it consists of a pair of cylindrically shaped centrioles surrounded by fibrous pericentriolar material . before or during dna replication in s phase , the centrioles split , and each cylinder serves as a template for the assembly of a new 
daughter  centriole . before mitosis , when cells contain two pairs of centrioles , each pair serves as a nucleation center for microtubules of the spindle apparatus .
defects in centrosome assembly or in centrosome separation can result in defective nucleation of spindle microtubules , and in several cases , in the formation of monopolar spindles and mitotic arrest ( sunkel et al .
several years ago , evidence was published that defective centrosome assembly can prevent cells from entering s phase . in particular , removal of the centrosome by microsurgery or by
laser ablation resulted in a cell cycle arrest , as did inhibition or silencing of several centrosome - associated proteins , such as dynactin , parp-3 , centriolin , or akap450 ( hinchcliffe et al .
, 2001 ; khodjakov and rieder , 2001 ; quintyne and schroer , 2002 ; augustin et al . , 2003 ; gromley et al . , 2003 ; keryer et al
the mechanism leading to this centrosome - dependent cell cycle arrest in g1 phase has been unclear ; it was proposed that a checkpoint control would prevent those cells with imperfect centrosomes from continuing the cell cycle , to prevent the assembly of defective spindles later in mitosis ( murray , 2001 ) . in this study , we followed cell cycle progress after inhibition of centrosome assembly by depleting the pericentriolar proteins pericentriolar material 1 ( pcm-1 ) and pericentrin .
these proteins have been shown to be necessary for the assembly of other centrosomal constituents ( dictenberg et al . , 1998 ; dammermann and merdes , 2002 ; kubo and tsukita , 2003 ) .
we found that depletion of pcm-1 or pericentrin activates the p38-dependent stress pathway and the p53-dependent cell cycle checkpoint .
we have previously shown that depletion of the protein pcm-1 leads to defects in the assembly of the centrosomal components centrin , ninein , and pericentrin , and to an altered organization of the microtubule network in interphase cells ( dammermann and merdes , 2002 ) . to investigate the consequences of pcm-1 depletion on the cell cycle , we performed rna silencing experiments in primary human fibroblasts , mrc-5 .
depleted cells were tested for incorporation of brdu into the nucleus , as an indicator of dna synthesis ( fig .
we determined that in pcm-1depleted cells only 15  4% incorporated brdu , as compared with 35  3% in controls , as expected for a normal cycling population ( fig .
this is consistent with previous reports on microinjection of pcm-1inhibiting antibodies ( balczon et al . , 2002 ) and on centrosome removal by microsurgery or laser ablation , which prevent cells from entering s phase ( hinchcliffe et al .
several years ago , experiments on cells treated with the microtubule drugs colcemid , nocodazole , and taxol indicated that untransformed cells are arrested in g1 phase , when microtubules are depolymerized or when microtubule dynamics are altered ( trielli et al . , 1996 ; di leonardo et al . , 1997
this raises the question of whether dna replication in pcm-1depleted cells is inhibited because of an altered microtubule network , or whether defects at the centrosome itself suffice to induce a cell cycle arrest .
1 a ) , which in contrast to pcm-1 , only slightly reduces microtubule density but seems to have no significant effect on microtubule anchoring at the centrosome ( dammermann and merdes , 2002 ) . consistently ,
( a ) immunofluorescence of mrc-5 fibroblasts treated with control rna or sirna against pcm-1 and pericentrin , respectively .
graphs depict the percentages of cells lacking pcm-1 or pericentrin expression after treatment with control or silencing rnas .
cells were scored as negative if they lacked centrosomal staining of pcm-1 or pericentrin , only displaying diffuse background fluorescence .
( b ) percentages of cells incorporating brdu after treatment with control rna or silencing rna against pcm-1 or pericentrin .
data were obtained from cell cultures that were double labeled for brdu and pcm-1 or pericentrin . among cell cultures treated with silencing rna , only cells that were visibly depleted of pcm-1 or pericentrin were scored .
( a ) immunoblots of mrc-5 cells treated with control rna or sirna against pcm-1 , prb , or both pcm-1 and prb simultaneously .
blots were probed with antibodies against pcm-1 , cyclin a , the licensing factor mcm3 , the dna replication factor pcna , prb , and -tubulin .
( b ) immunoblots of cells treated with control rna or sirnas against pcm-1 , pericentrin , or both simultaneously .
( c ) mrc-5 cells treated with control rna , sirna against pcm-1 , or sirna against pcm-1 and prb simultaneously .
the percentages of cells staining positively for the proliferation marker ki-67 or for incorporated brdu are shown .
( d ) flow - cytometric analysis of mrc-5 cells treated with control rna or sirna against pcm-1 .
( e ) immunoblots of mrc-5 cells treated with control rna or sirna against pcm-1 for 48 or 120 h or with sirna against pericentrin for 72 h. blots were probed with antibodies against p53 , -tubulin , or p21 . because these data indicated that cells failed to undergo s phase 
dependent dna replication when missing the full complement of centrosome proteins , we wanted to test in more detail at what stage cells are arrested .
immunoblotting of cell extracts of pcm-1depleted cultures indicated drastic reduction of cyclin a ( fig .
2 a ) , which is normally found expressed in cells during late g1 , s , and g2 phases .
furthermore , we detected that pcm-1depleted cells also showed reduced amounts of the proteins minichromosome maintenance deficient 3 ( mcm3 ) and proliferating cell nuclear antigen ( pcna ) acting in licensing and dna replication ( fig .
2 a ) and reduced percentages of cells expressing the cell proliferation marker ki-67 ( fig .
altogether , these data indicated that depletion of centrosome proteins reduces the number of cells entering s phase .
we verified this hypothesis by comparing profiles of cell cultures analyzed by flow cytometry ( fig . 2 d ) : consistent with our data on brdu incorporation
we then wanted to determine whether s phase entry was blocked because of checkpoint activation in cells depleted of pcm-1 or pericentrin .
we found that the overall levels of the retinoblastoma protein ( prb ) were reduced to 38  17% and that most of the remaining prb , normally hyperphosphorylated during late g1 and s phase , was present in its faster migrating , hypophosphorylated form ( 77  13% in depleted vs. 38  13% in control cells ; fig .
however , was found up - regulated , especially after prolonged depletion of pcm-1 or pericentrin ( fig .
in depleted cells , the cdk2 inhibitor p21 was found equally up - regulated ( fig .
these data suggested that depletion of the two centrosome - associated proteins pcm-1 and pericentrin leads to the activation of the p53-dependent checkpoint . in the next step , we wanted to determine whether cell cycle progress would be affected if the p53-dependent checkpoint control was abrogated
for this purpose , we attempted simultaneous depletion of p53 and pcm-1 by cotransfecting sirna oligomers against both .
3 a shows that p53 levels could not be reduced when pcm-1 was missing . we tried to refine this experiment by sequential depletion of mrc-5 cells , by first depleting > 90% of p53 after 72 h , followed by simultaneous sirna treatment against p53 and pcm-1 .
however , we observed that under these conditions , p53 levels increased back to 4050% in three different experiments ( unpublished data ) .
we concluded that p53 turnover is altered and that the residual p53 protein might be stabilized in the absence of an intact centrosome .
we therefore changed our experimental strategy and compared cell cycle progress after pcm-1 depletion in several cell lines lacking p53 .
we used mouse embryonic fibroblasts from p53 knockout mice ( unpublished data ) as well as the human lung carcinoma cell line h1299 . because of the loss of p53 checkpoint control , both lines displayed a relatively high basic rate of dna synthesis ( fig .
we found that in both p53/ cell lines , pcm-1 depletion did not inhibit cell cycle progress .
, we also tested the effect of pcm-1 depletion in the p53+/+ and p53/ lines of hct116 cells .
brdu incorporation in these was reduced from 42% in controls to 33% in partially depleted p53 + cells , whereas p53 cells showed brdu incorporation in 49% after partial pcm-1 depletion . consistently
, hela cells with functionally suppressed p53 checkpoint control do not arrest in the absence of centrosomes ( la terra et al . ,
in contrast to p53 , the removal of prb did not cause resumption of the cell cycle in pcm-1depleted mrc-5 cells , because the percentages of brdu - incorporating cells and ki-67expressing cells remained low , as did the expression of mcm3 protein ( fig .
on the other hand , the amounts of cyclin a and pcna were restored to nearly control levels .
a possible scenario would be that centrosome defects activate both prb and p53 in parallel , with p53 having feedback effects on prb phosphorylation and prb protein levels but not vice versa .
this would be consistent with our observation that prb levels drop after pcm-1 or pericentrin depletion and that depletion of prb itself does not fully restore s phase activity , which is probably blocked because of checkpoint control mechanisms directly dependent on p53 .
eventually , loss of prb might be compensated by rb - related  pocket proteins ,  such as p107 or p130 .
cell cycle arrest after pcm-1 depletion depends on the checkpoint protein p53 . ( a ) immunoblots of mrc-5 cells treated with control rna or sirnas against pcm-1 , p53 , or both pcm-1 and p53 combined .
( b ) graph depicts brdu incorporation in p53/ h1299 cells treated with control rna or sirna against pcm-1 .
( c ) immunoblots of h1299 and mrc-5 cells treated with control rna or sirna against pcm-1 ( pcm1d ) for 72 h. blots were probed with antibodies against pcm-1 , p53 , pcna , mcm3 , prb , or -tubulin .
finally , we addressed the question of how defects in centrosome assembly could activate the p53-dependent checkpoint .
it has been reported that p38 mapk is involved in the cell 's response to a range of stress factors , such as uv irradiation , osmotic shock , heat shock , starvation , and cytokine treatment ( zarubin and han , 2005 ) .
the response to these stress factors is mediated partly through p53 phosphorylation , which is believed to stabilize p53 and to increase the expression of the cdk inhibitor p21 , thereby blocking the cell cycle ( agarwal et al . , 1998 ) .
to assess the involvement of stress - activated p38 mapk in response to centrosome defects , we have treated pcm - depleted mrc-5 cells with the p38 mapk  specific inhibitor sb203580 .
we found that the inhibitor prevented cell cycle arrest , as indicated by resumption of dna synthesis ( fig .
p38 inhibition in pcm-1depleted cells also restored regular levels of cyclin a and hyperphosphorylated prb , as well as expression of ki-67 and mcm3 ( fig .
further , the inhibition of p38 led to decreased levels of p21 but only slightly decreased p53 ( fig .
this could be explained by a stabilization of p53 after centrosome inactivation , as discussed in the previous paragraph , which might occur independently of p38-dependent p53 activation .
alternatively , the centrosome - dependent cell cycle arrest might not simply be a linear consequence of p53 activation via p38 .
for example , p53 might be activated by other kinases in addition to p38 . moreover , although p38 has been shown in multiple experiments to mediate cell cycle arrest by phosphorylating various sites of p53 ( bulavin et al .
, 1999 ; huang et al . , 1999 ) , p38 is also known to phosphorylate and thereby inactivate cdc25a and cyclin d , which in turn arrests the cell cycle ( lavoie et al . , 1996 ; casanovas et al . , 2000
however , p38 mapk activation in the absence of p53 seems to be insufficient for centrosome - dependent cell cycle arrest because pcm-1 depletion in p53/ cells did not stop the cell cycle .
( a ) immunoblots of mrc-5 cells treated with control rna , sirna against pcm-1 , or sirna against pcm-1 together with the p38 inhibitor sb203580 ( pcm-1 + inh ) .
blots were probed with antibodies against prb , cyclin a ( cyc a ) , p53 , the cdk inhibitor p21 , active p38 , or -tubulin .
( b ) graphs depicting mrc-5 cells treated with control rna , sirna against pcm-1 , or sirna against pcm-1 together with the p38 inhibitor sb203580 ( pcm-1 + inh ) .
the staining of brdu incorporation ( after rna treatment for 72 or 96 h ) , ki-67 immunofluorescence ( after rna treatment for 96 h ) , or mcm3 immunofluorescence ( after rna treatment for 72 h ) is shown . in our pcm-1depletion experiments , we observed a significant decrease in the expression of proteins associated with cell proliferation , such as mcm3 , pcna , or ki67 .
however , because about half of the depleted cells were still entering s phase , we wanted to test whether centrosome defects only slowed down cell cycle activity , or whether depleted cells were predisposed to a stable arrest that did not affect all cells simultaneously . immunofluorescence of p53 after pcm-1 depletion revealed that p53 levels varied between individual cells , with some cells showing a very high increase but a small increase or no obvious response in others ( fig . 5 a ) .
we then tested whether the cells ' response to centrosome inhibition induces exit from the cell cycle via senescence .
senescence is marked by the acquisition of a permanent g0 state and an increase of cellular -galactosidase activity ( dimri et al . , 1995 ) .
a cytochemical analysis of pcm-1depleted cultures revealed a 10-fold increase of -galactosidase  positive cells ( fig . 5 b ) , compared with a basal level of 3% in controls .
consistently , we previously reported that cultures of u2os cells died after silencing of pcm-1 for long periods ( dammermann and merdes , 2002 ) , although the experimental conditions varied from the protocol used here .
senescence has been characterized as a cellular program activated as a result of physiological stresses preventing further cell proliferation ( ben - porath and weinberg , 2004 ) . instead of a specific centrosome - dependent cell cycle control as proposed by murray ( 2001 ) ,
the idea of the centrosome as a center for stress - related signaling seems plausible , considering its central location in the focus of the microtubule network and its ability to bind various molecules of signaling pathways and of cell cycle regulation .
for example , significant amounts of protein kinase a , polo - like kinase , and protein phosphatases 1 and 2a , as well as cyclin e and p53 , have all been localized to the centrosome ( fry et al . , 2000 ; morris et al . , 2000 ;
is mediated by large coiled - coil proteins such as pericentrin , akap450 , or akap220 ( diviani et al .
, 2000 ; reinton et al . , 2000 ; diviani and scott , 2001 ) . absence or inhibition of anchoring proteins could therefore disrupt cellular signaling pathways and elicit a stress response
. further research will be necessary to investigate the multiple interactions between centrosome proteins , cell signaling , and the complex regulation of the cell cycle .
( a ) fibroblasts treated with control and pcm-1 sirna , stained for pcm-1 ( red ) , dna ( blue ) , and p53 ( green ) .
( b ) -galactosidase assay on control and pcm-1depleted cells 96 h after transfection using 5-bromo-4-chloro-3-indolyl -d - galactopyranoside as a chromogenic substrate .
human fetal lung fibroblasts mrc-5 ( european collection of cell cultures ) and h1299 cells ( a gift from b. vojtesek , masaryk memorial cancer institute , brno , czech republic ) were cultured in dme ( invitrogen ) , supplemented with 10% fetal bovine serum ( perbio science ) , 2 mm l - glutamine , and antibiotics .
hct116 cells ( a gift from b. vogelstein , johns hopkins university , baltimore , md ) were cultured in mccoy 's medium containing the same supplements .
about 3  10 cells from early passages of mrc-5 and 0.5  10 cells of h1299 or hct116 were transfected with 9 or 18 g of sirna oligonucleotides , respectively , in a nucleofector electroporation device , using the appropriate transfection kit ( amaxa ) .
the cells were then plated onto two 10-cm petri dishes to obtain a confluency of 3040% on the next day . routinely , sirna treatment was for a total of 72 h before fixation or preparation of cell extracts , unless indicated otherwise .
p38 mapk inhibition was performed in cells that were transfected with oligos and cultured overnight before the specific inhibitor sb203580 ( calbiochem ) was added at a concentration of 10 m .
inhibitor - treated cells were left in culture for another 48 h and then fixed in methanol or extracted for sds - page .
cells were harvested using trypsin - edta , counted , and washed in ice - cold pbs .
amounts of extract equal to 200,000 cells were loaded and run on 7.5 , 10 , or 12.5% sds - page gels and blotted onto nitrocellulose .
the following antibodies were used for western blot analysis or immunofluorescence : affinity - purified rabbit anti 
pcm-1 ( dammermann and merdes , 2002 ) , anti - mcm3 mab clone 3a2 ( mbl international corporation ) , anti - prb mab clone 4h1 ( cell signaling ) , anti - pcna mab clone pc-10 ( sigma - aldrich ) , anti  cyclin e mab clone he12 ( zymed laboratories ) , anti  cyclin a mab clone cy - a1 ( sigma - aldrich ) , anti p53 mab clone do-1 ( novocastra ) , anti - p21 mab clone sx118 ( bd biosciences ) , rabbit anti - activep38mapk polyclonal antibody ( promega ) , anti-tubulin mab clone dm1a ( sigma - aldrich ) , goat anti 
rabbit igg polyclonal antibody hrp ( ge healthcare ) , rat anti - brdu mab ( harlan ) , anti 
ki-67 mab clone mm1 ( novocastra ) , rabbit anti - pericentrin polyclonal antibody ( covance ) , donkey anti  rabbit or anti  mouse igg conjugated with alexa 488 or alexa 594 ( invitrogen ) .
quantification of protein levels was performed by scanning immunoblots and analyzed using the photoshop ( adobe ) histogram tool .
quantification of cells expressing specific proteins was performed by counting sirna - treated cells that were double stained with pcm-1 antibodies to verify depletion .
the following sirna oligomers with dtdt overhangs ( qiagen ) were used : pcm-1.2 , corresponding to human pcm-1 ( ucagcuucgugauucucag ) ; peric , corresponding to human pericentrin ( gcagcugagcugaaggaga ; dammermann and merdes , 2002 ) ; prb ( gcccuuacaaguuuccuag ) ; and p53 ( cuacuuccugaaaacaacg ) .
all cells in a culture dish were harvested by trypsinization , washed in ice - cold pbs , and fixed in 80% ice - cold ethanol in pbs . before staining , the cells were spun down in a cooled centrifuge and resuspended in the cold .
bovine pancreatic rnase ( sigma - aldrich ) was added at a final concentration of 2 g / ml , and cells were incubated at 37c for 30 min , followed by an incubation in 20 g / ml of propidium iodide ( sigma - aldrich ) for 20 min at room temperature .
cells grown on coverslips were fixed in ice - cold methanol and stored at 20c until use .
antibody staining was performed using the reagents listed in the previous paragraphs , according to standard protocols .
the percentage of cells in s phase was assessed by adding 100 m brdu ( sigma - aldrich ) to the cultures 30 min before fixation .
double labeling of brdu and pcm-1 was performed by probing first for pcm-1 , using rabbit anti 
pcm-1 and fluorescent anti - rabbit antibody , and coverslips were postfixed in pbs containing 3.7% paraformaldehyde , treated with 2 m hcl for 30 min , and stained with rat anti - brdu and fluorescent secondary anti - rat antibody .
cells were viewed with a fluorescence microscope ( axioskop 2 ; carl zeiss microimaging , inc . )
equipped with a camera ( axiocam ; carl zeiss microimaging , inc . ) and software ( axiovision ; carl zeiss microimaging , inc . ) .
human fetal lung fibroblasts mrc-5 ( european collection of cell cultures ) and h1299 cells ( a gift from b. vojtesek , masaryk memorial cancer institute , brno , czech republic ) were cultured in dme ( invitrogen ) , supplemented with 10% fetal bovine serum ( perbio science ) , 2 mm l - glutamine , and antibiotics .
hct116 cells ( a gift from b. vogelstein , johns hopkins university , baltimore , md ) were cultured in mccoy 's medium containing the same supplements .
about 3  10 cells from early passages of mrc-5 and 0.5  10 cells of h1299 or hct116 were transfected with 9 or 18 g of sirna oligonucleotides , respectively , in a nucleofector electroporation device , using the appropriate transfection kit ( amaxa ) .
the cells were then plated onto two 10-cm petri dishes to obtain a confluency of 3040% on the next day . routinely , sirna treatment was for a total of 72 h before fixation or preparation of cell extracts , unless indicated otherwise .
p38 mapk inhibition was performed in cells that were transfected with oligos and cultured overnight before the specific inhibitor sb203580 ( calbiochem ) was added at a concentration of 10 m .
inhibitor - treated cells were left in culture for another 48 h and then fixed in methanol or extracted for sds - page .
cells were harvested using trypsin - edta , counted , and washed in ice - cold pbs .
amounts of extract equal to 200,000 cells were loaded and run on 7.5 , 10 , or 12.5% sds - page gels and blotted onto nitrocellulose .
the following antibodies were used for western blot analysis or immunofluorescence : affinity - purified rabbit anti 
pcm-1 ( dammermann and merdes , 2002 ) , anti - mcm3 mab clone 3a2 ( mbl international corporation ) , anti - prb mab clone 4h1 ( cell signaling ) , anti - pcna mab clone pc-10 ( sigma - aldrich ) , anti  cyclin e mab clone he12 ( zymed laboratories ) , anti  cyclin a mab clone cy - a1 ( sigma - aldrich ) , anti p53 mab clone do-1 ( novocastra ) , anti - p21 mab clone sx118 ( bd biosciences ) , rabbit anti - activep38mapk polyclonal antibody ( promega ) , anti-tubulin mab clone dm1a ( sigma - aldrich ) , goat anti  mouse polyclonal antibody hrp ( promega ) , donkey anti  rabbit igg polyclonal antibody hrp ( ge healthcare ) , rat anti - brdu mab ( harlan ) , anti  ki-67 mab clone mm1 ( novocastra ) , rabbit anti - pericentrin polyclonal antibody ( covance ) , donkey anti  rabbit or anti  mouse igg conjugated with alexa 488 or alexa 594 ( invitrogen ) .
quantification of protein levels was performed by scanning immunoblots and analyzed using the photoshop ( adobe ) histogram tool .
quantification of cells expressing specific proteins was performed by counting sirna - treated cells that were double stained with pcm-1 antibodies to verify depletion .
the following sirna oligomers with dtdt overhangs ( qiagen ) were used : pcm-1.2 , corresponding to human pcm-1 ( ucagcuucgugauucucag ) ; peric , corresponding to human pericentrin ( gcagcugagcugaaggaga ; dammermann and merdes , 2002 ) ; prb ( gcccuuacaaguuuccuag ) ; and p53 ( cuacuuccugaaaacaacg ) .
all cells in a culture dish were harvested by trypsinization , washed in ice - cold pbs , and fixed in 80% ice - cold ethanol in pbs . before staining , the cells were spun down in a cooled centrifuge and resuspended in the cold . bovine pancreatic rnase ( sigma - aldrich )
was added at a final concentration of 2 g / ml , and cells were incubated at 37c for 30 min , followed by an incubation in 20 g / ml of propidium iodide ( sigma - aldrich ) for 20 min at room temperature .
cells grown on coverslips were fixed in ice - cold methanol and stored at 20c until use .
antibody staining was performed using the reagents listed in the previous paragraphs , according to standard protocols .
the percentage of cells in s phase was assessed by adding 100 m brdu ( sigma - aldrich ) to the cultures 30 min before fixation .
double labeling of brdu and pcm-1 was performed by probing first for pcm-1 , using rabbit anti 
pcm-1 and fluorescent anti - rabbit antibody , and coverslips were postfixed in pbs containing 3.7% paraformaldehyde , treated with 2 m hcl for 30 min , and stained with rat anti - brdu and fluorescent secondary anti - rat antibody .
cells were viewed with a fluorescence microscope ( axioskop 2 ; carl zeiss microimaging , inc . ) equipped with a camera ( axiocam ; carl zeiss microimaging , inc . ) and software ( axiovision ; carl zeiss microimaging , inc . ) .
 previous evidence has indicated that an intact centrosome is essential for cell cycle progress and that elimination of the centrosome or depletion of individual centrosome proteins prevents the entry into s phase . to investigate the molecular mechanisms of centrosome - dependent cell cycle progress , we performed rna silencing experiments of two centrosome - associated proteins , pericentriolar material 1 ( pcm-1 ) and pericentrin , in primary human fibroblasts . 
 we found that cells depleted of pcm-1 or pericentrin show lower levels of markers for s phase and cell proliferation , including cyclin a , ki-67 , proliferating cell nuclear antigen , minichromosome maintenance deficient 3 , and phosphorylated retinoblastoma protein . 
 also , the percentage of cells undergoing dna replication was reduced by > 50% . at the same time , levels of p53 and p21 increased in these cells , and cells were predisposed to undergo senescence . 
 conversely , depletion of centrosome proteins in cells lacking p53 did not cause any cell cycle arrest . 
 inhibition of p38 mitogen - activated protein kinase rescued cell cycle activity after centrosome protein depletion , indicating that p53 is activated by the p38 stress pathway . 
Introduction
Results and discussion
Materials and methods
Cell culture and transfection experiments
Antibodies and siRNA oligonucleotides
Flow cytometry analysis
-Galactosidase assay
Immunofluorescence microscopy