Báo cáo khoa học: Cleavage of focal adhesion proteins and PKCd during lovastatin-induced apoptosis in spontaneously immortalized rat brain neuroblasts ppt

Cleavage of focal adhesion proteins and PKCd during
lovastatin-induced apoptosis in spontaneously
immortalized rat brain neuroblasts
Lauro Gonza
´
lez-Ferna
´
ndez
1,
*, Maria Isabel Cerezo-Guisado
2,
*, Sonja Langmesser
1
,
Maria Julia Bragado
2
, Maria Jesu
´
s Lorenzo
2
and Luis Jesu
´
s Garcı
´
a-Marı
´
n
1
1 Departamento de Fisiologı
´
a and 2 Departamento de Bioquı

´
mica y Biologı
´
a Molecular y Gene
´
tica, Facultad de Veterinaria, Universidad de
Extremadura, Ca
´
ceres, Spain
The mevalonate pathway plays an important role in
cell growth and survival [1,2]. Mevalonate is intracellu-
larly synthesized from 3-hydroxy-3-methylglutaryl
coenzyme A (HMG-CoA), and this process is cata-
lysed by HMG-CoA reductase, the rate-limiting
enzyme in this pathway [2]. Mevalonate metabolism
yields a series of isoprenoid compounds which are
incorporated into cholesterol, isopentenyl adenine,
prenylated proteins and other end products essential
for cell growth [2,3]. Competitive inhibitors of HMG-
CoA reductase (statins), such as lovastatin, compactin,
simvastatin and pravastatin, not only block the biosyn-
thesis of mevalonate but, in addition, inhibit the prolif-
eration and induce apoptosis of both normal and
tumor cells [4–8].
HMG-CoA reductase activity [9] and cholesterol
biosynthesis [10,11] are very high in the early phase of
ontogenetic development of the brain. These data indi-
cate that the mevalonate pathway is essential to ensure
normal growth, differentiation and maintenance of
neuronal tissues. In accordance with this, we have

shown that lovastatin induces the apoptosis of sponta-
neously immortalized rat brain neuroblasts. Lovastatin
effects were associated with both cell morphological
Keywords
apoptosis; caspases; lovastatin; neuroblasts;
proteolysis
Correspondence
L. J. Garcı
´
a-Marı
´
n, Departamento de
Fisiologı
´
a, Facultad de Veterinaria, Avda.
Universidad s ⁄ n, E-10071 Ca
´
ceres, Spain
Fax: +34 927 257110
Tel: +34 927 257000 Ext. 1327
E-mail:
*Note
These authors contributed equally to this
work
(Received 13 July 2005, revised 27
September 2005, accepted 18 October
2005)
doi:10.1111/j.1742-4658.2005.05023.x
We have previously shown that lovastatin induces apoptosis in spontane-
ously immortalized rat brain neuroblasts. Focal adhesion proteins and pro-

tein kinase Cd (PKCd) have been implicated in the regulation of apoptosis.
We found that lovastatin exposure induced focal adhesion kinase, Crk-asso-
ciated substrate (p130
Cas
), PKCd cleavage and caspase-3 activation in a con-
centration-dependent manner. Lovastatin effects were fully prevented by
mevalonate. The cleavage of p130
Cas
was almost completely inhibited by
z-DEVD-fmk, a specific caspase-3 inhibitor, and z-VAD-fmk, a broad spec-
trum caspase inhibitor, indicating that cleavage is mediated by caspase-3.
In contrast, the lovastatin-induced cleavage of PKCd was only blocked
by z-VAD-fmk suggesting that PKCd cleavage is caspase-dependent but
caspase-3-independent. Additionally, z-VAD-fmk partially prevented lovast-
atin-induced neuroblast apoptosis. The present data show that lovastatin
may induce neuroblast apoptosis by both caspase-dependent and independ-
ent pathways. These findings may suggest that the caspase-dependent com-
ponent leading to the neuroblast cell death is likely to involve the cleavage
of focal adhesion proteins and PKCd, which may be partially responsible
for some biochemical features of neuroblast apoptosis induced by
lovastatin.
Abbreviations
Ac-DEVD-AMC, Acetyl-Asp-Glu-Val-Asp-7-amido-4methyl coumarin; FAK, focal adhesion kinase; HMG-CoA, 3-hydroxy-3-methylglutaryl
coenzyme A; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide; PKCd, protein kinase Cd;p130
Cas
, Crk-associated
substrate; z-DEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl
ketone.
FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS 1
changes and a decrease in the level of prenylation of

Ras and RhoA proteins [12].
Rho small GTPases are important regulators of the
actin cytoskeleton and the morphological heterogeneity
of vertebrate cells [13,14]. Among the family members
of the Rho GTPases, RhoA is one of the most studied
and has been implicated in the formation of stress
fibres and focal adhesion complexes [15]. Focal adhe-
sion complexes are protein aggregates linking actin
filaments to the cytoplasmic domain of integrins.
Maintenance of cell–matrix contact is an important
cell survival factor and the loss of cell–matrix and cell–
cell contact is a characteristic feature of apoptosis [16].
Focal adhesion complexes consist of integrin proteins,
the tyrosine kinases Src and focal adhesion kinase
(FAK), actin-binding structural proteins, and the
adaptor proteins paxillin and Crk-associated substrate
(p130
Cas
) [17]. Among these, FAK, p130
Cas
and paxil-
lin tyrosine phosphorylation are closely associated with
the reorganization of the actin cytoskeleton [18,19]. In
addition, the transition from flat to round cell mor-
phology, which is a characteristic feature in cells
undergoing apoptosis, is accompanied by cytoskeletal
rearrangement and changes in focal adhesion proteins.
Indeed, proteolytic cleavage of FAK [20–25] and
p130
Cas

[22,26,27] by caspases in many cell types
undergoing apoptosis further suggests the important
role played by the association of focal adhesion pro-
teins, the cytoskeleton and the extracellular matrix in
the maintenance of cell morphology and survival.
Previous studies have shown that several signalling
molecules including protein kinases are also cleaved
by caspases during apoptosis, examples include p21
(CDKN1A)-activated kinase 2 (PAK2) [28], MAPK
kinase kinase 1 (MEKK-1) [29], MAPK kinase kinase
kinase (Raf-1), protein kinase B (Akt) [30,31] and pro-
tein kinase C (PKC) [32]. PKC is a family of ser-
ine ⁄ threonine protein kinases that has been recently
implicated in apoptosis in a variety of cell types [33–
35]. At least 11 isoforms of PKC have been identified
and can be classified into conventional (a, bI, bII and
c), novel (d, e, g, and h), atypical (f and k ⁄ i) and
novel ⁄ atypical (l) based upon their cofactor require-
ments. PKC isozymes are known to be activated by
proteolytic separation of the regulatory domain from
the catalytic domain. While the classical and atypical
PKC isozymes are associated with cell survival, the
novel PKC isozymes are proapoptotic in function
[33–37]. Notably, proteolytic cleavage and activation
of PKCd by caspases have been shown to represent an
important step in apoptosis induced by many stimuli
[32,36–43]. Furthermore, expression of the catalytic
domain of PKCd is sufficient to induce apoptosis in
various cell types, suggesting that PKCd may be an
important effector of apoptosis [32].

As it has been previously mentioned, lovastatin pro-
motes growth suppression and apoptosis in rat brain
neuroblasts [12]. The involvement of caspase activity in
the neuronal apoptosis induced by lovastatin as well as
the relevance of the structural integrity of kinases and
focal adhesion proteins after lovastatin treatment in
neuroblasts have not been studied. Therefore, the main
objective of this work was to investigate the intracellu-
lar effects of lovastatin treatment in rat brain neuro-
blasts, i.e., activation of caspases and the cleavage of
focal adhesion proteins and PKCd. The elucidation of
intracellular mechanisms affected by lovastatin will
contribute to gaining insight in the growth inhibition
and apoptosis induced by this statin in neuroblasts,
and additionally it will contribute to establish the role
of the mevalonate pathway in the physiological growth
and development of cells from the central nervous
system.
Results
Effect of lovastatin on the cleavage of focal
adhesion proteins
We have previously shown that lovastatin induces
apoptosis of rat brain neuroblasts; its effect being asso-
ciated with a change in cell morphology from a flat to
a round shape [12]. Recent findings on apoptosis indi-
cate that this morphological change is accompanied by
proteolytic cleavage of focal adhesion proteins. There-
fore, in the present work, we first examined the integ-
rity of FAK and p130
Cas

proteins by western blot in
our experimental conditions. Incubation of neuroblasts
with different lovastatin concentrations for 24 h caused
the cleavage of both FAK (Fig. 1A) and p130
Cas
(Fig. 1B) proteins in a concentration-dependent man-
ner. The cleavage induced by lovastatin was significant
at 10 lm for both proteins, with 58 ± 12% of intact
FAK and 36 ± 11% of intact p130
Cas
, respectively,
remaining at this concentration (expressed as percent-
age of noncleavaged proteins in untreated cells). Pro-
teolysis of FAK generated two main cleavage products
of about 100 and 80 kDa, whereas proteolysis of
p130
Cas
generated three cleavage products of about 80,
60 and 31 kDa. The appearance of FAK and p130
Cas
proteolytic products correlated well with the decrease
in the level of the respective full-length proteins. Under
the same experimental conditions, the level of the cyto-
skeletal protein actin remained constant (Fig. 1C), sug-
gesting that the effect of lovastatin on FAK and
p130
Cas
cleavage was specific for these proteins and
Caspase-dependent effects in neuroblast apoptosis L. Gonza
´

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´
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2 FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS
that an equal amount of proteins were loaded in each
lane.
We have shown that mevalonate prevents the mor-
phological and biochemical features of neuroblast
apoptosis induced by lovastatin [12]. Therefore, to test
the ability of mevalonate at preventing the effect of
lovastatin on FAK and p130
Cas
cleavage, neuroblasts
were incubated with 10 lm lovastatin in the absence or
presence of mevalonate (100 lm) for 24 h. As shown
in Fig. 2, mevalonate completely prevented the clea-
vage of both FAK (106 ± 12% of intact FAK) and
p130
Cas
(91 ± 11% of intact p130
Cas
) proteins. Meval-
onate alone had no effect on the integrity of focal
adhesion proteins (101 ± 11% of intact FAK and
96 ± 5% of intact p130
Cas
). Actin levels remained
constant (Fig. 2C).
Effect of lovastatin on the cleavage of PKCd
Previous studies have shown that PKCd is cleaved into

a 41 kDa catalytically active and a 38 kDa regulatory
fragment in cells undergoing apoptosis. Furthermore,
these studies indicated that proteolytic activation of
PKCd contributes to phenotypic changes associated
with apoptosis [32]. Therefore, we next studied if
PKCd cleavage also occurs during lovastatin-induced
neuroblast apoptosis. As shown in Fig. 3A (upper
panel), lovastatin did not appear to modify the expres-
sion of full-length PKCd but induced the generation of
a cleavage product of about 42 kDa in a concentra-
tion-dependent manner. Actin levels did not change
after lovastatin treatment (Fig. 3A, lower panel). In
order to know whether this effect was specific of
HMG-CoA reductase inhibition, cells were incubated
with lovastatin (10 lm) in the presence or absence of
mevalonate (100 lm) for 24 h. Cotreatment with me-
valonate prevented the cleavage of PKCd induced by
lovastatin (Fig. 3B, upper panel). Treatment of cells
with mevalonate alone did not modify PKCd expres-
sion. Actin levels did not change under the same
experimental conditions (Fig. 3B, lower panel).
Effect of lovastatin on caspase-3 activation
Previous studies have shown that proteolytic cleavage
of focal adhesion proteins and PKCd is mediated
by caspases, mainly by caspase-3 [20,25,32,37,44]. To
determine whether these proteins are cleaved by
caspase-3 under our experimental conditions, we first
examined the effect of lovastatin on caspase-3 activa-
tion.
Caspase-3 is expressed as a 32 kDa proenzyme,

which is activated by proteolytic cleavage into an active
12–17 kDa form. To determine whether lovastatin
induced the activation of caspase-3, neuroblasts were
exposed to different amounts of lovastatin for 24 h and
the appearance of the activated form of caspase-3
A
B
C
Fig. 1. Effect of lovastatin on FAK and p130
Cas
proteins in immor-
talized rat brain neuroblasts. Cells initially cultured for 24 h in Ham’s
F12 ⁄ 10% fetal bovine serum were incubated for an additional 24 h
in medium alone (0) or in the presence of different concentrations
of lovastatin. At the end of the experiment, cells were lysed and
total proteins (20 lg per lane) were subjected to SDS ⁄ PAGE and
western blotting using specific antibodies against FAK (A), p130
Cas
(B) and actin (C) as described under Experimental procedures.
Molecular mass (kDa) is indicated by lines on the left. Full-length
proteins and cleavage fragments of proteins are indicated by
arrows on the right. Each blot is representative of three independ-
ent experiments.
L. Gonza
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FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS 3
was evaluated by western blot. As seen in Fig. 4A,

lovastatin increased the amount of the activated form
of caspase-3 in a concentration-dependent manner.
A
B
Fig. 3. Effect of lovastatin alone or in combination with mevalonate
on PKCd in immortalized rat brain neuroblasts. Cells previously cul-
tured for 24 h in growth medium were incubated for an additional
24 h with different concentrations of lovastatin (A) or with 10 l
M
lovastatin in the absence or presence of 100 lM mevalonate (B).
Control cells received medium or mevalonate alone. Proteins from
cell lysates (20 lg) were separated by SDS ⁄ PAGE and analyzed by
western Blotting using anti-PKCd (upper panel) and anti-actin (lower
panel) as described. Molecular mass (kDa) is indicated by lines on
the left. Full-length PKCd and a cleavage fragment are indicated by
arrows on the right. Each blot is representative of three independ-
ent experiments.
A
B
C
Fig. 2. Mevalonate prevents FAK and p130
Cas
cleavage induced by
lovastatin in immortalized rat brain neuroblasts. Cells initially cul-
tured for 24 h in Ham’s F12 ⁄ 10% fetal bovine serum were incu-
bated with lovastatin (10 l
M) in the absence or presence of
mevalonate (100 l
M) for an additional 24 h. Control cells were incu-
bated in presence of medium or mevalonate alone. At the end of

the experiment, cells were lysed and total proteins (20 lg per lane)
were subjected to SDS ⁄ PAGE and western blotting using specific
antibodies against FAK (A), p130
Cas
(B) and actin (C) as described.
Molecular mass (kDa) is indicated by lines on the left. Full-length
proteins and cleavage fragments of proteins are indicated by
arrows on the right. Each blot is representative of three independ-
ent experiments.
Caspase-dependent effects in neuroblast apoptosis L. Gonza
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4 FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS
Mevalonate (100 lm) abolished caspase-3 processing
induced by lovastatin, indicating that lovastatin effect
was specific (Fig. 4B). Actin levels did not change under
the same experimental conditions (Fig. 4A,B).
To verify the results obtained above, we measured
caspase-3 activity using the synthetic substrate
Ac-DEVD-AMC. Lysates containing active caspase-3
cleave the substrate releasing the fluorescent molecule
AMC, which is detected in a spectrofluorimeter with
excitation wavelength of 380 nm and emission wave-
length of 440 nm. As shown in Fig. 4C, lovastatin
(10 lm) markedly enhanced the activity of caspase-3.
Again, cotreatment with mevalonate prevented the
effect of lovastatin and completely restored caspase-3
activity to control levels. Mevalonate alone did not

affect caspase-3 activation (Fig. 4B,C).
Effect of caspase inhibition on lovastatin-induced
protein cleavage and neuronal apoptosis
Once we showed that lovastatin induced caspase-3 acti-
vation, we next investigated whether lovastatin-induced
proteolytic cleavage of focal adhesion proteins and
PKCd was mediated by caspase-3. Cells were incuba-
ted with lovastatin in the absence or presence of the
specific inhibitor of caspase-3, z-DEVD-fmk (50 lm)
or the broad spectrum caspase inhibitor, z-VAD-fmk
(50 lm). We previously confirmed that the specific
inhibitor z-DEVD-fmk completely blocked caspase-3
activity induced by lovastatin at 30 min (0.1 ± 0 vs.
2.1 ± 0.1 nmol product per 100 lg protein, respect-
ively). On the other hand z-VAD-fmk is able to block
the appearance of the active fragment of caspase-3
induced by lovastatin (data not shown). Both inhibi-
tors z-DEVD-fmk (Fig. 5A) and z-VAD-fmk (Fig. 5B)
partially prevented p130
Cas
cleavage induced by lovast-
atin by 67% and 74%, respectively. Regarding PKCd,
the specific caspase-3 inhibitor z-DEVD-fmk only
prevented its proteolytic cleavage by 10% (Fig. 5C),
but surprisingly z-VAD-fmk prevented it by 100%
(Fig. 5D). Both caspase inhibitors partially prevented
the cleavage of FAK induced by lovastatin (data not
shown).
Because z-VAD-fmk could inhibit lovastatin-induced
protein cleavage, the ability of this peptide to prevent

lovastatin-evoked apoptosis was assessed by three
independent assays, neuroblast viability, internucleo-
somal DNA fragmentation, and quantification of the
percentage of neuroblasts undergoing apoptosis, by
flow cytometry. Morphology of the cells was also stud-
ied by phase contrast microscopy. As we have shown
previously, the treatment of neuroblasts with 10 lm
lovastatin during 24 h caused a significant decrease in
A
B
C
Fig. 4. Effect of lovastatin alone or in combination with mevalonate
on caspase-3 activation in immortalized rat brain neuroblasts. Cells
previously cultured for 24 h in growth medium were incubated for
an additional 24 h with different concentrations of lovastatin (A) or
with 10 l
M lovastatin in the absence or presence of 100 lM meval-
onate (B and C). Control cells received medium or mevalonate
alone. At the end of the experiment, cell extracts were obtained
and used either to analyze, by western blot, the proteolytic activa-
tion of pro-caspase-3 (A and B, upper panel) or actin levels (A and
B, lower panel) or the activity of caspase-3 (C) as described. Each
blot is representative of three independent experiments (A and B).
Data are represented as mean ± SEM and representative of three
independent experiments performed in duplicate (C).
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FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS 5
cell viability (Fig. 6A), the appearance of internucleo-
somal DNA fragmentation (Fig. 6B) and an increase
in the percentage of apoptotic neuroblasts (Fig. 6C).
These lovastatin effects were partially blocked when
z-VAD-fmk (50 lm) was present in the medium. How-
ever, this general caspase inhibitor failed to prevent
lovastatin-induced cell shape changes (Fig. 6D–G), and
z-VAD-fmk alone had no effect, either on cell survival
or on morphology (Fig. 6).
Discussion
We have recently shown that lovastatin, a competitive
HMG-CoA reductase inhibitor, induces apoptosis of
spontaneously immortalized rat brain neuroblasts, its
effect being associated with cell morphological changes
and a decrease in the level of prenylation of RhoA pro-
tein [12]. These findings suggest that lovastatin may trig-
ger apoptosis of neuroblasts by inducing cytoskeletal
A
C
B
D
Fig. 5. Effect of caspase inhibitors on lovastatin-induced p130
Cas
and PKC d cleavage in immortalized rat brain neuroblasts. Cells previously
cultured for 24 h in growth medium were incubated with 10 l
M lovastatin in the absence or presence of 50 lM z-DEVD-fmk (A and C) or
50 l
M z-VAD-fmk (B and D) for an additional 24 h. Control cells received media, z-DEVD-fmk or z-VAD-fmk alone. At the end of the experi-
ment, cells were lysed and total proteins (20 lg per lane) were subjected to SDS ⁄ PAGE and western blotting using specific antibodies

against p130
Cas
(A and B), PKCd (C and D, upper panel) and actin (C and D, lower panel) as described. Molecular mass (kDa) is indicated by
lines on the left. Full-length proteins and cleavage fragments of proteins are indicated by arrows on the right. Each blot is representative of
three independent experiments.
Caspase-dependent effects in neuroblast apoptosis L. Gonza
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6 FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS
A
D
E
F
G
B
C
Fig. 6. Effect of lovastatin alone or in combination with z-VAD-fmk on cell viability (A), internucleosomal DNA fragmentation (B), percentage
of apoptotic cells (C), and cell morphology (D–G) in immortalized rat brain neuroblasts. Cells previously cultured for 24 h in growth medium
were incubated with 10 l
M lovastatin in the absence or presence of 50 lM z-VAD-fmk for an additional 24 h. Control cells were kept in med-
ium alone or supplemented w ith z-VAD-fmk only. At the end of the experiment, cell viability was determined by the MTT assay (A); inter-
nucleosomal DNA degradation was analyzed by using electrophoresis on 2% agarose gel (B); the percentage of cells with hypodiploid DNA
content was evaluated by flow cytometry (C). Each value represents the mean ± standard error of three independent experiments made in
triplicate. ns, Not significant; *, P < 0.05; ***, P < 0.001; compared to untreated cells (A and C). A representative photograph of three inde-
pendent experiments is shown in (B) where M represents 100 bp molecular mass markers. Morphological changes (D–G) were determined
by using phase contrast microscopy and photographs were taken (Magnification: 200·). (D) medium alone; (E) 10 l
M lovastatin; (F) 10 lM
lovastatin + 50 lM z-VAD-fmk; (G) 50 lM z-VAD-fmk alone.

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FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS 7
rearrangement as a consequence of changes in the
expression and ⁄ or activation of proteins that control
the organization of the cytoskeleton, such as focal adhe-
sion proteins and PKCd [20,23,26,33,35]. However, the
effect of lovastatin on the integrity of focal adhesion
proteins and PKCd has not been reported previously.
In the present work, we show for the first time that
lovastatin induces the cleavage of FAK and p130
Cas
in spontaneously immortalized rat brain neuroblasts
undergoing apoptosis. Lovastatin effects were concen-
tration dependent and completely prevented by simul-
taneous exposure of cells to exogenous mevalonate,
demonstrating that the effect of lovastatin on FAK
and p130
Cas
cleavage was due to its specific inhibitory
action on intracellular mevalonate synthesis. The clea-
vage of FAK and p130
Cas
in response to various apop-
totic inducers in different normal and tumour cell lines
has been reported previously [20,21,23–26,44–49],
which indicates that cleavage of these proteins may

play an important role in the execution of apoptosis.
FAK and p130
Cas
cleavage patterns induced by lovast-
atin are very similar to those seen in cells exposed to
other apoptotic stimuli, however, we could not detect
FAK cleavage products of about 40 kDa and 30 kDa
that have been described in some reports [21,44,46],
possibly due to the different cell type or antibody
used.
We have also shown for the first time that lovastatin
specifically induces the cleavage of PKCd in a concen-
tration-dependent manner, producing a fragment of
about 42 kDa that corresponds in size to the catalytic
domain of the kinase. Our result is in good agreement
with previous works that show that PKCd is cleaved
in various cell types undergoing apoptosis, including
neuronal cells [32,36,37,40,43], and suggests that
lovastatin may induce the activation of PKCd in rat
brain neuroblasts by its proteolytic cleavage. In differ-
ence to other reports, we show that the appearance of
the PKCd cleavage product is not accompanied by a
decrease in the full-length protein levels. These data
suggest that lovastatin may be inducing PKCd expres-
sion in neuroblasts. In agreement with this, Kaasinen
et al. [40] have shown that the expression of PKCd
mRNA is strikingly up-regulated during kainite-
induced apoptosis in the cortex and the CA1 and CA3
hippocampal regions.
Previous works indicate that the proteolytic cleavage

of focal adhesion proteins and PKCd is mediated by
members of the caspase family of cysteine proteases,
particularly by caspase-3 [20,25,32,37,44]. Therefore,
we first studied whether caspase-3 is activated under
our experimental conditions. We show that lovastatin
induces proteolytic activation of caspase-3 in a concen-
tration-dependent manner, and that mevalonate pre-
vents this effect. The ability of lovastatin to induce
caspase-3 activation has recently been documented in
various non-neuronal cell lines [50–53], but, to our
knowledge, this is the first report that demonstrates
that lovastatin is a potent inducer of caspase-3 activity
in neuronal cells. Fo
¨
cking et al. [54] previously showed
that lovastatin was not able to activate caspase-3 in a
murine hippocampal cell line. Taken together, these
results suggest that lovastatin-induced caspase-3 activa-
tion in neuronal cells may be cell type specific.
To examine the contribution of caspases to cleavage
of focal adhesion proteins and PKCd induced by lovast-
atin, pharmacological caspase inhibitors were employed.
Our results suggest that the cleavage of FAK (data not
shown) and p130
Cas
in rat brain neuroblasts is likely to
be mediated partially by caspase-3, other caspases and
also by other proteases. In this regard, calpain-induced
FAK and p130
Cas

proteolysis has been reported in cells
undergoing apoptosis [27,46]. Whether or not lovastatin
induces the activation of calpain in these cells is cur-
rently under study. On the other hand, we also show
that the cleavage of PKCd during lovastatin-induced
neuroblast apoptosis is completely dependent of caspase
activity. However, our results suggest that activation of
other caspases different than caspase-3 may be involved
in this event. This finding differs from those reported in
other apoptotic models, in which z-DEVD-fmk com-
pletely prevented PKCd cleavage [36,37,39,55,56], but is
in agreement with the fact that caspase-3 did not cleave
PKCd in the colon cancer line, COLO205 [57]. At pre-
sent, we do not know which caspase may be mediating
PKCd cleavage in our experimental conditions. How-
ever, the fact that lovastatin induces caspase-7 activa-
tion in the prostate cancer cell line, LNCaP [58], and
that the fluorogenic peptide substrate Ac-DEVD-AMC
is also a substrate for caspase-7 [59] suggests that the
cleavage of PKCd induced by lovastatin in rat brain
neuroblasts undergoing apoptosis may be mediated by
caspase-7.
Because caspase inhibition could block lovastatin-
induced cleavage of focal adhesion proteins and PKCd,
we investigated whether it also blocks lovastatin-
induced neuroblast morphological changes and apop-
tosis. Our results show that z-VAD-fmk partially
prevent the biochemical features of apoptosis but fail
to block the morphological changes induced by lovast-
atin at the same concentration that blocked PKCd

cleavage and almost completely inhibited both FAK
and p130
Cas
degradation. Our findings differ from
those of other studies in which the pan-caspase inhib-
itor prevents not only these caspase-mediated clea-
vage events but also the morphological changes and
Caspase-dependent effects in neuroblast apoptosis L. Gonza
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8 FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS
apoptosis induced by different agents in various cell
types [21,22,24,36,37,43,45]. Taken together, our data
allow us to speculate that the cleavage of FAK,
p130
Cas
and PKCd could be, at least partially, medi-
ating the biochemical features of apoptosis induced by
lovastatin. Therefore, our work suggests that other
proteins have to mediate the morphological changes
observed. On the other hand, the fact that z-VAD-fmk
partially prevents the apoptosis suggests that lovastatin
may induce neuroblast death by both caspase-depend-
ent and -independent pathways. Caspase-independent
apoptosis has been extensively described during normal
cell physiology [60,61].
In summary, we have demonstrated for the first time
that HMG-CoA reductase inhibition by lovastatin sti-

mulates caspase activity in rat brain neuroblasts, which
may help explain its apoptotic effect. In a parallel way,
our data showed that lovastatin induces proteolysis of
FAK, p130
Cas
and PKCd in these cells. Lovastatin
effects were concentration-dependent and prevented by
the HMG-CoA reductase product, which indicates that
lovastatin effects were due to an inhibition of mevalo-
nate synthesis. While a general caspase inhibition par-
tially prevented both proteolytic processes and the
biochemical parameters of apoptosis, inhibition of ca-
spase activity seems unrelated to the morphological
changes associated to this type of cell death. Therefore,
our results suggest that lovastatin may induce neuro-
blast apoptosis by both caspase-dependent and -inde-
pendent pathways. In addition, our data allow us to
speculate that the caspase-dependent component lead-
ing to neuroblast cell death may involve the cleavage
of focal adhesion proteins and PKCd, which may be
partially responsible for some biochemical features of
neuroblast apoptosis induced by lovastatin.
These findings might contribute to elucidation of the
molecular mechanisms of some statin effects described
in the central nervous system, such as growth suppres-
sion or induction of neuroblast apoptosis.
Experimental procedures
Reagents
Lovastatin (Mevinolin, MK-803) was from Calbiochem (La
Jolla, CA, USA). Mevalonic acid, 3-(4,5-dimethyl-2-thiazo-

lyl)-2,5-diphenyl-2H tetrazolium bromide (MTT), propi-
dium iodide, ribonuclease A, proteinase K and crystal
violet were purchased from Sigma Chemical Company (Sig-
ma-Aldrich, St. Louis, MO, USA). The caspase-3 substrate
(Ac-DEVD-AMC), the caspase-3 inhibitor (z-DEVD-fmk)
and the general caspase inhibitor (z-VAD-fmk) were from
PharMingen (BD Biosciences Europe, Brussels, Belgium).
Complete protease inhibitor cocktail tablets were from
Roche Molecular Biochemicals (Indianapolis, IN, USA).
Ham’s F-12 medium, fetal bovine serum, l-glutamine,
streptomycin, penicillin and trypsin ⁄ EDTA solution were
from PAN Biotech (Aidenbach, Germany). Tissue culture
flasks and dishes were from TPP (Trasadingen, Switzer-
land). Other reagents were obtained from different commer-
cial sources and were of the highest purity available.
Cell line and culture
Spontaneously immortalized rat brain neuroblasts were
used in this study. The cell line was obtained by sponta-
neous immortalization from cultures of 18 day old fetal rat
cerebral cortices and was kindly provided by A. Mun
˜
oz
(Instituto de Investigaciones Biome
´
dicas, CSIC, Madrid,
Spain). Cells were grown in Ham’s F-12 supplemented with
10% fetal bovine serum, l-glutamine (2 mm), streptomycin
(100 lgÆmL
)1
) and penicillin (100 UÆmL

)1
). Cells were
seeded at 5 · 10
5
in 75 cm
2
tissue culture flask in 10 mL of
culture medium and incubated at 37 °C under a 5%
CO
2
⁄ 95% air atmosphere. Cultures were passaged twice
weekly by trypsinization using a trypsin ⁄ EDTA solution.
Cell treatments
Confluent cells in 75 cm
2
tissue culture flasks were trypsi-
nized and seeded in tissue culture dishes at a concentration
of 2 · 10
4
cells per cm
2
. Twenty-four hours later, the med-
ium was aspirated and replaced with fresh medium alone
or containing the indicated concentrations of lovastatin,
mevalonate and caspase inhibitors and the incubation was
continued for a further 24 h. Cell morphology was analyzed
by phase contrast microscopy in a Leica DMIL inverted
microscope (Wetzlar, Germany). Photographs were taken
at the end of each experiment.
Western blot analysis

Cells cultured under different experimental conditions were
washed with NaCl ⁄ P
i
and lysed in Hepes buffer [50 mm
Hepes pH 7.4, 150 mm NaCl, 5 mm MgCl
2
,25mm NaF,
10 mm Na
4
P
2
O
7
, 10% glycerol, 1% Triton
`
X-100, 0.5 mm
Na
3
VO
4
and 1 tablet (50 mL) of complete protease inhib-
itor cocktail]. After centrifugation at 10 000 g for 15 min at
4 °C, protein concentration in each sample was determined
by using the Bio-Rad Protein Assay (Munich, Germany),
according to the instructions of the manufacturer. Equal
amounts of protein (20 lg) were subjected to electrophoret-
ic separation on denaturing 10% polyacrylamide gels, by
SDS ⁄ PAGE, and transferred to nitrocellulose membranes
(Protran, Schleicher and Schuell, Dassel, Germany). Mem-
branes were blocked in Blotto [50 mm Tris ⁄ HCl pH 8.0,

2mm CaCl
2
,80mm NaCl, 0.05% (v ⁄ v) Tween 20 and 5%
L. Gonza
´
lez-Ferna
´
ndez et al. Caspase-dependent effects in neuroblast apoptosis
FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS 9
(w ⁄ v) nonfat dry milk] and incubated with primary anti-
bodies. Incubation conditions were: mouse monoclonal
anti-FAK (Transduction Laboratories, BD Bioscience Eur-
ope) 1 : 1000 for 90 min at room temperature, mouse
monoclonal anti-p130
Cas
(Transduction Laboratories, BD
Bioscience Europe) 1 : 750 for 90 min at room temperature,
rabbit polyclonal anti-PKCd (Santa Cruz Biotechnology,
Santa Cruz, CA, USA) 0.25 lgÆmL
)1
for 120 min at room
temperature, and mouse monoclonal anti-caspase-3, active
form (17 kDa) (Cell Signalling, Beverly, MA, USA)
1 : 1000 for 90 at room temperature. Membranes were
washed twice with Blotto and incubated with the appropri-
ate horseradish peroxidase-conjugated secondary antibody
(anti-mouse IgG 1 : 6000 or anti-rabbit IgG 1 : 10000,
Pierce, Rockford, Il, USA) for 45 min at room tempera-
ture. After three washes (10 min each) with 50 mm
Tris ⁄ HCl pH 8.0, 2 mm CaCl

2
,80mm NaCl, membranes
were incubated with Super SignalÒ West Pico Chemilumi-
nescent Substrate (Pierce) for 5 min at room temperature
and exposed to Hyperfilm ECL (Amersham, Piscataway,
NJ, USA). Blots were stripped and reprobed with rabbit
anti-actin (Sigma) 1 : 5000 for 90 min at room temperature,
to verify equal loading of protein in all lanes.
Analysis of caspase-3 activity
Caspase-3 activity was measured using the synthetic sub-
strate Ac-DEVD-AMC. Cells exposed to different treatments
were washed with cold NaCl ⁄ P
i
and lysed at 4 °Cin10mm
Tris ⁄ HCl, 10 mm NaH
2
PO
4
, pH 7.5, 130 mm NaCl, 1%
Triton X-100 and 10 mm NaPP
i
. Lysates were clarified by
centrifugation at 10 000 g for 10 min at 4 °C. After measur-
ing protein concentration, aliquots of 50 lg protein were
diluted in reaction buffer (20 mm Hepes pH 7.5, 10% gly-
cerol, 2 mm dithiothreitol) and mixed with 20 lm caspase-3
substrate (Ac-DEVD-AMC). The reactions were incubated
at 37 °C for the indicated period of time and product forma-
tion was monitored in a spectrofluorometer using excitation
and emission wavelengths of 380 nm and 440 nm, respect-

ively. Fluorescence readings were calibrated using a solution
of commercial AMC (Sigma) of known concentration.
Cell viability assay
Cell viability was determined by the colorimetric MTT
assay as described previously [12]. At the end of each treat-
ment, cells were incubated for 15 min at 37 °C with MTT
(500 lgÆmL
)1
) and formazan precipitates were solubilized
with acidic isopropanol (0.04–0.1 m HCl in absolute isopro-
panol). The absorbance of converted dye was measured at
a wavelength of 570 nm. In some experiments, cell viability
was evaluated by the crystal violet method. At the end of
each experiment, medium was discarded and cells were
stained with crystal violet (0.03% in 2% ethanol) for
5 min. Subsequently, dishes were rinsed with tap water,
allowed to dry, and 1% SDS was added to solubilize the
dye. The absorbance of dye was measured at a wavelength
of 560 nm. Viable cells were calculated as percent of
absorbance with respect to untreated cells. Results using
both methods were similar.
DNA fragmentation assay
At the end of each experiment, cells were washed twice in
ice-cold NaCl ⁄ P
i
without Ca
2+
and Mg
2+
and then

scraped and pelleted at 4 ° C. Cells were lysed in Tris buffer
(10 mm Tris, pH 7.4, 5 mm EDTA, and 0.5% Triton
X-100) for 60 min at 4 °C. After centrifugation at 10 000 g
for 30 min at 4 °C, the supernatants were incubated with
RNase A (0.1 mgÆmL
)1
)at37°C for 45 min, and then with
proteinase K (0.2 mgÆmL
)1
)at37°C for 45 min. DNA was
then extracted twice with phenol ⁄ chloroform (1 : 1) and
precipitated with 0.1 volumes of sodium acetate (3 m) and
2.5 volumes of ice-cold ethanol at )80 °C overnight. The
precipitated DNA was collected by centrifugation at
10 000 g for 20 min and resuspended in autoclaved water.
DNA was resolved on 2% agarose ⁄ 0.1 lgÆmL
)1
ethidium
bromide gels in TBE buffer (80 mmolÆL
)1
Tris ⁄ borate,
2 mmolÆL
)1
EDTA, pH 8.0). After electrophoresis, gels
were examined under ultraviolet light and photographed
using a ChemiDoc System (Documentation and Analysis
System, Bio-Rad, Hercules, CA, USA).
Analysis of cell DNA content by flow cytometry
The ploidy determination of neuroblasts was estimated by
flow cytometry DNA analysis as described previously [12].

After treatment, cells were trypsinized, washed with
NaCl ⁄ P
i
, fixed at 4 °C in 70% ethanol and treated at 37 °C
with RNase (10 lgÆmL
)1
) for 30 min. The DNA content per
cell was evaluated in a Cyan flow cytometer (DAKO Cyto-
mation, Glostrup, Denmark) after staining the cells with
propidium iodide (50 lgÆmL
)1
) for 30 min at room tempera-
ture in the dark. For cell cycle analysis, only signals from
single cells were considered (10 000 events per sample).
Statistical analysis
Each experiment was repeated at least three times, with
good agreement among the results of individual experi-
ments. All data are expressed as the mean ± standard error
of the mean. Results were analyzed by one-way analysis of
variance (anova) followed by Student’s t-test. P-values of
less than 0.05 were considered significant.
Acknowledgements
The authors thank J. Ricardo Argent for his excellent
technical assistance and Dr Alberto Alvarez Barrientos
Caspase-dependent effects in neuroblast apoptosis L. Gonza
´
lez-Ferna
´
ndez et al.
10 FEBS Journal 273 (2006) 1–13 ª 2005 The Authors Journal Compilation ª 2005 FEBS

for his kind and valuable help on the flow cytometry
experiments. This work has been supported by Grants,
SAF 2001-0154 from the Ministerio de Ciencia y Tecn-
ologia, and 2PR01B007 from the Junta de Extrema-
dura, Spain. L. Gonza
´
lez-Ferna
´
ndez was supported by
a fellowship from Fundacio
´
n Fernando Valhondo
Calaff, Spain, M.I. Cerezo-Guisado is supported by a
doctoral fellowship from Junta de Extremadura, Spain
and S. Langmesser was supported by a fellowship from
the Studienstiftung des deutschen Volkes, Germany.
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