From the
Division of Biological Sciences and the Cancer Center, University of California, San Diego, La Jolla, California 92093-0322, the
Instituto di Medicina Interna, Malattie Endocrine del Metabolismo Università di Catania, Catania 95125, Italy, and the
Dottorato di Ricerca in Oncologia, Università di Catanzaro "Magna Graecia," Catanzaro 88100, Italy
Received for publication, February 25, 2003 , and in revised form, April 24, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The TP73 gene encodes several alternatively spliced variants, including the N-p73 (11, 13, 14). The
N-p73 is transcribed from an internal promoter in intron 3 and lacks the N-terminal transactivation domain that is required for p73 to induce apoptosis (11, 13, 14). The
N-p73 can interfere with the function of p53 and p73, and its expression has been implicated in tumor development (5, 15, 16). In sporadic human cancer cells, the TP73 gene is seldom mutated (5). Suppression of TP73 gene expression by hypermethylation has been described in neuroblastoma and leukemia cells (1719). However, TP73 expression is observed in breast, lung, bladder, and liver cancers (2025). This raises the question of whether alternative mechanisms other than the repression of TP73 expression are used by cancer cells to control the tumor suppression function of p73.
Previous studies (68, 26) have established c-Abl to be an obligatory activator of p73 in genotoxic response. The murine c-Abl gene was identified as the cellular homologue of the v-Abl oncogene of the Abelson murine leukemia virus (27). The oncogenic potential of human c-ABL is demonstrated by BCRABL in human chronic myelogenous leukemia (28). The c-Abl gene encodes a non-receptor tyrosine kinase that can shuttle between the cytoplasmic and the nuclear compartments (29). In the cytoplasm, c-Abl interacts with actin (3032) and regulates the F-actin dynamics in response to extracellular signals (33, 34). In the nucleus, c-Abl interacts with retinoblastoma 1 (3537), ataxia telangiectasia mutated (38, 39), p73 (68), breast cancer 1 (40), and RNA polymerase II (4143). Activation of the nuclear c-Abl tyrosine kinase by DNA damage occurs in S-phase cells (44) through ataxia telangiectasia mutation (38, 39), and can lead to the induction of apoptosis (45). That the nuclear c-Abl can stimulate apoptosis is best illustrated by the ability of BCR-ABL kinase to kill cells when this oncogenic protein is trapped in the nucleus (46).
In this study, we have found that the p73 protein is expressed in cells derived from anaplastic thyroid cancer, which is the most malignant form of thyroid carcinoma. In these p73
-expressing cancer cells, c-Abl is excluded from the nucleus. These results show that the apoptotic function of the c-Abl/p73 pathway can be controlled by the subcellular segregation of these two proteins and suggest an alternative mechanism to inactivate the p73 tumor suppression function.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoprecipitation and ImmunoblottingCell lysates were prepared in radioimmune precipitation assay buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1.0% Triton X-100, 1% deoxycholate, 5 mM EDTA). For immunoprecpitations, 1 mg of total protein was used. Immunoprecipitates were fractionated on SDS-PAGE and then transferred to polyvinylidene difluoride, which were immunoblotted with primary antibodies and horseradish peroxidase-conjugated secondary antibodies and visualized by chemiluminescence. The following antibodies were used: anti-p73 clones 429 and 1288 from Imgenex, clone ER-15 from Neomarker; anti-Abl 8E9 (BD Biosciences); anti-actin (Sigma) anti-phosphotyrosine 4G10 (USB); and anti-tubulin, anti-p21Cip1, anti-p53, and anti-histone H2B were from Santa Cruz Biotechnology Inc.
ImmunofluorescenceCells were fixed in 4% formaldehyde, permeabilized with phosphate-buffered saline/0.1% Triton X-100, blocked with phosphate-buffered saline/10% normal goat serum, and incubated with primary antibodies (anti-HA, anti-p21Cip1, or anti-Abl) for 1 h. Cells were then incubated with Cy3 or fluorescein isothiocyanate-conjugated secondary antibodies for 12 h. To visualize actin stress fibers, cells were incubated with Alexa Fluor 488-conjugated phalloidin for an additional 30 min. Cells were finally counterstained with Hoechst to visualize the nuclei. Deconvolution microscopy was performed with a Delta Vision System.
Transcript Analysis by RT-PCRTotal RNAs were prepared from cultured cells using Trizol (Invitrogen). RT-PCR was performed with a One-Step RT-PCR kit (Invitrogen) using forward primer 5'-CGGGACGGACGCCGATG-3' and reverse primer 5'-CTTGGCGATCTGGCAGTAG-3' annealing to human TP73 exon 1 and 5, respectively.
Subcellular FractionationCells were incubated with or without 10 nM leptomycin B (LMB) overnight. Cell pellets for fractionation were resuspended in hypotonic buffer (10 mM Tris, pH 8.0, 10 mM KCl, 2 mM phenylmethylsulfonyl fluoride plus protease inhibitor mixture) to allow cell swelling for 2 min at 4 °C. Then, Nonidet P-40 was added to a final concentration of 0.4%. Samples were centrifuged at 2,000 rpm for 5 min at 4 °C and the supernatants collected as the cytoplasmic fractions. Pellets containing cell nuclei were washed once with hypotonic buffer and then extracted with high salt lysis Buffer (50 mM Tris pH 8.0, 5 mM EDTA, pH 8.0, 1% Nonidet P-40, 1% sodium deoxycolate, 0.025% SDS, 400 mM NaCl, 2 mM phenylmethylsulfonyl fluoride plus protease inhibitor mixture). Equal amount of proteins was loaded onto a 7.5% SDS-acrylamide gel, transferred onto polyvinylidene difluoride membranes, and blotted with anti-Abl, anti-tubulin, and anti-histone-2B antibodies.
Plasmid ConstructionTo construct AblNuk, Abl nuclear export signal was inactivated by a point mutation as described in Ref. 47. Fv, a modified version of the FK506 binding domain of FK506-binding protein (FKBP) was derived from pC4M-Fv2E vector provided by ARIAD Inc. Two copies of Fv were fused in-frame to the C terminus of c-Abl. An HA tag was placed in-frame at the end of the last copy of Fv. Two complementary oligonucleotides containing three contiguous copies of SV40 NLS (PKKKRAKV) were annealed and digested with SalI. The digested oligonucleotides were then cloned in-frame to the SalI site of mouse c-Abl cDNA.
Apoptosis Assay in Transiently Transfected CellsCells were seeded onto coverslips and either transfected with empty vector or with the indicated constructs. A plasmid expresing H2B-GFP was included in all transfection mixtures to mark the transfected cells. The transfected cells were either left untreated () or treated (+) with 50 nM chemical dimerizer AP20187 (ARIAD Inc., Cambridge, MA) for up to 24 h. Cells were then fixed and stained with Hoechst 33342. Apoptotic cells were scored based upon their fragmented nuclei and condensed chromosomes. Percentages of apoptotic cells among GFP-positive cell population were scored.
Stable Expression of AblNuk-FKBP and Treatment with Dimerizer ARO and KAK cells were infected with vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovius expressing the pMSCV-vector or AblNuk-FKBP. Infected cultures were expanded without antibiotics selection (see "Results"). Immunofluorescence using anti-HA showed between 4050% of cells in the infected cultures to express AblNuk-FKBP. Mock and AblNuk-FKBP-infected cultures were seeded onto cover slips and treated with 50 nM dimerizer AP20187 (ARIAD Inc.) for 24 h. Cells were then fixed and stained with monoclonal anti-HA antibody (BabCO, Richmond, CA), then with Hoechst 33342. Anti-HA-positive nuclei were counted, and the percent of HA (+) nuclei was determined for each treatment condition.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Expression of the p73 protein was confirmed with three monoclonal anti-p73 antibodies, directed at the transactivating domain (clone 429), the DNA binding domain (clone 1288), and the C-terminal region of the /
isoforms (clone ER15) (not shown) (48). Reactivity of a 73 kDa protein band with these three antibodies plus size comparison with recombinant p73
, p73
, p73
, and p73
proteins established that p73
is expressed in KAK and ARO cells (Fig. 1, a and b and data not shown). The p73
protein was not detected in primary thyroid epithelial cells (Fig. 1a, panels AC) or in follicular and papillary thyroid cancer cells (Fig. 1b, lanes 14). Indirect immunofluorescence staining with anti-p73 showed it to be localized in the nucleus of ARO and KAK cells (Fig. 1c). We also found p73
to be expressed in two of four anaplastic cancer tissues examined (Fig. 1d). These results showed that p73
is up-regulated in a significant fraction of anaplastic thyroid cancer.
Tumor Suppression Function of p73 Is Repressed in Anaplastic Thyroid Cancer CellsTo determine whether the anaplastic thyroid cancer cells have developed strategies to restrain the tumor suppression function of p73
, we tested if these cells can respond to ectopically expressed p73
(HA-p73
). As a control, we also expressed HA-p73
in Saos-2, an osteosarcoma cell line that does not express p73 (not shown). Consistent with previous results (49), HA-p73
caused apoptosis in Saos-2 cells (Fig. 2a). However, HA-p73
did not induce apoptosis in ARO or KAK cells (Fig. 2a). We also found that HA-p73
caused the cleavage of pro-caspase 3 in Saos-2 and CA301 cells, but not in ARO or KAK cells (not shown). The ectopic expression of HA-p73
induced the expression of p21Cip1 in every transfected Saos-2 cell (Fig. 2, b and c). By contrast, induction of p21Cip1 was observed in only 30% of ARO cells transfected with HA-p73
(Fig. 2, b and c). These results suggest that the function of p73
in transactivating p21Cip1 and in inducing apoptosis is compromised in the ARO and KAK cells.
|
Reduced Nuclear Entry of c-Abl in Anaplastic Thyroid Cancer CellsPrevious studies (7, 8) have shown that the apoptosis function of p73 is compromised in cells derived from the Abl-knockout mice. We therefore examined the expression, the tyrosine kinase activity, and the subcellular distribution of c-ABL in thyroid cells. The c-ABL protein is expressed at similar levels in primary thyroid epithelial cells and thyroid cancer cells (Fig. 3a). Immune complex kinase assays showed that c-ABL isolated from the different thyroid cancer cells to contain similar levels of kinase activity (not shown). Indirect immunofluorescence staining of primary thyroid cells and thyroid cancer cells revealed a predominantly cytoplasmic distribution of the c-ABL protein (Fig. 3b, LMB panels). The c-ABL protein contains nuclear localization and nuclear export signals (29, 47). We have previously shown that the nuclear export of Abl can be inhibited by Leptomycin B (LMB) (29, 46), which inactivates the nuclear export protein Exportin-1. Treatment with LMB led to the nuclear accumulation of c-ABL in primary thyroid cells and WRO cells derived from a follicular thyroid carcinoma (Fig. 3b, +LMB panels). These results showed that c-ABL enters the nucleus of thyroid cells, and its predominant cytoplasmic localization is due to nuclear export. Interestingly, c-ABL did not accumulate in the nucleus following LMB treatment of ARO and KAK cells (Fig. 3b, +LMB panels). Thus, the rate of ABL nuclear import was negligible in ARO and KAK cells.
|
The immunofluorescence results were confirmed by cell fractionation experiments (Fig. 3c). When normalized to histone H2B, it was estimated that only 15% of the total ABL was present in the nuclear fraction of WRO and KAK cells prior to LMB treatment (Fig. 3c). With WRO cells, the amount of ABL in the nuclear fraction was significantly increased after LMB treatment. With KAK cells, however, LMB treatment did not alter the low level of ABL in the nuclear fraction. The restriction on ABL nuclear import was similarly observed by fractionation experiments with ARO cells (not shown). The mechanism underlying the reduced nuclear import of ABL in KAK and ARO cells is presently unknown.
Enforced Nuclear Entry with AblNukTo overcome the restriction of nuclear import, we engineered "AblNuk" by inserting three tandem copies of the SV40 NLS in the C-terminal region of mouse c-Abl. We also inactivated the nuclear export signal to prevent export (Fig. 4a). In transient transfection experiments, we demonstrated that AblNuk, but not Abl, accumulated in the nucleus of ARO and KAK cells (not shown). Thus, the SV40 NLS is functional in these cancer cells. These results also suggested that the three NLS of ABL are specifically inactivated in ARO and KAK cells.
|
Dimerization of AblNuk-FKBP Induces Apoptosis in p53(/) but Not p73(/) MEFsWe developed a way to conditionally activate the kinase activity of AblNuk. Previous studies (50, 51) have shown that oligomerization of the Abl protein can activate its kinase activity, observed with BCRABL. The FK506-binding protein, FKBP, has been engineered as an inducible dimerization domain (52). An N-terminal fusion of Abl with FKBP has been shown to cause the inducible dimerization and activation of Abl kinase (53). We used a modified FKBPv domain (Ariad Inc.) to engineer AblNuk-FKBP so that we could use a synthetic dimerizer (AP20187) that does not interact with the abundant endogenous FKBP protein (54). Two copies of the FKBPv domain, tagged with the HA-epitope, were fused in-frame with Abl or AblNuk at the C terminus (Fig. 4a). In transient co-transfection experiments with AblNuk-FKBP and HA-p73, we observed an increase in p73 tyrosine phosphorylation following the addition of AP20187 (not shown). Thus, AblNuk-FKBP retains its ability to interact and phosphorylate p73
.
We then expressed AblNuk-FKBP in p73(/) and p53(/) MEFs and determined its ability to induce apoptosis in these cells. The endogenous Abl protein was mostly cytoplasmic in these MEFs, but it accumulated in the nucleus following treatment with LMB (Fig. 4b). Hence, nuclear import of endogenous c-Abl was not blocked in these MEFs. The AblNuk-FKBP protein was constitutively localized to the nucleus of transfected MEFs, as expected (not shown). Expression of AblNuk-FKBP did not induce apoptosis of p73(/) MEFs, either in the absence or the presence of the dimerizer (Fig. 4c). Transfection with HA-p73 did not cause a significant death response in the p73(/) MEFs. However, co-expression of HA-p73
with AblNuk-FKBP caused dimerizer-dependent apoptosis in p73(/) MEFs (Fig. 4c). To further demonstrate the interdependence between c-Abl tyrosine kinase and p73 in inducing apoptosis, we tested the HA-p73
(Y99F) mutant lacking the Abl phosphorylation site (6). In p73(/) MEFs, co-expression of AblNuk-FKBP with HA-p73
(Y99F) did not induce apoptosis with or without dimerizer (Fig. 4c). These results showed that dimerization of AblNuk-FKBP causes p73-dependent apoptosis. When expressed in p53(/) MEFs, AblNuk-FKBP induced apoptosis in the presence of dimerizer (Fig. 4d). Coexpression of HA-p73
with AblNuk-FKBP in p53(/) MEFs caused apoptosis in the absence of the dimerizer most likely due to the combination of increased p73
and dimerizer-independent Abl kinase activity, but apoptosis was significantly increased following dimerization (Fig. 4d). In p53(/) MEFs, HA-p73
(Y99F) suppressed apoptosis induced by AblNuk-FKBP (Fig. 4d), suggesting a possible dominant negative effect of p73
(Y99F) on the Abl/p73-dependent apoptosis. These results established that AblNuk-FKBP, when activated by dimerization, caused p73-dependent apoptosis that required tyrosine phosphorylation of the p73 protein.
Transient Expression of AblNuk-FKBP Induces Apoptosis of p73-positive Thyroid Cancer Cells upon DimerizationWhen transiently expressed, AblNuk-FKPB preferentially localized to the nucleus, whereas Abl-FKBP localized mainly to the cytoplasm of ARO (p73-positive) and C643 (p73-negative) anaplastic thyroid cancer cells (Fig. 5a). The subcellular distribution of these fusion proteins was not altered by dimerizer treatment (Fig. 5a). We then examined whether the enforced activation of AblNuk-FKBP could stimulate apoptosis by counting transfected cells (marked with a co-transfected GFP-histone H2B fusion protein) with condensed chromatin typical of apoptotic cell death. The basal level of apoptosis (
7%) in vector-transfected cultures was not altered by the addition of dimerizer (Fig. 5b). The transient expression of Abl-FKBP, which localizes to the cytoplasm, did not increase apoptosis either with or without the dimerizer (Fig. 5b). Transient expression of AblNuk-FKBP did not increase apoptosis. However, addition of AP20187 to AblNuk-FKBP transfected cultures resulted in a 3-fold increase in apoptosis (Fig. 5b). Co-expression of a dominant negative p73DD fragment (9) diminished the apoptosis response to dimerized AblNuk-FKBP. Co-expression of the baculovirus p35 protein, a potent inhibitor of caspases (55), also reduced this apoptotic response (Fig. 5b). Dimerizer did not cause an increase in apoptosis when it was added to C643 cells (p73-negative) transfected with Abl-FKBP or AblNuk-FKBP (Fig. 5b). These results showed that dimerization of AblNuk-FKBP, under conditions of transient transfection, could cause p73 and caspase-dependent chromatin condensation in anaplastic thyroid cancer cells.
|
Activation of Stably Expressed AblNuk-FKBP Induces p73 in Thyroid Cancer CellsWe also stably expressed AblNuk-FKBP in ARO and KAK cells through retroviral-mediated gene transfer (Fig. 6a). Initially, we selected for the infected cells by hygromycin resistance and found AblNuk-FKBP localized mostly to the cytoplasm of cells that survived the antibiotics selection. Thus, a selective pressure exists to exclude the stably expressed AblNuk-FKBP from the nucleus. To avoid this selective pressure, we infected ARO and KAK cells and expanded the infected populations without selection. Under this condition, we observed the exclusive nuclear localization of AblNuk-FKBP in the infected cells, which represented approximately half of the population (Fig. 6a). The AblNuk-FKBP was expressed at a level that was comparable with the endogenous ABL protein (Fig. 6b, KAK). Because only 4050% of the cells were infected (Fig. 6e), the level of AblNuk-FKBP could be 2-fold higher than that of the endogenous ABL on a per cell basis. With the ARO cells, AblNuk-FKBP was likely to be at the same level as the endogenous ABL on a per cell basis (Fig. 6b, ARO). A low level of phosphotyrosine (Ptyr) was detected on AblNuk-FKBP in the absence of dimerizer (Fig. 6c, lane 4). Addition of AP20187 caused an increase in Ptyr of AblNuk-FKBP as early as 15 min (Fig. 6c). Importantly, AP20187 caused an increase in the levels of p73
, and a concomitant increase of p21Cip1 without altering the levels of p53 (Fig. 6d). Again, the actual increase in p73
and p21Cip1 on a per cell basis could be higher than depicted by the immunoblots, because only 4050% of the cells expressed AblNuk-FKBP. The in vivo half-life of AP20187 is estimated to be around 12 h (not shown). Therefore, the increase in p73
and p21Cip1 peaked around 48 h after the addition of AP20187 and declined thereafter. Despite the increase in p73 and the induction of p21Cip1, treatment with AP20187 did not inhibit DNA synthesis (not shown), nor did it cause a preferential loss of AblNuk-FKBP-positive cells (Fig. 6e). Thus, stably expressed AblNuk-FKBP can induce p73 and p21Cip1, but it is not sufficient to trigger growth arrest or apoptosis. In other experiments with repeated addition of AP20187 for 48 h, we still did not observe the induction of apoptosis. Retrovirus-mediated AblNuk-FKBP protein expression was significantly lower than the level that can be achieved by plasmid-mediated transient expression. Therefore, our failure to demonstrate an effect of stably expressed AblNuk-FKBP on thyroid cancer cell apoptosis may be ascribed to the insufficient levels of its expression.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The up-regulation of p73 in a malignant form of thyroid cancer is unexpected and appears to be at odds with its proposed function in tumor suppression. Our finding that ARO and KAK cells have developed strategies to accommodate the up-regulation of p73
is also perplexing. These observations imply that p73
may confer some advantage to these cancer cells. Whether the up-regulation of p73
, combined with the nuclear exclusion of c-Abl, can contribute to the development of anaplastic thyroid cancer cells will await further investigation.
![]() |
FOOTNOTES |
---|
¶ These authors made equal contributions to this study.
|| Holds a fellowship from AIRC.
** Supported by a fellowship from the American-Italian Cancer Foundation (AICF).
Supported by a fellowship from the Leukemia Research Foundation (LRF).
¶¶ To whom correspondence should be addressed: Bonner Hall 3326, UCSD, 9500 Gilman Dr., La Jolla, CA 92093-0322. Tel.: 858-534-6253; Fax: 858-822-2002; E-mail: jywang{at}ucsd.edu.
1 The abbreviations used are: p21Cip1, cyclin-dependent kinase inhibitor 1A (p21); c-Abl, normal cellular homologue of the Abelson murine leukemia oncogene; AblNuk, a constitutively nuclear Abl mutant; FKBPv, an engineered AP20187-binding domain that is different from the natural FKBP; AP20187, a synthetic chemical dimerizer inducing homodimerization of Fv-domain containing fusion proteins; LMB, leptomycin B; NLS, nuclear localization sequence; GFP, green fluorescent protein; FKBP, FK506-binding protein; MEF, mouse embryo fibroblasts; HA, hemagglutinin; RT, reverse transcription.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|