| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 276, Issue 22, 18649-18652, June 1, 2001
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,, and**
From the Chugai Research Institute for Molecular
Medicine, 153-2 Nagai, Niihari-Mura, Ibaraki 300-41, Japan, the
§ Institute of Molecular and Cell Biology, National
Institute of Advanced Industrial Science and Technology (AIST),
1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan, the
¶ Graduate School of Biological Sciences, Nara Institute of
Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan,
and the Children's Medical Research Institute, Westmead, New
South Wales 2145, Australia
Received for publication, January 11, 2001, and in revised form, March 1, 2001
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
We isolated a 33-kDa protein, Pex19p/HK33/HsPXF,
as a p19ARF-binding protein in a yeast two-hybrid screen. We
demonstrate here that Pex19p interacts with p19ARF in the cell
cytoplasm and excludes p19ARF from the nucleus, leading to a concurrent
inactivation of p53 function. Down-regulation of Pex19p by its
antisense expression resulted in increased levels of p19ARF, increased
p53 function, and a p53/p21WAF1-mediated senescence-like cell cycle
arrest. The data demonstrated a novel mechanism of down-regulation of the p19ARF-p53 pathway.
The INK4a (MTS1, CDKN2) locus on
chromosome 9p21 is frequently altered in human cancers. It encodes two
unrelated tumor suppressor proteins: p16INK4a, an inhibitor of the
cyclin D-dependent kinases that acts upstream
of pRb, and p19ARF, an alternative reading frame protein that acts
upstream of p53 (1-3). Both of these proteins have roles in
replicative senescence and ras-induced premature
senescence of primary cells (4-7). Analysis of p19ARF knock-out mice
suggested that this protein functions as a tumor suppressor (3,
8, 9). Recently, it has been shown that p19ARF acts by obstructing
degradation and transcriptional silencing of p53 by
mdm2 (10, 11). It retains Mdm2 in the nucleolus, preventing its export to the cytoplasm, which is required for mdm2-mediated p53 degradation (12-16). Because p19ARF
shares no amino acid homology with known proteins and lacks any
decisive functional protein motifs, other cellular factors that might
regulate its activity and thereby its execution of growth arrest via
the p19ARF-p53 pathway remain poorly defined. Using a yeast interactive screen, we have identified the farnesylated protein Pex19p/HK33/HsPXF (essential for peroxisomal biogenesis) (17-19) as a p19ARF-binding protein. In the present study, we report that the two proteins interact
in the cell cytoplasm leading to exclusion of p19ARF from the nucleus
and inactivation of p53 function, which constitutes a novel mechanism
of down-regulation of the p19ARF-p53 pathway. Neutralization of the
Pex19p function by its antisense expression led to an accelerated
activation of p19ARF function and p53-p21WAF1-mediated cell cycle
arrest that resembled cellular senescence.
Yeast Two-hybrid Screen--
cDNAs encoding full-length
p19ARF (p19ARF-F), amino-terminal 80 amino acids (p19ARF-N) and
carboxyl-terminal 89 amino acids (p19ARF-C) were cloned into the
BamHI, SalI site of the yeast expression vector
pODB8 (a kind gift from O. Louvet) (20). For library screening the
yeast reporter strain PJ69/2A
(Trp Cell Culture and Transfections--
Mouse embryonic fibroblasts
and monkey kidney cells were cultured in Dulbecco's modified Eagle's
minimal essential medium supplemented with 10% fetal bovine serum.
Transfections were performed using LipofectAMINETM (Life Technologies,
Inc.). Typically, 1 µg of plasmid DNA was used per well of a 24-well
dish, and 3 µg was used per 6-cm dish.
Plasmid Constructions--
Full-length Pex19p was cloned
from mouse testis by reverse transcription-polymerase chain
reaction using sense (5'-gaa ttc atg gcg gct gct gag gaa ggt-3')
and antisense (5'-gtc gac tca cat gat cag aca ctg ttc-3') primers with
EcoRI and SalI sites, respectively. Polymerase
chain reaction amplification product (94 °C for 30 s, 55 °C
for 30 s, and 72 °C for 3 min) was purified and sequentially
ligated to pGEM-T Easy (Promega), pEGFPC1
(CLONTECH), and pcDNA4/HisMax (Invitrogen)
vectors. Mouse p19ARF and its deletion mutants were cloned into the
indicated vectors by a similar strategy. The integrity of the plasmids
was confirmed by sequencing.
Mammalian Two-hybrid Analysis--
COS-7 cells were
seeded at 50-60% confluence in 24-well plates and transfected with 1 µg of DNA containing pG5 reporter plasmid, pM/mHK33, VP16/p19ARF, and
pM or VP16 control vectors as indicated in the relevant figure legends.
After 3 h of transfections, cells were refed with fresh medium and
then lysed in universal lysis buffer (Promega) after 48 h.
Luciferase activity was measured using the
Dual-LuciferaseTM reporter assay system (Promega). The
results presented are the means of at least three transfections.
In Vivo Coimmunoprecipitation--
Cell lysates (400 µg of
protein) in 400 µl of Nonidet P-40 lysis buffer were incubated
at 4 °C for 1-2 h with an antibody used for immunoprecipitation, as
indicated in figure legends. Immunocomplexes were separated by
incubation with protein A/G-Sepharose, Western blotting was performed
with the indicated antibodies by standard procedures, and detection was
done using ECL chemiluminescence.
Reporter Assays--
NIH 3T3, NIH-ARF,
NIH-ARF/pcDNA4-HisMAX- Pex19p (sense), and
NIH-ARF/pcDNA4-HisMAX-Pex19p (antisense) derivatives (selected in 1 mg/ml zeocine, Invitrogen) were transfected with the p53-responsive luciferase reporter plasmid, PG-13luc (kindly provided by Dr. Bert
Vogelstein). As a control, pRL-TK vector (Promega) was co-transfected in each assay to correct for variations in transfection efficiency. Cells were lysed and measured for luciferase activity as described above.
To isolate p19ARF interacting proteins, a Gal4BD (Gal4 binding
domain)-p19ARF fusion protein was used as a bait to screen a library of
human cDNAs cloned into a Gal4AD (activation domain) yeast
two-hybrid plasmid. One of the five clones isolated was strongly
positive as determined by His prototrophy and induction of
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/Leu/His) was
sequentially transformed with the plasmid pODB8/p19ARF and a human
testis cDNA library (CLONTECH) according to the
manufacturer's protocol. The cDNA-derived plasmids were recovered
from yeast and reintroduced into the yeast reporter strain Y187 to
confirm specificity of the interactions. To determine 
-galactosidase activity in yeast, five colonies of simultaneously transformed Y187
yeast cells were grown overnight in Leu/Trp
plates. Cell extracts were prepared using standard conditions, and
enzyme activity was determined using the GAL-Tropix kit according to
the manufacturer's protocol (Tropix Inc.). The clones were sequenced
using an ABI sequencer (PerkinElmer Life Sciences).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-galactosidase expression. The nucleotide sequence of this clone
revealed its identity to the 33-kDa farnesylated protein Pex19p/HK33/HsPxF/mPxF, which is essential for peroxisomal biogenesis (17-19, 21). The amino- and carboxyl-terminal halves of p19ARF were
cloned into the Gal4BD vector. Pex19p, either full-length or carboxyl-
but not amino-terminal, strongly activated the -galactosidase reporter, indicating that it is the carboxyl terminus of p19ARF that
interacts with Pex1p (Fig.
1A). Pex19p and p19ARF
interacted strongly in a mammalian two-hybrid assay (Fig.
1B). To further detect their interaction in mammalian cells,
we transfected COS-7 cells with expression plasmids encoding tagged
Pex19p and p19ARF and found that the proteins coimmunoprecipitated
(Fig. 1C).
GFP1-tesmin and MPD-myc were
used as respective unrelated negative controls. As seen in Fig.
1C (left panel), GFP-Pex19p coprecipitated with
p19ARF-myc (immunoprecipitated with anti-myc antibody) but not with
MPD-myc or with isotype-matched control antibody (Fig. 1C,
cf. lanes 1 and 4). GFP-tesmin (negative control)
did not precipitate with p19ARF-myc. Furthermore, immunoprecipitation of hemagglutinin (HA)-tagged p19ARF from NIH-ARF cells with anti-HA tag
antibody resulted in coprecipitation of a protein at ~35-kDa, along
with several others including Mdm2 (22). It is likely that the
35-kDa p19ARF coprecipitate is Pex19p.
View larger version (53K):
[in a new window]
Fig. 1.
p19ARF interacts with Pex19p.
A, activation of yeast two-hybrid - galactosidase
reporter by Pex19p and either the full-length p19ARF or its
carboxyl-terminal half. Yeast cells were transformed with plasmids
encoding Pex19p and full-length (F), amino-terminal
(N), or carboxyl-terminal (C) p19ARF.
-Galactosidase (-Gal) activity was measured
by liquid assay. B, activation of mammalian two-hybrid
luciferase reporter by transfection of cDNAs encoding mouse Pex19p
and p19ARF in COS-7 cells. C, in vivo
coimmunoprecipitation of Pex19p and p19ARF. Cells were transfected with
plasmids encoding a GFP-Pex19p fusion protein or GFP-tesmin (a negative
control) and myc epitope-tagged p19ARF
(p19ARF-myc) or MPD-myc (a negative control).
Immunoprecipitation was performed with a polyclonal anti-myc
antibody, and the myc immunocomplexes were analyzed for the
presence of GFP-Pex19p or GFP-tesmin by Western blotting with a
monoclonal anti-GFP antibody. GFP-Pex19p coprecipitated with p19ARF-myc
(lane 1) but not in the absence of p19ARF-myc (lane
3), and not with MPD-myc (lanes 4 and 5) or
control isotype-matched antibody (con.IgG). GFP-tesmin was
not precipitated with p19ARF-myc. Input panel shows the
signal from 10% of the lysate used for immunoprecipitation.
To determine whether Pex19p and p19ARF colocalize within intact cells,
we visualized the proteins by immunostaining COS-7 (not shown) and NIH
3T3 cells transfected with tagged p19ARF and/or Pex19p. p19ARF
localized mainly in the nucleolus, but there was some diffuse staining
in the cytoplasm (Fig. 2A,
a and b). Pex19p was seen only in the cytoplasm
(Fig. 2A, c). Most notably, cells expressing both
p19ARF-myc and GFP-Pex19p showed p19ARF in the cytoplasm in more than
90% of cells (Fig. 2A, d) where it colocalized with Pex19p (Fig. 2A, d-f) suggesting that the
overexpression of Pex19p causes nuclear exclusion of p19ARF (Fig.
2A). As p19ARF has been shown to bind to and inactivate
mdm2 in the nucleus (11, 16) resulting in activation of p53,
we predicted that exclusion of p19ARF from the nucleus would lead to
inactivation of p53 function via increased mdm2-mediated
degradation.
|
NIH 3T3 cells lack endogenous p19ARF because of biallelic loss of the INK4a locus. As expected, transfection of these cells with p19ARF resulted in a dose-dependent increase in p53 activity (Fig. 2B, a). Cotransfections of Pex19p with p19ARF blocked p53 activation (Fig. 2B, b). We ruled out the possibility that p53 might be inactivated by direct interaction with Pex19p by performing in vivo coimmunoprecipitation of Pex19p and p53 wherein no Pex19p was seen to precipitate with p53 (data not shown). We next used stably transfected NIH 3T3 cells (NIH-ARF) that express exogenous HA-tagged p19ARF under the control of the heavy metal-inducible metallothionein promoter (22). The addition of 100 µM ZnSO4 to the culture medium resulted in the expression of HA-p19ARF (detectable by Western blotting) and a concurrent increase in p53-dependent luciferase reporter activity (Fig. 2B, c). NIH-ARF cells were stably transfected with expression plasmids encoding sense (S) and antisense (AS) His-tagged Pex19p protein and were selected in medium supplemented with zeocine (1 mg/ml). These derivatives were analyzed for endogenous p53 activity when cultured in the presence of increasing amounts of ZnSO4; 100 µM induced p19ARF expression detectable by Western blotting. As predicted and consistent with the results of transient transfections (Fig. 2B, b), Pex19p transfectants (NIH-ARF/Pex19p-S) showed down-regulation of p53 function and notably, the antisense derivative (NIH-ARF/Pex19p-AS) showed dramatic up-regulation (Fig. 2B, c). NIH 3T3 cells that lacked p19ARF did not show any effect of transfections of Pexp19-S and-AS constructs on p53-dependent reporter activity (Fig. 2B, d). Taken together these results demonstrated that: (i) an overexpression of Pex19p blocks p19ARF enhancement of p53-mediated transcriptional activation; (ii) this occurs most likely because of nuclear exclusion of p19ARF and abrogation of its interactions with Mdm2, resulting in active degradation of p53; and (iii) such an effect of Pex19p on the transcriptional activity of p53 is mediated by p19ARF.
We next analyzed the effect of Pex19p-p19ARF interactions on the
expression of p21WAF1, a gene that is transactivated by p53. Induction of p19ARF led to up-regulation of p21WAF1 expression in
NIH-ARF cells as has been described (22). Notably, Western blot
analysis of p19ARF and Pex19p with respective tag-specific antibodies revealed that the induction of p19ARF led to stabilization of Pex19p (Fig. 3A,
a). On the other hand, antisense derivatives of
NIH-ARF/Pex19p had more p19ARF than sense derivatives when cultured in
the presence of ZnSO4 for an equal time (Fig.
3A, a and b) suggesting that Pex19p
may cause destabilization/degradation of p19ARF by a mechanism that
remains to be defined. Accordingly, NIH-ARF/Pex19p antisense
derivatives showed a high level of p21WAF1 expression, whereas Pex19p
sense derivatives showed a lower level as compared with the control
NIH-ARF cells (Fig. 3A, a). The data suggested
that antisense Pex19p decreased the level of endogenous Pex19p,
resulting in activation of the p19ARF-p53-p21WAF1 pathway. This
interpretation was supported by the finding that transfection of the
antisense construct into NIH-ARF cells that stably express His-tagged
Pex19p resulted in decreased levels of His-Pex19p protein (Fig.
3A, c). We next studied the growth of NIH-ARF
cells and their Pex19p sense and antisense derivatives for Pex19p
expression when cultured with and without ZnSO4. As
expected, the induction of p19ARF in NIH-ARF cells led to growth
retardation (Fig. 3, B and C). Increased
expression of Pex19p decreased p19ARF-induced growth retardation (Fig.
3, B and C), seemingly because of inactivation of
the p53 function via nuclear exclusion of p19ARF by Pex19p, as
demonstrated above. NIH-ARF expressing antisense Pex19p showed severe
retardation of growth, exhaustion of their replicative potential, and a
senescence-like morphology (Fig. 3, B and C). On
the other hand, growth of NIH 3T3 cells that lack p19ARF expression was
not affected by transfections of Pex19p-S and -AS, constructs demonstrating that the effect was mediated by p19ARF-Pex19p
interactions (Fig. 3C and data not shown). This data implies
that p19ARF function is blocked, at least in part, by its interactions
with endogenous Pex19p, and abrogation of these interactions by
antisense expression of Pex19p led to activation of p19ARF-p53-p21WAF1
pathway and execution of a senescence-like growth arrest.
|
As p19ARF and its human homologue p14ARF are important mediators of
cellular senescence (5, 6, 9, 23, 24), understanding its precise
mechanism of action and how it is controlled is clearly important. p53
has a central role in many aspects of the cell's response to its
environment and control of proliferation (25), in part because of
transcriptional control of effectors such as p21WAF1; understanding the
factors that regulate its activity is also of critical importance. We
have described here a novel mechanism of down-regulation of the
p19ARF-p53-p21WAF1 pathway.
| |
FOOTNOTES |
|---|
** To whom correspondence should be addressed. Tel.: 81-298-30-6211; Fax: 81-298-30-6270; E-mail: renu@cimmed.com.
Published, JBC Papers in Press, March 19, 2001, DOI 10.1074/jbc.C100011200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GFP, green fluorescent protein; HA, hemagglutinin; MPD, mevalonate pyrophosphate decarbonylase; ARF, alternative reading frame.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
| 1. | Quelle, D. E., Zindy, F., Ashmun, R. A., and Sherr, C. J. (1995) Cell 83, 993-1000 |
| 2. | Serrano, M., Lee, H., Chin, L., Cordon-Cardo, C., Beach, D., and DePinho, R. A. (1996) Cell 85, 27-37 |
| 3. | Kamijo, T., Zindy, F., Roussel, M. F., Quelle, D. E., Downing, J. R., Ashmun, R. A., Grosveld, G., and Sherr, C. J. (1997) Cell 91, 649-659 |
| 4. | Vogt, M., Haggblom, C., Yeargin, J., Christiansen-Weber, T., and Haas, M. (1998) Cell Growth & Differ. 9, 139-146 |
| 5. | Huschtscha, L. I., and Reddel, R. R. (1999) Carcinogenesis 20, 921-926 |
| 6. | Carnero, A., Hudson, J. D., Price, C. M., and Beach, D. H. (2000) Nat. Cell Biol. 2, 148-155 |
| 7. | Jacobs, J. J., Kieboom, K., Marino, S., DePinho, R. A., and van Lohuizen, M. (1999) Nature 397, 164-168 |
| 8. | Sharpless, N. E., and DePinho, R. A. (1999) Curr. Opin. Genet. Dev. 9, 22-30 |
| 9. | Kamijo, T., van de Kamp, E., Chong, M. J., Zindy, F., Diehl, J. A., Sherr, C. J., and McKinnon, P. J. (1999) Cancer Res. 59, 2464-2469 |
| 10. | Stott, F. J., Bates, S., James, M. C., McConnell, B. B., Starborg, M., Brookes, S., Palmero, I., Ryan, K., Hara, E., Vousden, K. H., and Peters, G. (1998) EMBO J. 17, 5001-5014 |
| 11. | Kamijo, T., Weber, J. D., Zambetti, G., Zindy, F., Roussel, M. F., and Sherr, C. J. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8292-8297 |
| 12. | Zhang, Y., Xiong, Y., and Yarbrough, W. G. (1998) Cell 92, 725-734 |
| 13. | Honda, R., and Yasuda, H. (1999) EMBO J. 18, 22-27 |
| 14. | Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J., and Bar-Sagi, D. (1999) Nat. Cell Biol. 1, 20-26 |
| 15. | Weber, J. D., Kuo, M. L., Bothner, B., DiGiammarino, E. L., Kriwacki, R. W., Roussel, M. F., and Sherr, C. J. (2000) Mol. Cell. Biol. 20, 2517-2528 |
| 16. | Tao, W., and Levine, A. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6937-6941 |
| 17. | Braun, A., Kammerer, S., Weissenhorn, W., Weiss, E. H., and Cleve, H. (1994) Gene 146, 291-295 |
| 18. | Kammerer, S., Arnold, N., Gutensohn, W., Mewes, H. W., Kunau, W. H., Hofler, G., Roscher, A. A., and Braun, A. (1997) Genomics 45, 200-210 |
| 19. | Gotte, K., Girzalsky, W., Linkert, M., Baumgart, E., Kammerer, S., Kunau, W. H., and Erdmann, R. (1998) Mol. Cell. Biol. 18, 616-628 |
| 20. | Louvet, O., Doignon, F., and Crouzet, M. (1997) BioTechniques 23, 816-818 |
| 21. | Fujiki, Y. (2000) FEBS Lett. 476, 42-46 |
| 22. | Kurokawa, K., Tanaka, T., and Kato, J. (1999) Oncogene 18, 2718-2727 |
| 23. | Dimri, G. P., Itahana, K., Acosta, M., and Campisi, J. (2000) Mol. Cell. Biol. 20, 273-285 |
| 24. | Jacobs, J. J., Scheijen, B., Voncken, J. W., Kieboom, K., Berns, A., and van Lohuizen, M. (1999) Genes Dev. 13, 2678-2690 |
| 25. | Levine, A. J. (1997) Cell 88, 323-331 |
This article has been cited by other articles:
|
|
 |
|
  C. L. Bristow, R. Wolkowicz, M. Trucy, A. Franklin, F. Di Meo, M. T. Kozlowski, R. Winston, and R. R. Arnold NF-{kappa}B Signaling, Elastase Localization, and Phagocytosis Differ in HIV-1 Permissive and Nonpermissive U937 Clones J. Immunol., January 1, 2008; 180(1): 492 - 499. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Korgaonkar, J. Hagen, V. Tompkins, A. A. Frazier, C. Allamargot, F. W. Quelle, and D. E. Quelle Nucleophosmin (B23) Targets ARF to Nucleoli and Inhibits Its Function Mol. Cell. Biol., February 15, 2005; 25(4): 1258 - 1271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Bertwistle, M. Sugimoto, and C. J. Sherr Physical and Functional Interactions of the Arf Tumor Suppressor Protein with Nucleophosmin/B23 Mol. Cell. Biol., February 1, 2004; 24(3): 985 - 996. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Rizos, E. Diefenbach, P. Badhwar, S. Woodruff, T. M. Becker, R. J. Rooney, and R. F. Kefford Association of p14ARF with the p120E4F Transcriptional Repressor Enhances Cell Cycle Inhibition J. Biol. Chem., February 7, 2003; 278(7): 4981 - 4989. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Md. K. Hasan, T. Yaguchi, T. Sugihara, P. K. R. Kumar, K. Taira, R. R. Reddel, S. C. Kaul, and R. Wadhwa CARF Is a Novel Protein That Cooperates with Mouse p19ARF (Human p14ARF) in Activating p53 J. Biol. Chem., September 27, 2002; 277(40): 37765 - 37770. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Wadhwa, T. Sugihara, Md. K. Hasan, K. Taira, R. R. Reddel, and S. C. Kaul A Major Functional Difference between the Mouse and Human ARF Tumor Suppressor Proteins J. Biol. Chem., September 20, 2002; 277(39): 36665 - 36670. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Marcotte and E. Wang Replicative Senescence Revisited J. Gerontol. A Biol. Sci. Med. Sci., July 1, 2002; 57(7): B257 - 269. [Abstract] [Full Text]
|
|
|
|
|
|
C. Korgaonkar, L. Zhao, M. Modestou, and D. E. Quelle ARF Function Does Not Require p53 Stabilization or Mdm2 Relocalization Mol. Cell. Biol., January 1, 2002; 22(1): 196 - 206. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
) of 100 µM ZnSO4 were
fixed, stained with Giemsa and photographed just after the addition of
ZnSO4 (Day 0) or after 3 days of culture
(Day 3). Note the senescence-like morphology of
NIH-ARF/Pex19p-AS cells when cultured in the presence of
ZnSO4. C, equal numbers of cells of the types
indicated were plated and cultured with (+) or without (