Inhibition of the MEK/ERK signaling pathway by the novel
antimetastatic agent NAMI-A down regulates c-
myc
gene expression
and endothelial cell proliferation
Gianfranco Pintus
1,2
, Bruna Tadolini
1,2
, Anna Maria Posadino
1,2
, Bastiano Sanna
1,2
, Marcella Debidda
1,2
,
Federico Bennardini
2,3
, Gianni Sava
4
and Carlo Ventura
1,2
1
Department of Biomedical Sciences, Division of Biochemistry, Laboratory of Cardiovascular Research,
2
Division of Cell Biology,
National Institute of Biostructures and Biosystems, and
3
Department of Drug Sciences, University of Sassari, Italy;
4
Callerio

elicited by PMA in serum-free cells. These results suggest
that inhibition of MEK/ERK signaling by NAMI-A may
have an important role in modulating c-myc gene expression
and ECV304 proliferation.
Keywords: ruthenium compound; signal transduction; gene
expression; cell proliferation; cancer.
Uncontrolled cell proliferation, as well as neoplastic growth,
consistently associate with the functional abrogation of
different intracellular checkpoint pathways. Among these,
the mitogen-activated protein kinase (MAPK)/extracellular
signal-regulated kinase (ERK) pathway has been proposed
to play a crucial role in the modulation of cellular process
such as proliferation, differentiation and development [1].
Interestingly, a MAPK-related pathway has also been
reported to be involved in neoplastic transformation [2], and
an increase in MAPK expression and activity reported in
carcinoma cells suggest that its overexpression may be of
critical relevance in the maintenance of tumor cell growth
[3]. The finding that the ERK signaling pathway is involved
in tumor cell migration and invasion [4,5], as well as in
tubular formation induced by insulin in human endothelial
cells [6], suggests a crucial role for the MAPK/ERK
pathway in the modulation of extracellular stimuli leading
to angiogenesis and metastatic growth. Within this context,
the c-myc protooncogene emerged as a major conductor
among the different genes involved in both primary and
metastatic tumor growth. This protooncogene is intimately
implicated in the control of cell proliferation and its
overexpression is detected in many tumor cell types [7].
Amplified c-myc expression has also been reported to

antitumoral or antimetastatic arsenal. NAMI-A (imidazo-
lium trans-imidazoledimethyl sulfoxide-tetrachlororuthe-
nate) is a new ruthenium-based compound active against
lung metastasis in vivo [13] and tumor cell invasion in vitro
[14]. So far, the molecular mechanisms by which this novel
ruthenium complex exerts its antimetastatic activity are
largely unknown.
In this study, we attempted at elucidating the molecular
target(s) and the possible mechanism(s) involved in
NAMI-A action, by assessing its effects on cell proliferation,
ERK1/2 activation and c-myc gene expression in ECV304, a
spontaneously transformed human endothelial cell line.
MATERIALS AND METHODS
Cell culture
ECV304 is a spontaneously transformed, immortal endo-
thelial cell line established from the vein of an apparently
normal human umbilical cord. This line displays high
proliferation rates along with the capability to induce tumor
in nude mice and has been proposed as a suitable model for
providing novel insights into the mechanisms governing
angiogenesis under both physiological and pathological
conditions [6,15]. ECV304 were provided by the European
Collection of Animal Cell Cultures (Salisbury, UK). Cells
were grown in medium M199 supplemented with 10% fetal
bovine serum (Life Technologies, Paisley, UK),
100 UÆmL
)1
penicillin, and 100 lgÆmL
)1
streptomycin

NaCl, 2.5 m
M
KCl, 8.5 m
M
NaH
2
PO
4
,1.5m
M
KH
2
PO
4
) pH 7.3, exposed to 5% trichloroacetic acid
(500 lL) for 5 min and then fixed in methanol (500 lL).
Finally, the cells were digested by the addition of 25
M
formic acid (500 lL). Each formic acid digest was trans-
ferred with one rinse of NaCl/P
i
(1 mL) to a scintillation vial
containing 3.5 mL of INSTA-GEL scintillation fluid (Pack-
ard instrument Co., Meriden, CT, USA), and radioactivity
was determined by liquid scintillation counting using a
Wallac 1215 RackBeta liquid scintillation counter (LKB
Instrument Inc., Gaithersburg, MD, USA) [16].
Immunoblot analysis
Serum-starved or growing ECV304 cells were treated as
described in the figure legends. Immunoblotting analysis

)1
leupeptin, and 10 l
M
pepstatin). The samples were sonicated for 10 s (Branson,
sonifer B-12, setting 3) and incubated at 4 °Cfor15min.
Lysates were then centrifuged at 10 000 g for 15 min (4 °C)
and analyzed for the protein content. Each sample was
added with Laemmli sample buffer and boiled for 4 min.
Equal amounts of sample protein (5–10 lg per lane) and
prestained molecular mass markers (Santa Cruz Biotech-
nology, Inc., Santa Cruz, CA, USA) were fractionated by
SDS/PAGE with a 12% acrylamide gel. Proteins were
transferred to nitrocellulose in 25 m
M
Tris/HCl, 192 m
M
glycine, and 10% methanol at 4 °C for 12–16 h at a
constant current of 50 mA or for 2 h at 300 mA with
similar results. Nitrocellulose membranes were incubated in
20 m
M
Tris/HCl, pH 7.6, 137 m
M
NaCl, 0.2% Tween 20
with 5% nonfat dried milk for 1 h, washed three-times in
Tween 20 (3, 3, 5 min) and incubated for 1 h with primary
antibody in Tween 20 containing 1% milk. Incubation was
performed at room temperature for nonphospho-antibodies
and overnight at 4 °C for phospho-specific antibodies.
Proteins of interest were detected using specific antibodies

M
sodium vanadate, 50 m
M
sodium fluoride,
20 m
M
2-glycerophosphate, 0.1 l
M
okadaic acid, 1 m
M
phenylmethanesulfonyl fluoride, 20 lgÆmL
)1
aprotinin,
5862 G. Pintus et al. (Eur. J. Biochem. 269) Ó FEBS 2002
50 lgÆmL
)1
leupeptin, and 10 l
M
pepstatin). After 15 min
on ice, insoluble material was removed by sedimentation for
20 min at 100 000 g, and ERK1/2 was recovered by
immunoprecipitation with anti-(ERK1/2) Ig [17]. Briefly,
each cell extract (400 lg) was mixed with 10 lLofanti-
(ERK1/2) Ig for 1 h, and then 30 lL of 50% protein A–
Sepharose in lysis buffer was added for an additional 1 h.
MAPK/ERK activity was estimated as previously described
[18], using the serine/threonine kinase SPA assay kit
(Amersham Pharmacia Biotech). The immune complex
was recovered by sedimentation for 5 min in a micro-
centrifuge, washed three times with 0.5 mL NaCl/P

Pharmacia Biotech). Samples were then incubated for 1 h at
37 °C, pelleted by centrifugation at 14 000 g and the
resulting pellet was transferred to a scintillation vial. The
radioactivity was determined by liquid scintillation
counting.
Determination of c-
myc
gene expression by RT-PCR
analysis
Serum-starved or growing ECV304 cells cultured in T25
culture flasks (Falcon, Oxnard, CA, USA), were treated as
described in figure legends. At the indicated time points,
total RNA was extracted, reverse transcribed and amplified
according to a previously described procedure [19]. Total
RNA (1 lg) from ECV304 cells was reverse-transcribed for
45 min at 37 °C. The reaction was performed in a solution
of 25 lL containing, 50 m
M
Tris/HCl pH 8.3, 75 m
M
KCl,
3m
M
MgCl
2
,10m
M
dithiothreitol, 0.2 m
M
of each dNTP,

and establishing the point at which exponential accumula-
tion plateaus. Using 30 PCR cycles, the products of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
and c-myc amplification were all within the linear phase of
the reaction. Indeed, similar conditions have been previ-
ously reported for semiquantitative analysis of gene expres-
sion [19]. The position of PCR fragments was evaluated by
comparisonwithaDNAmolecularmassmarker(Gibco
BRL). GAPDH mRNA was used for each sample as an
internal control for mRNA integrity and equal loading. The
levels of radioactivity incorporated into c-myc product were
normalized by comparison with the levels of radioactivity
incorporated into the GAPDH product from the same
sample. Specific primers directed against human sequences
for c-myc and GAPDH and PCR conditions were as
previously described [20].
Analysis of c-
myc
gene transcription by nuclear runoff
assay
To prepare ECV304 nuclei, cells were washed with ice-cold
NaCl/P
i
and lysed with 0.5% Nonidet P-40 solution [10 m
M
Tris HCl, 10 m
M
NaCl, 3 m
M
MgCl

(3000 CiÆmmol
)1
), followed by incubation at room tem-
perature for 15 min. DNA was digested by incubating the
transcription mixture for 5 min at room temperature in the
presence of 1 lL of 20 000 UÆmL
)1
RNase-free DNase.
Nuclear RNA was isolated by using the Trizol reagent
(Amersham Pharmacia Biotech, Buckinghamshire, UK),
followed by purification on RNAMATRIX
TM
(BIO 101,
inc. Vista, CA, USA). Radiolabeled nuclear RNA was then
subjected to a solution hybridization RNase protection
assay, as previously described [21]. Briefly, a 354-base pair
fragment amplified from the human genomic c-myc gene
[20] was inserted into pCRII-TOPO (Invitrogen Ltd,
Paisley, UK). Equal counts of
32
P-labeled RNA
( 5 · 10
6
c.p.m.) were hybridized for 12 h at 55 °Cin
the presence of an unlabeled antisense c-myc RNA probe
generated by transcription of the plasmid linearized with
BamHI. Samples were then incubated with a combination
of RNase A and T1 and exposed to proteinase K. The
protected fragments were recovered after phenol chloro-
form extraction and electrophoretically separated in a

48 and 72 h, indicating the capability of NAMI-A to
markedly affect cell proliferation. In these time course
experiments we utilized 100 l
M
NAMI-A since it proved to
be the most effective concentration in counteracting DNA
synthesis over a 1–200 l
M
range (Fig. 1B).
Further analysis of the inhibitory response elicited by
NAMI-A on ECV304 growth was accomplished by inves-
tigating the effect of NAMI-A on proliferating cell nuclear
antigen (PCNA), a cell growth-related protein. Growing
cells were exposed to the drug for the indicated times and
PCNA expression was evaluated by Western blotting
analysis using specific anti-PCNA Ig. The current experi-
mental data show that cell exposure to NAMI-A
time-dependently inhibited PCNA protein expression, as
compared to untreated cells This effect was detectable after
24 h of cell treatment and reached the maximal amplitude at
72 h (Fig. 1C).
NAMI-A inhibits both ERK1/2 activation and activity
The MAPK/ERK cascade, including ERK1/2, normally
promotes cell proliferation, as indicated by the strong
correlation between ERK1/2 activation and both DNA
synthesis and PCNA expression [22,23]. Here, we assessed
both ERK1/2 activation and activity in either serum- or
phorbol 12-myristate 13-acetate (PMA)- stimulated
ECV304 cells that had been exposed to NAMI-A. Cells
weretreatedwith100l

Using a serine/threonine kinase SPA assay kit, the
capability of NAMI-A to affect the ERK1/2 phosphoryla-
tion activity was also assessed. Consistent with immunoblot
analyses, a 15-min exposure to 100 l
M
NAMI-A signifi-
cantly but not completely inhibited serum-stimulated kinase
activity, which was still detected after 1 h of drug treatment.
In contrast, the exposure of ECV304 cells to 100 l
M
NAMI-A completely prevented the phorbol ester-induced
ERK1/2 activity at all times assessed (Fig. 4A). The effect
elicited by NAMI-A on both serum- and PMA-induced
ERK1/2 activity was dose-dependent and 100 l
M
was the
most effective concentration in down regulating ERK1/2
activity (Fig. 4B,C).
NAMI-A-induced ERK1/2 inhibition down regulates
c-
myc
gene expression
To investigate whether NAMI-A-induced inhibition of
ERK1/2 signaling could affect the expression of a cell
growth-related gene, we assessed c-myc gene expression in
growing cells treated either with NAMI-A or the MAPK/
ERK inhibitor PD98059. The addition of 100 l
M
NAMI-A
to the incubation medium significantly inhibited serum-

shows the dose–response effect of NAMI-A on ECV304
c-myc mRNA expression. As reported for DNA synthesis
and ERK1/2 activity, 100 l
M
NAMI-A resulted to be the
concentration most effective in inhibiting c-myc gene
expression (Fig. 5C). Furthermore, NAMI-A addition to
PD98059-treated cells failed to produce any additive
inhibition of the residual c-myc gene expression induced
by serum, suggesting the possibility that both NAMI-A and
PD98059 might have exerted their effect by following the
same signal transduction pathway (Fig. 5C).
We next investigated whether NAMI-A may also induce
c-myc down-regulation in serum-free cells exposed to PMA,
an experimental condition, which has been previously
shown to elicit a NAMI-A inhibitable increase in ERK1/2
activity. Figure 6A shows that in serum-free cells, 100 n
M
PMA increased c-myc gene expression. Such an effect was
evident at 1 h, peaked after 3 h of treatment, thereafter
progressively declined and returned to the control value at
12 h. PMA-induced increase in c-myc gene expression
Fig. 2. NAMI-A and PD98059 down regulate serum-induced ERK1/2
phosphorylation. (A) Growing cells were stimulated for the indicated
times with medium M199, containing 10% fetal bovine serum in the
absence (j) or presence (s)of100l
M
NAMI-A. * Significantly
different from fetal bovine serum. (B) Serum-starved ECV304 cells
were stimulated for the indicated times with medium M199 containing

PMA (B) in the presence (+) or absence (–) of 5 l
M
GF109203X. (C,D) Serum-starved ECV304 cells were pretreated for
1h with 100l
M
NAMI-A (+) and then stimulated for 1 h with
medium M199 containing 10% fetal bovine serum (C) or 100 n
M
PMA (D) in the presence (+) or absence (–) of 100 l
M
NAMI-A. *
Significantly different from fetal bovine serum; §, significantly different
from PMA. The upper part of each panel shows representative auto-
radiograms corresponding to the immunoreactivity of ERK1/2 and
phospho ERK1/2 (A,B), and MEK1/2 and phospho MEK1/2 (C,D).
The lower part of each panel reports the quantitative analysis of
phospho ERK1/2 (A,B), and phospho MEK1/2 (C,D) immunodensity
expressed as percentage of control (individual densities from ERK1
and 2 bands were added up to generate one single ERK1/2 value).
Ó FEBS 2002 NAMI-A affects c-myc gene expression and MEK/ERK phosphorylation (Eur. J. Biochem. 269) 5865
occurred in a dose-dependent fashion, reaching the maximal
amplitude at 100 n
M
over a concentration range of
10–200 n
M
PMA (Fig. 6B). Both NAMI-A and PD98059
dose-dependently counteracted and ultimately abolished the
increase in c-myc mRNA expression elicited by PMA
(Fig. 6C,D), suggesting a close relationship between the

NAMI-A. (B) Growing cells were stimulated with medium M199
containing 10% fetal bovine serum in the absence (j) or presence (half
shaded square) of 50 l
M
PD98059. (C) Growing cells were stimulated
for 3 h with medium M199 containing 10% fetal bovine serum in the
absence (0 l
M
)orpresence(d) of the indicated concentration of
NAMI-A. Cells were also treated with 100 l
M
NAMI-A + 50 l
M
PD98059 for 3 h. The upper part of Panel A shows representative
ethidium bromide stained gels of the reaction products obtained using
5 lL of the RT products after 30 cycles of PCR amplification. The
lower part of panel A, and panels B, C report the expression of c-myc
mRNA levels detected by[
32
P]dCTP-PCR. Individual results were
normalizedtoGAPDHmRNAdetectedineachsampleandexpressed
as a ratio to GAPDH. *, Significantly different from fetal bovine serum.
Fig. 4. NAMI-A partially inhibits serum-induced ERK 1/2 activity but
completely counteracts the activity induced by PMA. (A) Serum-starved
ECV304 cells were left untreated (U) or pretreated for 1 h with 100 l
M
NAMI-A (N). Cells were then stimulated for the indicated times with
medium M199 containing either 10% fetal bovine serum (open bars)
or 100 n
M

both normal and pathological conditions [6,15]. A recent
investigation reports that ERK1/2 is constitutively active in
ECV304 cells and that such a condition plays a key role in
the alteration of the growth behavior of these cells [24].
Constitutively active components of the ERK pathway have
been shown to be associated with neoplastic growth [3] and
an increase in ERK-related signaling has been found to
represent a common outcome for a number of molecular
events leading to angiogenesis [6] and metastatic growth
[25,26]. These observations prompt the hypothesis that
pharmacological manipulation of the ERK pathway may
prove rewarding in counteracting such pathological process.
A number of extracellular stimuli, including serum, have
been reported to activate ERK via Ras/Raf/MEK/signaling
[27,28]. This activation is mediated, at least in part, by
G-protein coupled receptors (GPCRs), stimulation of
membrane phospholipid hydrolysis, and activation of the
diacylglycerol sensitive isoforms of protein kinase C (PKC)
[28]. Thus, phorbol esters such PMA also powerfully
activated the ERK cascade either by c-raf-mediated phos-
phorylation [29] or in a Ras-dependent manner [30]. The
Fig. 7. NAMI-A inhibits c-myc gene transcription and protein expres-
sion. (A) Nuclear run-off transcription assay. Growing cells were sti-
mulated for the indicated times with medium M199 containing 10%
(v/v) fetal bovine serum in the absence (–) or presence (+) of 100 l
M
NAMI-A and then processed as described in materials and methods.
Representative autoradiograms of the ribonuclease protection analysis
of c-myc mRNA are shown in panel A. Autoradiographic exposure
was for 2 days on Kodak X-Omat film with an intensifying screen.

The upper part of panels A and B, shows representative ethidium
bromide stained gels of the reaction products obtained using 5 lLof
the RT products after 30 cycles of PCR amplification. The lower part
of panels A, B and panels C, D report the expression of c-myc mRNA
levels detected by [
32
P]dCTP-PCR (30 cycles). Individual results were
normalized to GAPDH mRNA detected in each sample and expressed
as a ratio to GAPDH.
Ó FEBS 2002 NAMI-A affects c-myc gene expression and MEK/ERK phosphorylation (Eur. J. Biochem. 269) 5867
present results showing the ability of NAMI-A to com-
pletely abolish ERK1/2 activation elicited by PMA in
serum-free cells, clearly indicate the specificity of the drug in
inhibiting phorbol ester-generated signals leading to
ERK1/2 phosphorylation. Whereas, the finding that expo-
sure of serum-cultured cells to NAMI-A markedly,
although not completely, inhibited ERK1/2 phosphoryla-
tion indicates that the wide-scale profiling of intracellular
signals leading to ERK1/2 activation may have not been
entirely encompassed by the drug-treatment. Such a hypo-
thesis is further confirmed by the observation that, differ-
ently from the effect of serum, the stimulatory effect of
PMA on ERK1/2 activity could also be totally abolished by
NAMI-A. Within this context, the present observation that
the level of phospho-ERK1/2, as well as the peak increase in
ERK1/2 activity, was significantly lower in the presence of
PMA than in serum-stimulated cells suggests, as previously
reported [31,32], that multiple signaling mechanisms
occurred in ERK1/2 activation induced by serum. The
finding that PKC inhibition only partially down regulated

this new compound. The present observation that the
selective MEK/ERK inhibitor PD98059 significantly
reduced c-myc mRNA level in serum-cultured cells indicates
that c-myc gene expression may be at least in part mediated
by the activation of this signal transduction pathway. In the
current study, a number of interrelated observations suggest
that the inhibitory effect elicited by NAMI-A and PD98059
on c-myc mRNA expression might have occurred through a
common intracellular pathway. First, both ERK1/2 activa-
tion and c-myc gene expression were markedly inhibited
following the exposure of serum-feeded cells to NAMI-A.
Second, both the MEK/ERK inhibitor PD98059 and
NAMI-A inhibited c-myc gene expression with a similar
time-course and by superimposable magnitudes. Third,
NAMI-A failed to further decrease c-myc mRNA expres-
sion in PD98059-treated, serum-cultured cells. Since both
PD98059 and NAMI-A could not completely inhibit c-myc
gene expression in serum-cultured cells, it is possible that
protooncogene expression may also have been induced by
serum through signal(s) unrelated to ERK1/2.
The finding that, in serum-free cells PD98059 completely
suppressed PMA-induced c-myc mRNA expression,
strongly indicates a crucial role of the Ras/Raf/MEK/
ERK pathway in modulating phorbol ester-activated
mechanism(s) leading to c-myc gene expression. Consistent
with the present results is the finding that the c-myc gene
promoter contains conserved binding sites for the Ets family
[34] and that both phorbol ester and Ras are able to induce
expression from Ap-1/Ets-driven promoters via the
upstream MEK/ERK effector Raf [35]. The finding that

subject for future investigations. Nevertheless, the cellular
targets and signaling mechanisms uncovered in the present
study are associated with the modulation of critical growth
regulatory decisions in different cell types. Moreover,
growing evidence supports the view that unplugging the
currently dissected pathways from regulatory control may
associate with abnormalities in the architectural plans of cell
growth and differentiation, ultimately ensuing in the
appearance of a malignant phenotype [5,11,25,26]. These
findings, along with the recent observation that NAMI-A
inhibited tumor cell invasion of matrigel [14], strongly
suggest that the down regulation of ERK1/2 currently
elicited by NAMI-A may be a part of the molecular
mechanism by which this drug exerts its antimetastatic
activity in vivo. Such a view may be further inferred by the
following observations. The NAMI-induced inhibition of
5868 G. Pintus et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ERK1/2 activation and activity was attained in a trans-
formed endothelial cell line and it is now evident that the
endothelial cell plays a crucial role in angiogenesis [39] and
that it might affect the growth and differentiation of
neighboring cells [40]. The endothelial cell is also the target
cell for tumor neovascularization, a process that closely
associates with tumor growth and metastasis [12], and in
this context, NAMI-A has been shown to inhibit angiogen-
esis [41] and to induce apoptosis in endothelial cells [42]. In
conclusion, the inhibitory effects of NAMI-A on ERK1/2
activation and c-myc gene expression observed here may
represent a prominent feature in the control of pathological
processes of the blood vessel wall and in the modulation of

Extracellular signal-regulated kinase (ERK) signaling pathway
and MCF-7 cell migration by a mechanism that requires focal
adhesion kinase Src, and Shc. Rapid dissociation of GRB2/
Sps-Shc complex is associated with the transient phosphorylation
of ERK in urokinase-treated cells. J. Biol. Chem. 275, 19382–
19388.
5. Kobayashi, H., Suzuki, M., Tanaka, Y., Hirashima, Y. & Terao,
T. (2001) Suppression of urokinase expression and Invasiveness by
urinary trypsin inhibitor is mediated through inhibition of protein
kinase C- and MEK/ERK/c-Jun – dependent signaling pathways.
J. Biol. Chem. 276, 2015–2022.
6. Shigematsu, S., Yamauchi, K., Nakajima, K., Iijima, S., Aizawa,
T. & Hashizume, K.D. (1999) Glucose and insulin stimulate
migration and tubular formation of human endothelial cells
in vitro. Am.J.Physiol.40, E433–E438.
7. Spencer, C.A. & Groudine, M. (1991) Control of c-myc regulation
in normal and neoplastic cells. Adv. Cancer Res. 56, 1–48.
8. Bover, L., Barrio, M., Bravo, A.I., Slavutsky, I., Larripa, I.,
Bolondi, A., Ayala, M. & Mordoh, J. (1998) The human breast
cancer cell line IIB-BR-G has amplified c-myc and c-fos oncogenes
in vitro and is spontaneously metastatic in vivo. Cell. Mol. Biol. 44,
493–504.
9. Pavelic, Z.P., Pavelic, L., Lower, E.E., Gapany, M., Gapany, S.,
Barker, E.A. & Preisler, H.D. (1992) c-myc, c-erbB-2, and Ki-67
expression in normal breast tissue and in invasive and noninvasive
breast carcinoma. Cancer Res. 52, 2597–2602.
10. Kerkhoff, E., Houben, R., Loffler, S., Troppmair, J., Lee, J.E. &
Rapp, U.R. (1998) Regulation of c-myc expression by Ras/Raf
signaling. Oncogene 16, 211–216.
11. Welch, D.R., Sakamaki, T., Pioquinto, R., Leonard, T.O.,

alpha-induced low density lipoprotein receptor expression in
HepG2 cells. J. Biol. Chem. 273, 15742–15748.
18. McDonald, O.B., Chen, W.J., Ellis, B., Hoffman, C., Overton, L.,
Rink, M., Smith, A., Marshall, C.J. & Wood, E.R. (1999) A
scintillation proximity assay for the Raf/MEK/ERK kinase cas-
cade: high-throughput screening and identification of selective
enzyme inhibitors. Anal. Biochem. 268, 318–329.
19. Pintus, G., Tadolini, B., Maioli, M., Posadino, A.M., Gaspa, L. &
Ventura, C. (1999) Heparin down-regulates the phorbol ester-
induced protein kinase C gene expression in human endothelial
cells: enzyme-mediated autoregulation of protein kinase C-alpha
and -delta genes. FEBS Lett. 449, 135–140.
20. Hsieh, S.C., Huang, M.H., Tsai, C.Y., Tsai, Y.Y., Tsai, S.T. &
Sun, K.H., Yu, H.S., Han, S.H. & C.L. (1997) The expression of
genes modulating programmed cell death in normal human
polymorphonuclear neutrophils. Biochem. Biophys. Res. Commun.
233, 700–706.
21. Ventura,C.,Pintus,G.,Fiori,M.G.,Bennardini,F.,Pinna,G.&
Gaspa, L. (1997) Opioid peptide gene expression in the primary
hereditary cardiomyopathy of the Syrian hamster. I. Regulation of
prodynorphin gene expression by nuclear protein kinase C. J. Biol.
Chem. 272, 6685–6692.
22. Ishizuka, S., Yano, T., Hagiwara, K., Sone, M., Nihei, H., Ozasa,
H. & Horikawa, S. (1999) Extracellular signal-regulated kinase
mediates renal regeneration in rats with myoglobinuric acute renal
injury. Biochem. Biophys. Res. Commun. 254, 88–92.
23. Sergeant, N., Lyon, M., Rudland, P.S., Fernig, D.G. &
Delehedde, M. (2000) Stimulation of DNA synthesis and cell
proliferation of human mammary myoepithelial-like cells by
hepatocyte growth factor/scatter factor depends on heparan

30. Chiloeches, A., Paterson, H.F., Marais, R., Clerk, A., Marshall,
C.J. & Sugden, P.H. (1999) Regulation of Ras. GTP loading and
Ras–Raf association in neonatal rat ventricular myocytes by G
protein-coupled receptor agonists and phorbol ester. Activation of
the extracellular signal-regulated kinase cascade by phorbol ester
is mediated by Ras. J. Biol. Chem. 274, 19762–19770.
31. Han, X.B. & Conn, P.M. (1999) The role of protein kinases A and
C pathways in the regulation of mitogen-activated protein kinase
activation in response to gonadotropin-releasing hormone
receptor activation. Endocrinology 5, 2241–2251.
32. Lerosey,I.,Pizon,V.,Tavitian,A.&deGunzburg,J.(1991)The
cAMP-dependent protein kinase phosphorylates the rap1 protein
in vitro as well as in intact fibroblasts, but not the closely related
rap2 protein. Biochem. Biophys. Res. Commun. 175, 430–2436.
33. Luttrell, L.M., Daaka, Y. & Lefkowitz, R.J. (1999) Regulation of
tyrosine kinase cascades by G-protein-coupled receptors. Curr.
Opin. Cell. Biol. 11, 177–183.
34. Roussel, M.F., Davis, J.N., Cleveland, J.L., Ghysdael, J. &
Hiebert, S.W. (1994) Dual control of myc expression through a
single DNA binding site targeted by ets family proteins and E2F-1.
Oncogene. 2, 405–415.
35. Bruder, J.T., Heidecker, G. & Rapp, U.R. (1992) Serum-, TPA-,
and Ras-induced expression from Ap-1/Ets-driven promoters
requires Raf-1 kinase. Genes Dev. 6, 545–556.
36. Lang, J.C., Whitelaw, B., Talbot, S. & Wilkie, N.M. (1988)
Transcriptional regulation of the human c-myc gene. Br. J.
Cancer. 9, 62–66.
37. Leonetti, C., Biroccio, A., Candiloro, A., Citro, G., Fornari, C.,
Mottolese, M., Del Bufalo, D. & Zupi, G. (1999) Increase of cis-
platin sensitivity by c-myc antisense oligodeoxynucleotides in a