 at day 4 (Student’s
of the enzyme. Therefore, the distinction between activation and down-regulation of PKC is critical to the understanding of TPA action. The system of the migration of tumor cells from spheroids was chosen since it reminds in vivo situation where tumor ceils usual grow in form a spheroid both in primary or secondary places. Our study was performed following the addition of TPA to the medium. This treatment stimulated cells movement and also led to PKC down-regulation. Since proliferation of glioma BT5C cells was inhibited by TPA treatment, the increase in migration area was independent of cell multiplication and may have resulted from protein kinase C down regulation. In addition, BTSC-R cells, which lack PKC in their membranes, migrated better then non-treated parental glioma cells regardless of TPA presence in the medium. Altogether, it looks like PKC negatively correlates with migration of studied cells. This finding could be supported by the results showing that the higher level of phosphorylation of I-IMM by protein kinase C caused a progressive inhibition of actin-activated ATPase [4]. BT5C-R cells which were exposed to TPA for a prolonged period of more than 20 passages lost their sensitivity to growth inhibition mediated by TPA. It could be only speculated that the role of PKC was overridden by different kinases.
zt 27.7’ 50 806.1 f 48.1’ n = 43 n =
t-test)
An-overall conclusion from these studies is that the lack of protein kinase C facilitates the migration of glioma cells. If this finding could be extended to other experimental models (e.g. multistage skin carcinogenesis) then the role of tumor promoting phorbol esters could be considered, which by inducing PKC downcreates a permissive event for regulation, vigorous migration. This in turn may allow a cell escape from environmental control and give rise to a tumor. References Bradford,
M.M.
quantitation
(1976):
A rapid sensitive method for the
of micrograms
quantities of protein
the principle of protein day binding. Anal.
utilizing
Biochem..
72,
248 - 254. Catagna,
Y, (1982);
activated phospholipid or
Sane, K.,
M., Takai, Y., Kaibuchi, K.,
U. and Nishizuka, promoting
dependent
phorbol
Kikkawa,
Direct activation of calcium
ester.
protein kinase by tumJ.
Biol.
Chem.,
257,
7847 - 7851. Dugina V.B., I.M.
Svitkina T.M.,
(1987).
by phorbol
Special ester.
Vasiliev,
type
Proc.
of
Natl.
J.M.
and Gelfant,
reorganization Acad.
Sci.
induced
U.S.A.,
84.
4112-4125. Ikebe,
M..
Hartshorne,
Phosphorylation
of
D.J.
the
and
Elzinga.
20 000.dalton
M.
light
(1987): chain
of
smooth muscle myosine by the calcium activated phospholipid-dependent
protein
kinase.
J.
Biol.
Chem..
262,
9569 - 9574. Kraft, S.A.,
Anderson W.B.
(1983)
the amount of Ca, phospholipid associated 621-623.
with
plasma
Phorbol ester increase
dependent
membrane.
protein kinase Nature,
301,
170 6
7
8
9
10
Laerum, O.D. and Rajewski M.F. (1976) Neoplastic transformation of fetal rat brain cells in culture after exposure to ethylnitroso-urea. J. Natl. Cancer Inst., 55. 1177- 1187. Miioszewska, J., Trawicki, W., Jamik, P., Moraczewski, J., Przybyszewska, M. and Szaniawska B. (1986): Protein kinase C translocation in relation to proliferative status of C3H lOTU.2 cells. FEBS Lett., 206, 283-286. Nishinuka, Y. (1984): The role of protein kinase C in ceil surface signal transduction and tumor promotion. Nature, 308, 693 - 698 Rosen, E.M., Jaken, S., Carley, W., Lucket, P.M., Setter, E., Bhargava, M. and Goldberg, J.D. (1991) Regulation of motility in bovin brain endothelial cells. J. Cell Physiol., 146, 325 - 335. Swartz, G.K., Redwood,
S.M.,
Ohmuna,
T., Holland,
11
12
13
J.F.. Droler, M.J. and Lin, B.C.-S. (1990): Inhibition of invasion human bladder carcinoma cells by protein kinase inhibitor Staurosporine. J. Natl. Cancer Inst., 22, 1753 - 1757. Rodrigez-Pena. A. and Rozengurt, E., (1984) Disappearance of Ca sensitive, phospholipid-dependent protein kinase C activity in phorbol ester treated 3T3 cells. Biochem. Biophys. Res. Commun., 120, 1053- 1059 Szaniawska, B., Bjerkvig, R. and Laerum O.D. (1986): Growth arrest and polyploidisation induced by metahalone microtubule inhibitors on rat glioma cells in culture. Anticancer Res., 6. 71- 77. Yuhas, J.M., Li, A.P. Martinez, A.D. and Ladman, A.J. (1986): A simplified method for production and growth of multtcellular tumor spheroids. Cancer Res., 37, 3639 - 3643.
Cancer Letters, 63 (1992) 167 - 170 Elsevier Scientific Publishers Ireland Ltd.
The role of protein kinase c in migration of rat glioma cells from spheroid cultures Przemyslaw Kowalczyk Deportment (Received (Accepted
Janik, Bozena Szaniawska,
of Tumor Biology,
Cancer
Center
Joanna
Institute of Oncology,
The role of protein kinase C in migration of tumor cells from spheroid cultures was inuestigated using parental rat glioma cells and their TPA resistant counterparts. These two lines differed in their PKC content as judged by the histone phosphorylation method. Also 4 days of treatment with IRA led to PKC downregulation. Cells hauing a drastically decreased PKC level migrated better then those having a normal PKC content.
Keywords: glioma
protein
kinase C; cell migration;
Introduction Protein kinase C (PKC) plays an important role in the transduction of extra cellular signals for cell proliferation and function [8]. It is well recognized that cells treated with promoting phorbol ester (TPA) change their shape and become more elongated [3]. Since TPA specifically binds and stimulates protein kinase C [2] it is reasonable to expect that the enzyme Correspondence
Warsaw,
Wawelska
Ewa Pietruszewska,
I&00-973
Dariusz.
Warsaw (Poland)
27 January 1992) 10 February 1992)
Summary
Cancer
Miloszewska,
Center
to: P. Janik, Department of Tumor Biology, Institute of Oncology, Wawelska 15.00-973
Poland.
0304.3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific in Ireland
Publishers
may have an impact on migration of cells. Prolonged treatment with TPA results in down regulation of PKC [ 111. These two opposite effects of TPA treatment allows us to analyse cells that have the same origin but differing protein kinase C content. In the experiments reported here we have examined the role of protein kinase C on tumor cell migration from spheroid cultures. This has been done by using normal glioma cells treated with TPA as well as their counterparts which have been exposed to TPA for a prolonged period. Materials
and Methods
Cells The BT5C rat glioma cell line obtained from fetal brain cells which underwent transformation after in vitro exposure to ethylnitrosourea [6] was used in the experiments. BT5C-R (PKC depleted) cell line was obtained after prolonged cultivation with 200 ng/ml medium of TPA (more then 20 passages). Plating efficiency Two hundred per dish of BT5C and BT5CR cells were plated at the presence or absence of TPA in culture medium. Cells were allowed to grow for 7 days and colonies were scored after fixing and staining. Ireland Ltd
168
Migration assay Multicellular tumor spheroids were initiated by using the liquid over-lay culture [13]. A 3 x lo6 quantity of cells were seeded into a 10 cm* Petri dish coated with 0.75% agar dissolved in medium. In order to study the directional migration the 7-day-old spheroids were transferred by Pasteur pipette to multiwell dishes not coated with agar and covered with medium with or without TPA. After 4 days cultures were fixed and stained in situ using crystal violet. The area of migration was calculated from two orthogonal diameters as described previously [ 121. PKC actioity PKC was assayed by the method of histone phosphorylation, as described earlier [5,7]. Protein concentrations was determined by the method of Bradford [l]. Results Plating efficiency Table 1 shows that 6 days of culture in the presence of 200 ng/ml TPA inhibits plating efficiency of BT5C cells, whereas BT5C-R cells chronically propagated with TPA are resistant to the inhibitory effect of TPA. PKC study Table 2 shows results of PKC determinations in glioma cells. The original BT5C cell line displayed PKC activity in membranous and cytosolic fractions. When these cells were treated for 4 days with TPA they show very
Table 2. PKC activity (pmol 32P/min per mg protein) in cytosol and membrane fractions of BT5C cells and their TPA resistant clone-BT5C-R.
BT5C BTSC zt TPA BT5C-R
Membranes
Cytosol
375.2 zt 31.9 8.3 zt 4.1 1.9 l 0.9
415.0 34.2 27.0
f f f
19.8 17.1 13.0
Mean from 5 experiments =t S.D. Cells were treated for 4 days with TPA (200 ng/ml medium).
of
low levels of PKC activity in their membranes and cytosol. The membrane fraction of the TPA resistant line (BT5C-R) had barely detectable PKC activity. These results confirm that resistance to TPA treatment is associated with depletion of PKC from the membrane fraction. Furthermore, the resistant BT5C-R cell line can grow independently of protein kinase C. Migration study Table 3 shows the results of 4 days of TPA treatment on the migration of BT5C cells and their TPA resistant line. The most interesting finding was that cells, depleted of PKC by either prolonged cultivation with TPA or by 4 days treatment with TPA migrated better then control untreated parental BT5C cells. As could be expected, TPA treatment of the resistant BT5C-R line had no effect on migration. Discussion
Table 1. Effect of TPA treatment on plating efficiency of BT5C cells and TPA resistant clone BT5C-R. TPA ng/ml of medium
BT5C BT5C-R
0
100
200
46.3 zt 2.7’ 47.8 f 3.3
40.4 f 5.2 49.1 f 2.9
27.4 f 6.3’ 51.5 jz 5.1
Mean of 10 dishes (Student’s t-test).
*
S.D.
‘Statistically
significant
It has been presented before that promoting phorbol esters like TPA change the shape of cells [3] and stimulate migration of brain endothelial cells [9]. Also staurosporin, a protein kinase C inhibitor reduced the rate of migration of bladder carcinoma cells [lo]. However, staurosporin is also an inhibitor of various kinases including CAMP and cGMP. TPA on one hand is a stimulator of PKC but on the other hand it also induces the down-regulation
169
Table 3.
Effect of TPA treatment
on directional
Area of migration TPA (ng/ml)
0 200
migration
BT5C and BT5C-R cells from spheroid.
(Fm*) BT5CR
BT5C Day 0
Day 4
Day 0
Day 4
9.2 n = 8.9 n =
653.6 f 10.3 n = 134 835.2 zt 22.4’ n = 36
9.4 f 3.1 n = 95 9.6 z+z2.6 n = 42
772.0
f
3.0 180 f 2.2 40
Mean f S.E.; n, number of spheroids used. *Statistically significant from TPA 0 at day 4 (Student’s
of the enzyme. Therefore, the distinction between activation and down-regulation of PKC is critical to the understanding of TPA action. The system of the migration of tumor cells from spheroids was chosen since it reminds in vivo situation where tumor ceils usual grow in form a spheroid both in primary or secondary places. Our study was performed following the addition of TPA to the medium. This treatment stimulated cells movement and also led to PKC down-regulation. Since proliferation of glioma BT5C cells was inhibited by TPA treatment, the increase in migration area was independent of cell multiplication and may have resulted from protein kinase C down regulation. In addition, BTSC-R cells, which lack PKC in their membranes, migrated better then non-treated parental glioma cells regardless of TPA presence in the medium. Altogether, it looks like PKC negatively correlates with migration of studied cells. This finding could be supported by the results showing that the higher level of phosphorylation of I-IMM by protein kinase C caused a progressive inhibition of actin-activated ATPase [4]. BT5C-R cells which were exposed to TPA for a prolonged period of more than 20 passages lost their sensitivity to growth inhibition mediated by TPA. It could be only speculated that the role of PKC was overridden by different kinases.
zt 27.7’ 50 806.1 f 48.1’ n = 43 n =
t-test)
An-overall conclusion from these studies is that the lack of protein kinase C facilitates the migration of glioma cells. If this finding could be extended to other experimental models (e.g. multistage skin carcinogenesis) then the role of tumor promoting phorbol esters could be considered, which by inducing PKC downcreates a permissive event for regulation, vigorous migration. This in turn may allow a cell escape from environmental control and give rise to a tumor. References Bradford,
M.M.
quantitation
(1976):
A rapid sensitive method for the
of micrograms
quantities of protein
the principle of protein day binding. Anal.
utilizing
Biochem..
72,
248 - 254. Catagna,
Y, (1982);
activated phospholipid or
Sane, K.,
M., Takai, Y., Kaibuchi, K.,
U. and Nishizuka, promoting
dependent
phorbol
Kikkawa,
Direct activation of calcium
ester.
protein kinase by tumJ.
Biol.
Chem.,
257,
7847 - 7851. Dugina V.B., I.M.
Svitkina T.M.,
(1987).
by phorbol
Special ester.
Vasiliev,
type
Proc.
of
Natl.
J.M.
and Gelfant,
reorganization Acad.
Sci.
induced
U.S.A.,
84.
4112-4125. Ikebe,
M..
Hartshorne,
Phosphorylation
of
D.J.
the
and
Elzinga.
20 000.dalton
M.
light
(1987): chain
of
smooth muscle myosine by the calcium activated phospholipid-dependent
protein
kinase.
J.
Biol.
Chem..
262,
9569 - 9574. Kraft, S.A.,
Anderson W.B.
(1983)
the amount of Ca, phospholipid associated 621-623.
with
plasma
Phorbol ester increase
dependent
membrane.
protein kinase Nature,
301,
170 6
7
8
9
10
Laerum, O.D. and Rajewski M.F. (1976) Neoplastic transformation of fetal rat brain cells in culture after exposure to ethylnitroso-urea. J. Natl. Cancer Inst., 55. 1177- 1187. Miioszewska, J., Trawicki, W., Jamik, P., Moraczewski, J., Przybyszewska, M. and Szaniawska B. (1986): Protein kinase C translocation in relation to proliferative status of C3H lOTU.2 cells. FEBS Lett., 206, 283-286. Nishinuka, Y. (1984): The role of protein kinase C in ceil surface signal transduction and tumor promotion. Nature, 308, 693 - 698 Rosen, E.M., Jaken, S., Carley, W., Lucket, P.M., Setter, E., Bhargava, M. and Goldberg, J.D. (1991) Regulation of motility in bovin brain endothelial cells. J. Cell Physiol., 146, 325 - 335. Swartz, G.K., Redwood,
S.M.,
Ohmuna,
T., Holland,
11
12
13
J.F.. Droler, M.J. and Lin, B.C.-S. (1990): Inhibition of invasion human bladder carcinoma cells by protein kinase inhibitor Staurosporine. J. Natl. Cancer Inst., 22, 1753 - 1757. Rodrigez-Pena. A. and Rozengurt, E., (1984) Disappearance of Ca sensitive, phospholipid-dependent protein kinase C activity in phorbol ester treated 3T3 cells. Biochem. Biophys. Res. Commun., 120, 1053- 1059 Szaniawska, B., Bjerkvig, R. and Laerum O.D. (1986): Growth arrest and polyploidisation induced by metahalone microtubule inhibitors on rat glioma cells in culture. Anticancer Res., 6. 71- 77. Yuhas, J.M., Li, A.P. Martinez, A.D. and Ladman, A.J. (1986): A simplified method for production and growth of multtcellular tumor spheroids. Cancer Res., 37, 3639 - 3643.
