Cytotoxicity of okadaic acid and kinetic characterization of protein tyrosine phosphatase activity in V79 fibroblasts

Article abstract of DOI:10.1211/146080800128736150

Protein phosphorylation and dephosphorylation mediate signal transduction events that control several important cellular processes. The aim of this work was to determine some kinetic properties of a protein phosphatase obtained from non-metabolizing V79 Chinese hamster cells. The effect of okadaic acid, a tumour promoter, on these cell cultures was also determined. Phosphatase activity was assayed in V79 cells which were lysed with 0.1 M imidazole buffer (pH 7.4). Enzyme activity was determined using p-nitrophenylphosphate and tyrosine phosphate (TyrP) as substrates. Maximum phosphatase activity was obtained at pH7·4. The apparent K(m) (Michael's constant) and specificity constant for TyrP were 0·06 nm and 57, respectively. Phosphatase activity was inhibited by 100μM pCMB (p-chloro-mercuribenzoate; 70%), 10 mM fluoride (30%), 10 mM phosphate (40%), 100 μM m-vanadate (80%) and 100 μM o-vanadate (90%). Tartrate (5 mM) had no effect. The low K(m) value for TyrP and the high level of inhibition by vanadate suggest that the V79 phosphatase is a protein tyrosine phosphatase. The cytotoxic effect of okadaic acid was evaluated and the IC50 was 15, 20, 35 and 45 nM for nucleic acid content, neutral red uptake, MTT and protein phosphatase assays, respectively. These results agree with the kinetic data, indicating that the V79 phosphatase is a protein tyrosine phosphatase, as enzyme activity was stimulated when the cells were treated with okadaic acid. Serine/threonine protein phosphatases are completely inhibited by okadaic acid. The phosphatase enzyme system may be useful for studying cellular adaptation to apoptosis, oxidative stress and other conditions.

Full text of DOI:10.1211/146080800128736150

Pharm. Pharmacol. Commun. 2000, 6: 331±334
Received April 4, 2000
Accepted June 6, 2000
# 2000 Pharm. Pharmacol. Commun.
Cytotoxicity of Okadaic Acid and Kinetic Characterization of
Protein Tyrosine Phosphatase Activity in V79 Fibroblasts
H. AOYAMA, P. S. MELO, P. A. GRANJEIRO, M. HAUN AND C. V. FERREIRA
Departamento de Bioqu Âõ mica, Instituto de Biologia, Universidade Estadual de Campinas, 13083-970
Campinas, S aÄ o Paulo, Brazil
Abstract
Protein phosphorylation and dephosphorylation mediate signal transduction events that
control several important cellular processes. The aim of this work was to determine some
kinetic properties of a protein phosphatase obtained from non-metabolizing V79 Chinese
hamster cells. The effect of okadaic acid, a tumour promoter, on these cell cultures was
also determined.
Phosphatase activity was assayed in V79 cells which were lysed with 0Á1 M imidazole
buffer (pH 7Á4). Enzyme activity was determined using p-nitrophenylphosphate and tyr-
osine phosphate (TyrP) as substrates. Maximum phosphatase activity was obtained at
pH 7Á4. The apparent K (Michael's constant) and speci®city constant for TyrP were
m
0
Á06 nM and 57, respectively. Phosphatase activity was inhibited by 100 mM pCMB (p-
chloro-mercuribenzoate; 70%), 10 mM ¯uoride (30%), 10 mM phosphate (40%), 100 mM m-
vanadate (80%) and 100 mM o-vanadate (90%). Tartrate (5 mM) had no effect. The low Km
value for TyrP and the high level of inhibition by vanadate suggest that the V79 phos-
phatase is a protein tyrosine phosphatase. The cytotoxic effect of okadaic acid was eval-
uated and the IC50 was 15, 20, 35 and 45 nM for nucleic acid content, neutral red uptake,
MTT and protein phosphatase assays, respectively. These results agree with the kinetic
data, indicating that the V79 phosphatase is a protein tyrosine phosphatase, as enzyme
activity was stimulated when the cells were treated with okadaic acid. Serine=threonine
protein phosphatases are completely inhibited by okadaic acid.
The phosphatase enzyme system may be useful for studying cellular adaptation to
apoptosis, oxidative stress and other conditions.
Protein tyrosine phosphatases (PTPases) are sig-
nalling molecules that act in concert with protein
tyrosine kinases to regulate a variety of funda-
mental cellular processes such as cell growth,
mitogenesis, metabolism, gene transcription, cell
cycle control and immune and stress responses
and the cytoplasmic forms, such as PTP1B (Fau-
man & Saper 1996; Jia 1997).
Protein phosphatases are classi®ed according to
their structure and inhibition by speci®c com-
pounds. Threonine and serine phosphatases are
inhibited by okadaic acid and tyrosine phosphatases
are inhibited by vanadate (Young et al 1993; Maier
et al 1995).
Cultured mammalian cells can be used to evalu-
ate the cytotoxicity of compounds with potential
therapeutic activity (Garthoff 1995). The endpoints
for these assays include cell lysosomal and mito-
chondrial membrane integrity, which provides
information about the susceptibility of cellular
organelles and compartments (Jones et al 1999;
Rodriguez & Haun 1999).
(Cohen 1988; Johnson & Barford 1993; Trow-
bridge 1991; Denu et al 1996). The activity of
protein kinases or phosphatases acting on a parti-
cular site are closely regulated (Jia 1997).
Tyrosine phosphorylation plays a critical role in
the signal transduction that controls many diverse
cellular processes. Protein tyrosine phosphatases
are divided into two groups: the receptor-like
forms, such as CD45 (leukocyte common antigen),
Correspondence: C. V. Ferreira, Departamento de Bioqu Âõ -
mica, Instituto de Biologia, Universidade Estadual de Campi-
nas, Cidade Universit a ria, 13083-970 Campinas, S aÄ o Paulo,
Brazil.
The trypan blue exclusion test allows evaluation
of the structural integrity of the cell membrane
(Loveland et al 1992). Neutral red uptake measures
lysosomal integrity by re¯ecting the ability
E-Mail: carmenv@unicamp.br

3
32
H. AOYAMA ET AL
of cells to incorporate dye into their lysosomes
Babich & Borefreund 1990, 1991; Forsby et al
991; Repetto & Sanz 1993). It can also serve as
added to each well to ®x the cells and extract the
neutral red incorporated into the lysosomes. The
plates were shaken for 20 min on a plate shaker and
the absorbance was measured at 540 nm (Riddell
et al 1986; Renzi et al 1993).
(
1
an indicator of cell membrane integrity (Bore-
freund & Puerner 1984; Olivier et al 1995). The
reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) assesses
mitochondrial function through the activity of
succinate dehydrogenase (Mosmann 1983; Scu-
diero et al 1988). The protein content or nucleic
acid content provides an index of the total cell
number based on a determination of the cellular
macromolecule content (Combrier et al 1990; Haun
et al 1992).
MTT assay
The medium containing okadaic acid was removed
� 1
and 1 mL MTT solution (1 mg mL culture med-
ium) was added to each well. After incubation for
ꢀ
4
h at 37 C, the medium was removed and the
formazan solubilized in 1 mL ethanol. The plate
was shaken for 20 min on a plate shaker and the
absorbance was measured at 570 nm (Mosmann
In this work we evaluated some kinetic para-
meters of protein phosphatase in V79 cells and the
cytotoxic effect of okadaic acid on these cells.
1983; Denizot & Lang 1986).
Nucleic acid content
The cell monolayer was solubilized with 1 mL
Materials and Methods
ꢀ
0
Á5 M NaOH at 37 C for 1 h. The absorbance of the
solution in each well was measured at 260 nm and
the results expressed as a percentage of the control
Materials
V79 Chinese hamster lung ®broblasts from a clone
M8) were generously supplied by Rog e rio Mene-
(
Cingi et al 1991).
(
ghini, University of S aÄ o Paulo, S aÄ o Paulo, Brazil.
All other chemicals were of the highest purity
available.
Protein phosphatase assay
The reaction mixture in a ®nal volume of 1 mL
contained 100 mM imidazole buffer at pH 7Á4, 5 mM
p-nitrophenylphosphate (pNPP) or tyrosine phos-
phate (TyrP) as substrate and cell extract enzyme.
Cell culture
V79 cells were routinely grown as monolayers in
Dulbecco's modi®ed Eagle's medium contain-
ꢀ
After 30 min incubation at 37 C, the reaction was
�
1
stopped by adding 1 mL 1 M NaOH. The amount of
pNP and Tyr produced were calculated using molar
extinction coef®cients of 18 000 M cm and
ing antibiotics (100 IU mL
penicillin, 100 mg
�
1
mL streptomycin) and supplemented with 10%
heat-inactivated foetal calf serum, in a 5% CO2
�
1
� 1
�
1
� 1
ꢀ
The cultures were routinely stained with Hoeschst
2411 M cm , respectively. One unit of enzyme
activity was de®ned as the amount of enzyme that
converted one mole substrate to product in one
minute.
humidi®ed atmosphere at 37 C (Renzi et al 1993).
3
3258, a ¯uorescent dye that binds speci®cally to
DNA (Chen 1977) to verify possible contamination
by mycoplasma. For the MTT, nucleic acid content
and neutral red uptake assays, 24-well tissue cul-
4
� 1
Effect of potential inhibitors
ture plates were inoculated with 3 6 10 cells mL
ꢀ
The assay conditions were as previously described
by Ferreira et al (1988), using pNPP as substrate
and cell extract enzyme. The potential inhibitors
were added to the assay medium and enzyme
activity determined. In the absence of inhibitors the
activity was considered to be 100%.
and incubated at 37 C for 48 h. The cells were then
incubated for 24 h with okadaic acid at concentra-
tions of 10±100 nM which produced 0±100% cell
death.
Neutral red uptake
After removing the medium from the plates, 1 mL
�
1
neutral red solution (50 mg mL culture medium)
was added to each well followed by incubation for
3
Results and Discussion
ꢀ
with phosphate-buffered saline containing calcium
h at 37 C. The cells were then washed rapidly
Protein phosphatase activity was maximum at
pH 7Á4. The V
(maximum velocity) and Km
max
and 1 mL 1% glacial acetic acid=50% ethanol was
(Michael's constant) values were calculated from

FIBROBLAST PROTEIN TYROSINE PHOSPHATASE AND OKADAIC ACID
333
Table 1. Effect of potential inhibitors on protein phosphatase
activity.
Rieber 1993), on V79 ®broblast viability. Cell
viability was evaluated by neutral red uptake, MTT
reduction, protein phosphatase activity and nucleic
acid content. Nucleic acid content is a useful assay
for quantifying the number of cells as DNA levels
can be measured by their absorbance at 260 nm,
Inhibitor
Concn
Relative activity (%)
m-Vanadate
o-Vanadate
Tartrate
Fluoride
Phosphate
pCMB
100 mM
100 mM
5 mM
10 mM
10 mM
100 mM
20Æ 3
13Á5Æ 1Á5
96Æ 6
�
1
with 50 mg mL DNA giving an absorbance of 1Á0.
The IC50 of okadaic acid was 15, 20, 35 and 45 nM
for nucleic acid content, neutral red uptake, MTT
and protein phosphatase assays, respectively (Fig-
ure 1). These results con®rmed kinetic data which
suggested that the phosphatase in V79 cells is a
protein tyrosine phosphatase because of the strong
inhibition by vanadate and the high af®nity for
TyrP. Protein phosphatase activity was not affected
when the ®broblasts were treated with okadaic acid.
Although the enzyme activity decreased with
increasing okadaic acid concentrations, this effect
was probably related to the signi®cant decrease in
cell numbers rather than to a direct effect on the
enzyme itself.
66Æ 6Á75
64Á5Æ 3
28Á5Æ 0Á75
Enzyme activity was assayed with p-nitrophenylphosphate
as substrate in the presence of the indicated concentrations of
inhibitors. Activity in the absence of inhibitors was considered
tmo e bn et s .1 00%. Values represent the meanÆ s.d. of three experi-
Lineweaver-Burk plots with varying concentrations
of substrate (0±10 mM pNPP and 0±1 mM TyrP).
The protein phosphatase had high speci®city
(
Vmax=K ) and af®nity constants for TyrP (57 and
m
0
Á06 mM, respectively), and was inhibited by
pCMB (p-chloro-mercuribenzoate), m-vanadate
and o-vanadate and to a lesser extent by ¯uoride
and phosphate (Table 1). Tartrate had no effect.
Vanadate is a potent inhibitor of phosphatase in-
vitro and acts as a phosphate analogue in the
transition state (Zhang et al 1997).
Acknowledgements
This study was supported by PADCT=FINEP,
FAPESP and CAPES.
Figure 1 shows the effect of okadaic acid, a
non-phorbol tumour promoter known to inhibit
serine=threonine protein phosphatase (Rieber &
References
Babich, H., Borefreund, E. (1990) Application of the neutral
red cytotoxicity assay to in vitro toxicology. ATLA 18:
1
29±144
Babich, H., Borefreund, E. (1991) Cytotoxicity and genotoxi-
city assays with cultured ®sh cells: a review. Toxicol. Vitr.
5
: 91±100
Borefreund, E., Puerner, J. A. (1984) A simple quantitative
procedure using monolayer cultures for cytotoxicity assays
(
HTD=NR 90). J. Tiss. Cult. Methods 9: 7±9
Chen, T. R. (1977) In situ detection of mycoplasma contam-
ination in cell cultures by ¯uorescent Hoescht 33258 stain.
Exp. Cell Res. 104: 255±262
Cingi, M. R., De Angelis, I., Fortunati, E., Reggiani,
D., Bianchi, V., Tiozzo, R., Zucco, F. (1991) Choice
and standardisation of test protocols in cytotoxicology:
a
multicenter approach. Toxicol. Vitr. 5: 119±
1
25
Cohen, P. (1998) Protein phosphorylation and hormone action.
Proc. R. Soc. Ser. B. 234: 115±144
Combrier, E., Metezeau, P., Ronot, X., Kiefer-Gachelin, H.,
Adolphe, M. (1990) Comparative assessment of in vitro
toxicity of xenobiotics using ¯ow cytometry and spectro-
photometry. Toxicol. Vitr. 4: 751±755
Denizot, F., Lang, R. (1986) Rapid colorimetric assay for cell
growth and survival. Modi®cations to the tetrazolium dye
procedure giving improved sensitivity and reliability.
J. Immunol. Methods 89: 271±277
Denu, J. M., Stuckey, J. A, Dixon, J. E. (1996) Form
and function in protein dephosphorylation. Cell 87: 361±
364
Figure 1. Effect of okadaic acid on cell viability in V79 cells.
Neutral red uptake (d); MTT reduction (m); nucleic acid
content (j); protein tyrosine phosphatase activity (!). Inhibi-
tion was expressed relative to normal cell viability (100%).
Each point represents the meanÆ s.d. of at least three experi-
ments run in quadruplicate.

3
34
H. AOYAMA ET AL
Fauman, E. B., Saper, M. A. (1996) Structure and function of the
protein tyrosine phosphatases. Trends Biochem. Sci. 21: 413±
Olivier, P., Testard, P., Marzin, D., Abbot, D. (1995) Effect of
high polyol concentrations on the neutral red absorption
assay and tetrazolium-MTT test of rat hepatocytes in pri-
mary culture. Toxicon. Vitr. 9: 133±138
Renzi, D., Valtolini, M., Forster, R. (1993) The evaluation
of a multi-endpoint cytotoxicity assay system. ATLA 21:
89±96
4
17
Ferreira, C. V., Granjeiro, J. M., Taga, E. M., Aoyama, H.
1988) Puri®cation and characterization of multiple forms of
soybean seed acid phosphatases. Plant Physiol. Biochem.
6: 487±494
(
3
Forsby, A., Andersson, M., Lewan, L., Sterner, O. (1991) The
cytotoxicity of 22 sesquiterpenoid unsaturated dialdehydes,
as determined by the neutral red absorption assay and by
protein determination. Toxicol. Vitr. 5: 9±14
Garthoff, B. (1995) Alternative testing drug research and devel-
opment ± the validation issue. Toxicol. Vitr. 9: 789±793
Haun, M., Pereia, M. F., Joyas, A., Campos, V., Filardi, L. D.
S., Castro, S. L., Dur a n, N. (1992) Bacterial Chemistry. VI.
Biological activities and cytotoxicity of 1,3-dihydro-2H-
Indol-zone derivatives. Biol. Res. 25: 21±25
Jia, Z. (1997) Protein phosphatases: structures and implica-
tions. Biochem. Cell Biol. 75: 17±26
Johnson, L. N., Barford, D. (1993) The effect of phosphoryla-
tion on the structure and function of proteins. Annu. Rev.
Biophys. Biomol. Struct. 22: 199±232
Jones, P. A., Bracher, M., Marenus, K., Kojima, H. (1999)
Performance of the neutral red uptake assay in the COLIPA
international validation study on alternatives to the rabbit
eye irritation test. Toxicol. Vitr. 13: 325±333
Loveland, B. E., Johns, T. G., Mackay, I. R., Vaillant, F.,
Wang, Z. X., Hertzog, P. J. (1992) Validation of the MTT
dye assay for enumeration of cells in proliferative and
antiproliferative assays. Biochem. Int. 27: 501±510
Maier, G. D., Wright, M. A, Lozano, Y., Djordjevic, A.,
Matthewa, J. P., Young, M. R. I. (1995) Regulation of
cytoskeletal organization in tumor cells by protein phospha-
tases 1A and 2A. Int. J. Cancer 61: 54±61
Repetto, G., Sanz, P. (1993) Neutral red uptake, cellular
growth and lysosomal function: in vitro effect of 24 metals.
ATLA 21: 501±507
Riddell, R. J., Clothier, R. H., Balls, M. (1986) An evaluation
of three in vitro cytotoxicity assays. Food Chem. Toxicol.
24: 469±471
Rieber, M., Rieber, M. S. (1993) Early inhibition of protein
phosphatases preferentially blocks phorbol ester-stimulated
mitogenic signalling in melanocytes: increase in speci®c
tyrosine phosphoproteins. Biochem. Biophys. Res. Com-
mun. 192: 483±491
Rodriguez, J. A., Haun, M. (1999) Cytotoxicity of trans-
dehydro-crotonin from Croton Cajucara on V779 cells
and rat hepatocytes. Plant Med. 65: 1±5
Scudiero, D. A., Shoemaker, R. H., Paull, K. D., Monks, A.,
Tierney, S., Nofziger, T. H., Currens, M. J., Seniff, D.,
Boyd, M. R. (1988) Evaluation of a soluble tetrazo-
lium=formazan assay for cell growth and drug sensitivity
in culture using human and other tumour cell lines. Cancer
Res. 48: 4827±4833
Trowbridge, I. S. (1991) A prototype for transmembrane
protein tyrosine phosphatases. J. Biol. Chem. 266: 23517±
23520
Young, M. R. I., Lozano, Y., Maier, G. D. (1993) Protein
phosphatase limit tumor mobility. Int. J. Cancer 55:
1036±1041
Zhang, M., Zhou, M., Van Etten, R. L., Stauffacher, C. V.
(1997) Crystal structure of bovine low molecular weight
phosphotyrosyl phosphatase complexed with the transition
state analog vanadate. Biochemistry 36: 15±23
Mosmann, T. (1983) Rapid colorimetric assay for cellular
growth and survival: application to proliferation and cyto-
toxicity assay. J. Immunol. Method. 65: 55±63