the ink4a locus on human chromosome 9p21 encodes the p14arf tumor suppressor protein , which uses an upstream promoter and a shifted frame in exon 2 ( compared to the p16 protein ) .
the ink4a locus is often inactivated by deletion or by methylation in human melanomas , lymphomas , and other tumors .
p14arf has the ability to suppress growth through multiple p53-dependent or p53-independent pathways [ 5 - 7 ] .
p53 is a binding partner and ubiquitination target of hdm2 [ 9 - 11 ] . in cells that harbour wild type p53 ,
p14arf can associate with p53 bound hdm2 forming tri - molecular complexes . in human tumor cell lines , p14arf is localized mainly to nucleoli .
nucleoli are assembled around clusters of repeated ribosomal genes , which are transcribed by rna polymerase i. the nucleoli are the sites of ribosomal biogenesis .
it was suggested that p14arf targets hdm2 protein to the nucleoli , blocking the shuttling of hdm2 to the cytoplasm , and consequently enriching the nucleus with p53 protein .
however , the literature data shows contradictory data about p14arf - hdm2 localization reporting that p14arf targets hdm2 to the nucleoli , hdm2 re - localizes p14arf to the nucleoplasm , or these two proteins are found in different compartments . in the present study ,
our goal is to verify the localization of p14arf protein and to compare it with the localization of other regulatory proteins , such as p53 , hdm2 , rb , p27 , hsp70 , and pml .
we transfected p14arf , cloned into three different vectors : pbabe that did not contain any tag ( pbabe - p14arf ) , and as a fusion with gfp at the n - terminus ( gfp - p14arf ) and with dsred at the c - terminus ( p14arf - dsred ) into mcf7 , saos-2 and nih3t3 cells lines .
the three different constructs produced proteins with very similar localization patterns in all the three cell lines .
pictures that were taken on mcf7 cells are used to illustrate the findings of this paper .
we found that p14arf protein accumulated in the nucleoli and/or in extra - nucleolar nucleoplasmic inclusions of variable sizes ( figure 1 ) .
these p14arf nuclear inclusions were negative for the nucleolar marker b23 ( figure 2 ) and were not surrounded by perinucleolar heterochromatin .
it was difficult to distinguish nucleoli from the extranucleolar inclusions using only phase contrast microscope .
the p14arf nuclear inclusions were visible on the phase contrast as dark bodies in similar manner as nucleoli .
we found however that staining of the perinucleolar heterochromatin with hoechst 33258 was a reliable marker to routinely discriminate between nucleoli and extranucleolar inclusions .
full - length p14arf expressed from the retroviral vector pbabe ( pbabe - p14arf ) is localized to the nucleoli ( full arrowheads ) and to extra - nucleolar inclusions of mcf7 cells ( hollow arrowheads ) .
double staining for b23 and pbabe - p14arf shows that pbabe - p14arf forms extranucleolar inclusions as well as localizes to nucleoli in mcf7 cells .
after p14arf transfection , we observed that pml bodies were re - distributed to the p14arf nuclear inclusions ( figure 3 ) .
here we demonstrated a striking difference in the properties of the nucleolar p14arf and p14arf that formed extranucleolar inclusions .
pml bodies are randomly scattered in nuclei where pbabe - p14arf exclusively localized to nucleoli ( top row ) but gradually associate with the p14arf positive extranucleolar inclusions in parallel with their expansion ( 25raw ) in mcf7 cells .
in order to investigate whether the p14arf nuclear inclusions could be targets of the degradation pathway , we have stained the transfected cells for the 20s core subunit of the proteasomes .
the 20s subunits of the proteasomes were localized mainly to the cytoplasm of mcf7 cells .
when p14arf was expressed in cells , the 20s core subunits of the proteasomes were targeted to the nucleus and co - localized with p14arf in the extra - nucleolar inclusions ( figure 4 ) .
remarkably , the proteasomes were not associated with p14arf when it was present in the nucleoli .
20s proteasomes are targeted to pbbae - p14arf positive nuclear inclusions ( hollow arrowheads ) but not to the nucleoli ( full arrowheads ) in mcf7 cells .
one of the stress response proteins , hsp70 , is often redistributed both to the nucleus and to the nucleolus during periods of cell stress .
here we show that in those p14arf transfected cells where p14arf accumulated in the extranucleolar inclusions hsp70 was targeted to the nucleus and showed a high grade of co - localization with p14arf extranuclear inclusions ( figure 5 ) .
hdm2 protein ( red ) is re - distributed to pbabe - p14arf ( green ) nuclear inclusions , but not to nucleoli that contain p14arf in mcf7 cells .
we stained the cells for both p53 and hdm2 because these two proteins were shown to form a tri - molecular complex with p14arf protein .
neither protein was detected in nucleoli , even if p14arf was localized to the nucleoli ( figures 6 and 7 ) .
formation of the p14arf inclusions has no effect on the distribution of other nuclear proteins such as prb or p27 ( data not shown ) .
transfection of p14arf into mcf7 cells that contain wild type p53 and hdm2 but lack endogeneous p14arf did not induce increased expression of p53 .
p53 protein ( red ) co - localizes with pbabe - p14arf ( green ) in the extra - nucleolar inclusions in mcf7 cells .
pbabe - p14arf extra - nucleolar inclusions , but not the nucleoli , are the sites for the accumulation of hsp70 protein in mcf7 cells .
we transfected p14arf , cloned into three different vectors : pbabe that did not contain any tag ( pbabe - p14arf ) , and as a fusion with gfp at the n - terminus ( gfp - p14arf ) and with dsred at the c - terminus ( p14arf - dsred ) into mcf7 , saos-2 and nih3t3 cells lines .
the three different constructs produced proteins with very similar localization patterns in all the three cell lines .
pictures that were taken on mcf7 cells are used to illustrate the findings of this paper .
we found that p14arf protein accumulated in the nucleoli and/or in extra - nucleolar nucleoplasmic inclusions of variable sizes ( figure 1 ) .
these p14arf nuclear inclusions were negative for the nucleolar marker b23 ( figure 2 ) and were not surrounded by perinucleolar heterochromatin .
it was difficult to distinguish nucleoli from the extranucleolar inclusions using only phase contrast microscope .
the p14arf nuclear inclusions were visible on the phase contrast as dark bodies in similar manner as nucleoli .
we found however that staining of the perinucleolar heterochromatin with hoechst 33258 was a reliable marker to routinely discriminate between nucleoli and extranucleolar inclusions .
full - length p14arf expressed from the retroviral vector pbabe ( pbabe - p14arf ) is localized to the nucleoli ( full arrowheads ) and to extra - nucleolar inclusions of mcf7 cells ( hollow arrowheads ) .
double staining for b23 and pbabe - p14arf shows that pbabe - p14arf forms extranucleolar inclusions as well as localizes to nucleoli in mcf7 cells .
after p14arf transfection , we observed that pml bodies were re - distributed to the p14arf nuclear inclusions ( figure 3 ) .
here we demonstrated a striking difference in the properties of the nucleolar p14arf and p14arf that formed extranucleolar inclusions .
pml bodies are randomly scattered in nuclei where pbabe - p14arf exclusively localized to nucleoli ( top row ) but gradually associate with the p14arf positive extranucleolar inclusions in parallel with their expansion ( 25raw ) in mcf7 cells .
in order to investigate whether the p14arf nuclear inclusions could be targets of the degradation pathway , we have stained the transfected cells for the 20s core subunit of the proteasomes .
the 20s subunits of the proteasomes were localized mainly to the cytoplasm of mcf7 cells . when p14arf was expressed in cells ,
the 20s core subunits of the proteasomes were targeted to the nucleus and co - localized with p14arf in the extra - nucleolar inclusions ( figure 4 ) .
remarkably , the proteasomes were not associated with p14arf when it was present in the nucleoli .
20s proteasomes are targeted to pbbae - p14arf positive nuclear inclusions ( hollow arrowheads ) but not to the nucleoli ( full arrowheads ) in mcf7 cells .
one of the stress response proteins , hsp70 , is often redistributed both to the nucleus and to the nucleolus during periods of cell stress . here
we show that in those p14arf transfected cells where p14arf accumulated in the extranucleolar inclusions hsp70 was targeted to the nucleus and showed a high grade of co - localization with p14arf extranuclear inclusions ( figure 5 ) .
hdm2 protein ( red ) is re - distributed to pbabe - p14arf ( green ) nuclear inclusions , but not to nucleoli that contain p14arf in mcf7 cells .
we stained the cells for both p53 and hdm2 because these two proteins were shown to form a tri - molecular complex with p14arf protein .
neither protein was detected in nucleoli , even if p14arf was localized to the nucleoli ( figures 6 and 7 ) .
formation of the p14arf inclusions has no effect on the distribution of other nuclear proteins such as prb or p27 ( data not shown ) .
transfection of p14arf into mcf7 cells that contain wild type p53 and hdm2 but lack endogeneous p14arf did not induce increased expression of p53 .
p53 protein ( red ) co - localizes with pbabe - p14arf ( green ) in the extra - nucleolar inclusions in mcf7 cells .
pbabe - p14arf extra - nucleolar inclusions , but not the nucleoli , are the sites for the accumulation of hsp70 protein in mcf7 cells .
p14arf protein is considered to be mainly nucleolar protein that directly interacts with hdm2 and regulates the expression of the tumor suppressor protein p53 .
experiments on cell hybrids suggested that p14arf sequesters hdm2 in the nucleolus . on the other hand
it was also shown that overexpressed hdm2 protein could re - locate p14arf into the nucleoplasm in the presence of wild - type p53 .
we have detected the formation of nuclear inclusions by p14arf that was expressed from three different vectors , 2472 hours after transfection .
the nuclear inclusions were very similar in size , and in appearance in phase contrast microscope , to the nucleoli .
the lack of b23 protein and the absence of surrounding perinuclear heterochromatin clearly indicated that the inclusions were extranucleolar structures .
pml bodies are interferon inducible , multifunctional nuclear organelles that are involved in a number of cellular processes such as antiviral defence , mhc class i dependent antigen presentation and regulation of gene expression .
pml bodies also likely play a role in the regulation of the degradation of nuclear proteins .
for example mutant , misfolded influenza nucleoprotein accumulates in pml bodies in cells where proteasome mediated degradation is inhibited suggesting that pml bodies function as the nuclear analogues of the cytoplasmic aggresomes .
accumulation of mutant polyq - containing cellular proteins in polyglutamine ( polyq ) neurodegenerative diseases   leads to the relocalization of pml bodies to the polyq inclusions .
we also observed that the p14arf nuclear inclusions , but not the p14arf containing nucleoli , showed accumulation of hsp70 protein and were targeted by proteasomes . importantly hdm2 and p53 also accumulated in p14arf inclusions but when p14arf was localized to the nucleoli , p53 or hdm2 co - localization was not detected .
our data suggests that p14arf , although it often accumulates in the nucleolus , does not sequestrate hdm2 in the nucleolus .
p14arf / hdm2 complexes also contain p53 and are targeted for proteasome mediated degradation with the help of pml bodies .
overexpression of p14arf leads to the complete entrapment of hdm2 and p53 intro extranucleolar inclusion bodies .
our data show that co - localization between p53 , hdm2 and p14arf occurs at extranucleolar sites .
accumulation of pml bodies and proteasomes at these sites suggest that the components of the nuclear inclusions are targeted for proteasome - mediated degradation .
the following plasmids were used : full - length p14arf cdna ( encoding 132 residues ) , cloned in pbabe vector ( gift of klas g. wiman 
cck ki , stockholm ) ; and full - length p14arf fused to gfp in pegfp - c1 and to dsred in pdsred2 ( both clontech ) in c - terminal and n - terminal positions , respectively . in this study , mcf7 , a breast cancer cell line , saos-2 , an osteosarcoma cell line , and nih3t3 ,
mcf7 cells expressed wild - type p53 at low levels , hdm2 at moderate levels and had a deleted p14arf gene .
saos-2 cells had a homozygous deletion at the p53 loci and expressed moderate level of p14arf .
transfections were performed using the lipofectamine plus reagent and fugene ( life technologies ) according to the manufacturer 's protocol .
the following primary antibodies were used : mouse monoclonal antibody ( mab ) against b-23/nucleophosmin ( npm ) , a kind gift of p. k. chan ( baylor college of medicine , houston ) , mab against hdm2 ab-1 , clone smp14 ( dako ) , mab against heat shock protein 70 ( hsp70 ) ( w27 ) ( santa cruz biotechnology ) , mab against pml protein ( pg - m3 ) ( santa cruz biotechnology ) , mab against 20s proteasome ( clone hp810 ) ( affiniti research product ltd ) , mab against p14arf , 240 ( supernatant ) , a kind gift of j. bartek ( danish cancer society ) , mab against p53 do7 ( bd pharmingen ) , rabbit polyclonal anti - p14arf , a kind gift of k. wiman ( cck , ki , stockholm ) .
the following secondary antibodies were used : horse anti - mouse ig texas red conjugated ( vector lab ) , rabbit anti - mouse ig fitc ( dako ) , swine anti - rabbit fitc ( dako ) , swine anti - rabbit tritc ( dako ) .
bisbenzimide ( hoechst 33258 from sigma ) was added at a concentration of 0.4 g / ml to the secondary antibody for dna staining when needed .
cells were stained on coverslips after fixation in a mixture of methanol and acetone ( 1:1 ) at -20c .
images were captured using a das microscope leitz dm rb with a hamamatsu dual - mode cooled charge - coupled device ( ccd ) camera c4880 .
the 3d immunofluorescence images were generated from the reconstitution of a series of de - blurred optical sections .
briefly , the images were captured using a pxl cooled ccd camera ( photometrix ) on a zeiss axiophot microscope equipped with z - axis motor , external shutter and excitation filter - wheel , controlled by a mac2000 lep module .
the computer program , st - rfh - bin2 , that controlled the image acquisition hardware and produced the digital images was developed by us ( szekely , unpublished ) .
the program captured three times 1317 images from three excitation series ( rhodamine - fitc - hoechst ) with focal planes 0.3 m apart .
the incoming images were automatically corrected for dark current noise and optical shining through , and de - blurred using a nearest neighbour de - convolution algorithm .
the resultant image stacks were projected using the maximum intensity projection algorithm producing a single - direction and stereo - pair projected images as described .
the following plasmids were used : full - length p14arf cdna ( encoding 132 residues ) , cloned in pbabe vector ( gift of klas g. wiman 
cck ki , stockholm ) ; and full - length p14arf fused to gfp in pegfp - c1 and to dsred in pdsred2 ( both clontech ) in c - terminal and n - terminal positions , respectively .
in this study , mcf7 , a breast cancer cell line , saos-2 , an osteosarcoma cell line , and nih3t3 , a mouse immortalized fibroblasts were used .
mcf7 cells expressed wild - type p53 at low levels , hdm2 at moderate levels and had a deleted p14arf gene .
saos-2 cells had a homozygous deletion at the p53 loci and expressed moderate level of p14arf .
transfections were performed using the lipofectamine plus reagent and fugene ( life technologies ) according to the manufacturer 's protocol .
the following primary antibodies were used : mouse monoclonal antibody ( mab ) against b-23/nucleophosmin ( npm ) , a kind gift of p. k. chan ( baylor college of medicine , houston ) , mab against hdm2 ab-1 , clone smp14 ( dako ) , mab against heat shock protein 70 ( hsp70 ) ( w27 ) ( santa cruz biotechnology ) , mab against pml protein ( pg - m3 ) ( santa cruz biotechnology ) , mab against 20s proteasome ( clone hp810 ) ( affiniti research product ltd ) , mab against p14arf , 240 ( supernatant ) , a kind gift of j. bartek ( danish cancer society ) , mab against p53 do7 ( bd pharmingen ) , rabbit polyclonal anti - p14arf , a kind gift of k. wiman ( cck , ki , stockholm ) .
the following secondary antibodies were used : horse anti - mouse ig texas red conjugated ( vector lab ) , rabbit anti - mouse ig fitc ( dako ) , swine anti - rabbit fitc ( dako ) , swine anti - rabbit tritc ( dako ) .
bisbenzimide ( hoechst 33258 from sigma ) was added at a concentration of 0.4 g / ml to the secondary antibody for dna staining when needed .
cells were stained on coverslips after fixation in a mixture of methanol and acetone ( 1:1 ) at -20c .
images were captured using a das microscope leitz dm rb with a hamamatsu dual - mode cooled charge - coupled device ( ccd ) camera c4880 .
the 3d immunofluorescence images were generated from the reconstitution of a series of de - blurred optical sections .
briefly , the images were captured using a pxl cooled ccd camera ( photometrix ) on a zeiss axiophot microscope equipped with z - axis motor , external shutter and excitation filter - wheel , controlled by a mac2000 lep module .
the computer program , st - rfh - bin2 , that controlled the image acquisition hardware and produced the digital images was developed by us ( szekely , unpublished ) .
the program captured three times 1317 images from three excitation series ( rhodamine - fitc - hoechst ) with focal planes 0.3 m apart .
the incoming images were automatically corrected for dark current noise and optical shining through , and de - blurred using a nearest neighbour de - convolution algorithm .
the resultant image stacks were projected using the maximum intensity projection algorithm producing a single - direction and stereo - pair projected images as described .
ls conceived of the study , and participated in its design and coordination and together with ek drafted the manuscript all authors read and approved the final manuscript .
we thank klas g. wiman for the p14arf plasmids and anti - arf antibodies and p. k. chan for anti - b23 antibodies . this work was supported by cancerfonden and by a matching grant from the concern foundation , los angeles , the cancer research institute , new york and also by ssmf ( swedish society for medical research ) , karolinska institute and sven gard foundation .
 backgroundp14arf is a protein product of the alternative reading frame of the human ink4a locus . 
 it functions as a tumor suppressor protein . 
 p14arf suppresses growth through p53-dependent and p53-independent pathways.resultsp14arf protein localizes primarily to the nucleoli . 
 here we show that in transfected cells p14arf also appears in hsp70 positive extranucleolar inclusions . 
 the formation of p14arf inclusions induces the parallel re - localization p53 and hdm2 to these sites that are also targeted by pml bodies and proteasomes.conclusionour data show that co - localization between p53 , hdm2 and p14arf occurs at extranucleolar sites 
 . accumulation of pml and proteasomes at these sites suggest that the components of the nuclear inclusions are targeted for proteasome - mediated degradation . 