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

We found that lovastatin exposure induced focal adhesion kinase, Crk-asso-ciated substrate p130Cas, PKCd cleavage and caspase-3 activation in a con-centration-dependent manner.. These fin

lovastatin-induced apoptosis in spontaneously

immortalized rat brain neuroblasts

Lauro Gonza´lez-Ferna´ndez1,*, Maria Isabel Cerezo-Guisado2,*, Sonja Langmesser1,

Maria Julia Bragado2, Maria Jesu´s Lorenzo2and Luis Jesu´s Garcı´a-Marı´n1

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: lgarcia@unex.es

*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 (p130Cas), PKCd cleavage and caspase-3 activation in a con-centration-dependent manner Lovastatin effects were fully prevented by mevalonate The cleavage of p130Cas 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-dependindepend-ent 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; p130Cas, Crk-associated substrate; z-DEVD-fmk, benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone; z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.

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

(p130Cas) [17] Among these, FAK, p130Cas 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

p130Cas [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 p130Cas 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 p130Cas (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 p130Cas, respectively, remaining at this concentration (expressed as percent-age of noncleavpercent-aged proteins in untreated cells) Pro-teolysis of FAK generated two main cleavage products

of about 100 and 80 kDa, whereas proteolysis of p130Casgenerated three cleavage products of about 80,

60 and 31 kDa The appearance of FAK and p130Cas 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 p130Cas cleavage was specific for these proteins and

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 p130Cas 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 p130Cas(91 ± 11% of intact p130Cas) proteins Meval-onate alone had no effect on the integrity of focal adhesion proteins (101 ± 11% of intact FAK and

96 ± 5% of intact p130Cas) 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 experimindepend-ents.

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 l M 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 experimindepend-ents.

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 experimindepend-ents.

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 p130Cascleavage 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 l M

meval-onate (B and C) Control cells received medium or mevalmeval-onate

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).

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 PKCd 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 l M 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 p130Cas(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.

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 l M z-VAD-fmk for an additional 24 h Control cells were kept in med-ium alone or supplemented with 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 l M lovastatin + 50 l M z-VAD-fmk; (G) 50 l M z-VAD-fmk alone.

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 p130Cas

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 p130Cas cleavage was due to its specific inhibitory

action on intracellular mevalonate synthesis The

clea-vage of FAK and p130Casin 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 p130Cascleavage 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 p130Casin 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 p130Casproteolysis 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 p130Cas 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

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,

p130Cas 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 -independcaspase-depend-ent pathways Caspase-independcaspase-depend-ent

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, p130Cas 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· 105in 75 cm2tissue culture flask in 10 mL of culture medium and incubated at 37
C under a 5%

CO2⁄ 95% air atmosphere Cultures were passaged twice weekly by trypsinization using a trypsin⁄ EDTA solution

Cell treatments Confluent cells in 75 cm2 tissue culture flasks were trypsi-nized and seeded in tissue culture dishes at a concentration

of 2· 104cells per cm2 Twenty-four hours later, the med-ium was aspirated and replaced with fresh medmed-ium 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⁄ Pi and lysed in Hepes buffer [50 mm Hepes pH 7.4, 150 mm NaCl, 5 mm MgCl2, 25 mm NaF,

10 mm Na4P2O7, 10% glycerol, 1% Triton`X-100, 0.5 mm

Na3VO4 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,

2 mm CaCl2, 80 mm NaCl, 0.05% (v⁄ v) Tween 20 and 5%

(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-p130Cas (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-conjugappropri-ated 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 CaCl2, 80 mm NaCl, membranes

were incubated with Super Signal 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⁄ Piand lysed at 4
C in 10 mm

Tris⁄ HCl, 10 mm NaH2PO4, pH 7.5, 130 mm NaCl, 1%

Triton X-100 and 10 mm NaPPi 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 excitaforma-tion

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⁄ Pi without Ca2+ and Mg2+ 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) at 37
C for 45 min, and then with proteinase K (0.2 mgÆmL)1) at 37
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⁄ Pi, 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