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[Cancer Research 63, 6357-6362, October 1, 2003]
© 2003 American Association for Cancer Research

Regular Articles

PTEN Reverses MDM2-mediated Chemotherapy Resistance by Interacting with p53 in Acute Lymphoblastic Leukemia Cells1

Muxiang Zhou2, Lubing Gu, Harry W. Findley, Rong Jiang and William G. Woods

Division of Pediatric Hematology/Oncology/Bone Marrow Transplantation, Emory University School of Medicine, Atlanta, Georgia 30322


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The tumor suppressor PTEN has been associated with the cellular localization of MDM2 in regulation of apoptosis through inhibiting PI3k/Akt signaling. To investigate whether expression of PTEN is involved in MDM2-mediated chemoresistance, we examined a set of acute lymphoblastic leukemia (ALL) cell lines for the expression of PTEN and sensitivity to doxorubicin. Testing 9 ALL cell lines selected for wild-type p53 phenotype and uniformly high levels of MDM2 expression, we initially demonstrated that cell lines with high levels of PTEN expression were sensitive to doxorubicin, whereas lines lacking PTEN expression were generally resistant. Forced expression of PTEN in a PTEN-negative and doxorubicin-resistant ALL line (EU-1) resulted in decreased cell growth and enhanced sensitivity to doxorubicin. Examining the cellular localization of MDM2, we confirmed that the majority of MDM2 is localized in the nucleus in PTEN-negative doxorubicin-sensitive ALL cells, whereas MDM2 is expressed predominantly in the cytoplasm in either PTEN-positive or PTEN-transfected cells. Furthermore, by coimmunoprecipitaton and cotransfection assays, we found that PTEN physically binds p53 in vitro as well as in vivo. Binding of PTEN to p53 attenuated MDM2-mediated p53 inhibition. These results suggest that PTEN inhibits MDM2 and protects p53 through both p13k/Akt-dependent and -independent pathways. Furthermore, loss of PTEN can result in resistance to apoptosis by activating MDM2-mediated antiapoptotic mechanism.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The MDM2proto-oncogene is amplified and frequently overexpressed in a variety of human tumors including leukemia (1, 2, 3) . Overexpression of MDM2 in either transformed normal cells or neoplastic cells enhances tumorigenic potential and resistance to apoptosis (4 , 5) . The major role of MDM2 is to interact directly with the tumor suppressor p53, and block p53-mediated transactivation and apoptosis (6 , 7) . However, the p53 inhibitory property of MDM2 is abolished in certain circumstances in which MDM2 itself is functionally regulated within cells. Although MDM2 finally degrades p53 in the cytoplasm (8) , MDM2 must localize to the nucleus to bind p53, and repress p53-mediated transactivating and apoptotic activities (9) . When MDM2 is sequestrated in the cytoplasm by PTEN, its p53 binding and inhibitory functions are blocked (10) .

PTENis a recently identified tumor suppressor gene (11 , 12) that plays important roles not only in suppressing cancer but also in regulating apoptosis (13 , 14) . Loss of PTEN expression has been detected in many cancers, including those of human brain, breast, prostate, and lymphoid cells (11 , 15, 16, 17) . The major function of PTEN relies on its PI3k3 /Akt-inhibitory activity, and loss of PTEN function results in increased Akt activation (18) . Hyperactivation of Akt exerts antiapoptotic effects through phosphorylation of substrates that directly regulate the apoptotic machinery such as Bad (19 , 20) and caspase-9 (21) , or phosphorylation of substrates that indirectly inhibit apoptosis, such as inhibitor of NF{kappa}B kinases (22 , 23) and MDM2 (24) . PI3k/Akt signaling phosphorylates MDM2, which is necessary for translocation of MDM2 from the cytoplasm into the nucleus (25) . PTEN has been shown to repress the nuclear entry and the p53-inhibitory function of MDM2 through the PI3k/Akt signaling pathway (10) .

Because leukemic cells from a significant percentage of childhood ALL patients overexpress MDM2, and PTEN regulates MDM2 function, we examined the expression of PTEN and its association with MDM2, as well as with sensitivity to doxorubicin-induced apoptosis. By screening a panel of ALL cell lines with wt p53+, MDM2 overexpression, and ectopic PTEN expression, we confirmed that MDM2 was aberrantly localized to the cytoplasm in ALL lines that express high levels of PTEN and are sensitive to doxorubicin. In contrast, MDM2 was localized to the nucleus in lines that lost PTEN expression, and these lines were resistant to doxorubicin. Importantly, we observed that PTEN physically binds p53, and binding of PTEN to p53 diminished MDM2-mediated p53 inhibition. Thus, PTEN reverses MDM2-mediated chemotherapy resistance through both PI3k/Akt-dependent and -independent mechanisms.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines.
Ten cell lines established from children with B-cell precursor-ALL were studied. Three of these lines (EU-1, EU-3, and EU-4) were established in our laboratory at Emory University, and 6 (UOC-B1, UOC-B3, UOC-B4, UOC-B11, SUP-B13, and SUP-B15) were obtained from Stephen D. Smith (University of Kansas, Kansas City, KS). The Reh line was obtained from C. Rosenfeld (INSERM, Villejuif, France). Cultured cells resembled the primary leukemic cells in immunophenotypes as summarized in Table 1Citation and in previous publications (3 , 26) . Except for p53-null EU-4, all of the lines expressed wt p53 and high levels of MDM2 (3) . All of the lines were grown in RPMI 1640 containing 10% fetal bovine serum, 2 mM L-glutamine, 50 units penicillin, and 50 µg/ml streptomycin.


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  		Table 1 Sensitivity of pediatric ALL cell lines to apoptosis induced by IR and doxorubicin

 
Western Blot Assay.
Whole cell protein samples were prepared by lysing cells in a buffer composed of 150 mM NaCl, 50 mM Tris (pH 8.0), 5 mM EDTA, 1% (v/v) NP40, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 25 µg/ml leupeptin for 30 min at 4°C. For detecting the cellular localization of MDM2 and PTEN, nuclear and cytoplasmic fractions were isolated using the NE-PER kit (Pierce) according to the instructions of the manufacturer. Equal amounts of protein extracts were resolved by SDS-PAGE and transferred to nitrocellulose filter. After blocking with buffer containing 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% nonfat milk for 1 h at room temperature, the filter was incubated with specific antibodies for 1 h at room temperature followed by horseradish peroxidase-labeled secondary antibody. Blots were developed using a chemiluminescent detection system (ECL; Amersham Life Science, Buckinghamshire, England).

Gene Transfection and Reporter Assay.
For stable PTENgene transfection, EU-1 cells in exponential growth were transfected with a wt PTEN expression plasmid (pCMV-PTEN) and a catalytically inactive PTEN mutant plasmid (pCMV-PTEN-C124S) provided by Dr. Donald J. Tindall (Mayo Foundation, Rochester, MN). Transfection was performed by electroporation at 300 V, 950 µF. The cells were seeded 48 h after transfection into culture dishes for the selection of G418-resistant colonies. To test the physical interactions of PTEN, MDM2, and p53, EU-4 cells were transiently cotransfected with these plasmids (MDM2 and p53 expression plasmids provided by Dr. Bert Vogelstein, Johns Hopkins University, Baltimore, MD). The cells were lysed 48-h after transfection for coimmunoprecipitation assay as described below. To examine the effect of PTEN on p53- and MDM2-mediated p21 and p65 promoter activity, EU-4 cells were cotransfected with either the p21-promoter-luciferase plasmid (provided by Dr. Moshe Oren, Weizmann Institute of Science, Rehovot, Israel) or p65 promoter construct (pKBCAT; provided by Dr. Klaus Ueberla, Harvard Medical School, Boston, MA) plus different doses of p53, MDM2, and PTEN constructs. At 48 h after transfection, cell extracts were prepared, and CAT activity was evaluated with the CAT ELISA kit (Boehringer Mannheim, Mannheim, Germany). For luciferase assay, cell extracts were prepared with 1x lysis buffer, and then 20 µl aliquots of the supernatant were mixed with 100 µl of luciferase assay reagent (Promega) and analyzed on a Microplate Luminometer (Turner Designs). Both CAT and luciferase activities were normalized to ß-galactosidase activity as an internal control.

Coimmunoprecipitation.
EU-1 and EU-3 cells or EU-4 cells transiently transfected with PTEN, MDM2, and p53 were lysed in a buffer composed of 50 mM Tris (pH 7.6), 150 mM NaCl, 1% NP40, 10 mM sodium phosphate, 10 mM NaF, 1 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin. After centrifugation, 30 µg of the clarified cell lysate was incubated with 15 µl Protein G plus/Protein A-agarose and 1 µg MDM2, p53, or PTEN antibodies, respectively. After 24 h of incubation, the agarose was centrifuged, washed four times with ice-cold lysis buffer, suspended in electrophoresis sample buffer, and boiled for 5 min. The immunoprecipitated protein was additionally analyzed by Western blotting as described above.

Determination of Cell Growth Rate.
PTEN transfected EU-1 cells and control cells (EU-1 transfected with PTENm and EU-1 parental) were cultured in RPMI 1640 containing 10% fetal bovine serum at an initial concentration of 104/ml. Cells were then counted using a hemocytometer under light microscope. Triple flasks were counted for each time point to determine the growth rate.

Cytotoxicity and Apoptosis Assays.
The cytotoxic effect of doxorubicin on ALL cell lines or PTEN-transfected ALL line EU-1 was determined by the XTT assay as described previously (5) . Briefly, cells were cultured in 96-well microtiter plates with different concentrations of doxorubicin for 44 h. XTT (25 µg/well) was then added, and cells were incubated for an additional 4 h. The absorbance of the wells was then read with a microplate reader at a test wavelength of 450 nm and a reference wavelength of 620 nm. Appropriate controls lacking cells were included to determine background absorbance.

Flow cytometry to detect annexin-V staining using a kit from Oncogene (San Diego, CA) was performed to analyze doxorubicin-induced apoptosis in EU-1 cells transfected with PTEN. Cells with or without treatment were washed once with PBS and stained with FITC-annexin-V and propidium iodide according to the manufacturer’s instructions. Stained cells were analyzed using the FACScan (Becton Dickinson) and WinList software (Verity Software House Inc.).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sensitivity of ALL Cell Lines to DNA-damaging Agents.
We have tested previously the sensitivity of the 9 wt-p53 +, MDM2 overexpressing ALL cell lines to IR by DNA fragmentation assay (26) . As summarized in Table 1Citation , 3 lines (UOC-B1, Reh, and EU-1) were resistant to IR as demonstrated by lack of DNA fragmentation after treatment with IR at 10 Gy for 5 h. Similar treatment produced DNA fragments in the other 6 lines, and significant DNA ladder formation was observed in 2 (UOC-B11 and EU-3) of these 6 lines. In the present study, we additionally examined the response of these ALL lines to doxorubicin. Similar results were obtained as shown in Table 1Citation . IR-resistant lines were also resistant to doxorubicin (IC50 >1.5 µM after 48 h of treatment), and 2 lines with significant DNA fragmentation after IR treatment were very sensitive to doxorubicin (IC50 <0.15 µM).

Expression of PTEN, PI3k, and Akt in ALL Cell Lines.
The results of Western blot analysis for MDM2, PTEN, PI3k, and Akt protein expression are shown in Fig. 1Citation . Similar to our previous report that wt-p53+ ALL lines expressed high levels of MDM2 mRNA (3) , all 9 of the lines expressed high levels of MDM2 protein. The expression of PTEN was absent in 3 of the 9 ALL lines including Reh and EU-1 that were resistant to IR and doxorubicin. Two lines (UOC-B11 and EU-3) that were very sensitive to IR and doxorubicin expressed relatively high levels of PTEN compared with those lines that were less sensitive to IR and doxorubicin. Interestingly, the expression of PI3k and Akt (including the phosphorylated form of Akt, Akt-p) in these lines was correlated with the expression of PTEN in a functionally sequential manner. Cell lines without PTEN expression expressed higher levels of PI3k, lower levels of Akt, and constitutively higher levels of Akt-p than lines with PTEN expression. UOC-11 and EU-3 lines that expressed relative high levels of PTEN showed elevated expression of Akt, although the expression of PI3k and Akt-p was similar to that in lines with lower levels of PTEN expression.



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  		Fig. 1. Western blot assay for expression of MDM2, PTEN, and PTEN-associated gene products PI3k, Akt, and phosphorylated Akt (Akt-p) in 9 ALL cell lines. Protein (20 µg) from cell lysates was electrophoresed in SDS-PAGE gels, transferred to nitrocellulose, and probed with antibodies as indicated. Actin serves as a control for equal protein loading and protein integrity.

 
Effect of Transfected PTEN on Cell Growth and Sensitivity to Doxorubicin.
As described above, the expression of PTEN appeared to be associated with sensitivity of ALL cells to apoptosis induced by DNA-damaging agents. To additionally confirm the role of PTEN in regulating cell growth and apoptosis, we transfected the PTEN gene into EU-1 cell lines without endogenous PTEN expression. Transfection of a phosphatase-inactive PTENm plasmid into EU-1 was also performed as a control. As shown in Fig. 2Citation A, both transfected PTEN and PTENm proteins were stably expressed. We compared the growth rate of EU-1 cells expressing PTEN to EU-1 expressing PTENm and EU-1 transfected with empty neo plasmid. EU-1/PTEN cells showed significantly decreased growth rate compared with EU-1/neo cells. As shown in Fig. 2Citation B, the growth rate of EU-1/PTEN was only half of that for EU-1/neo after 4 or 5 days of culture. Unlike wt PTEN, the expression of PTENm in EU-1 cells partially decreased the growth rate of these cells.



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  		Fig. 2. Transfection of PTEN genes and their effects on cell growth in EU-1 ALL cell line. A, EU-1 cells lacking PTEN expression were transfected with the expression plasmid PTEN and control plasmids, including a catalytically inactive PTENm and a empty neo plasmid. Stable expression of transfected PTEN was detected by Western blot assay. EU-3 cells expressing high levels of endogenous PTEN served as control. B, effect of enforced PTEN expression on growth rate of EU-1 cells. Cells transfected with PTEN gene and control cells transfected either with PTENm construct or empty neo plasmid were cultured in 10 ml of RPMI 1640 supplemented with 10% fetal bovine serum at an initial concentration of 104/ml. Cells were counted every day. Data for the total number of cells (mean for triplicate cultures) are shown; bars, ±SD.

 
Furthermore, we evaluated the sensitivity of PTEN-transfected cells to doxorubicin. XTT assay showed that EU-1/PTEN cells were more sensitive to doxorubicin than were the EU-1/neo cells. Sensitivity to doxorubicin of EU-1/PTENm cells was less than EU-1/PTEN but higher than EU-1/neo. As shown in Fig. 3Citation A, a significant difference was noted in mean cell survival after 48-h treatment between EU-1/PTEN and EU-1/neo cells at doxorubicin concentrations of >=1 µM (P < 0.05; t test). Consistent with these observations, a flow-cytometric apoptosis assay showed that an increased percentage of EU-1/PTEN cells (from 10% to 19%) were annexin-V positive at 6 h after doxorubicin treatment as compared with EU-1/neo cells (from 4% to 6%; Fig. 3BCitation ).



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  		Fig. 3. Comparison of PTEN-transfected and control-transfected EU-1 cells for doxorubicin (Dox) sensitivity. A, EU-1 cells stably transfected with either PTEN or control plasmids (PTENm and neo vector only) were cultured with different concentrations of Dox for 48 h, and cell survival was determined by XTT assay. B, time course of apoptosis induced in EU-1 PTEN and EU-1 neo cells by Dox. Cells were treated with Dox (1.5 µM) for the indicated time, and apoptotic cells were detected by flow cytometry to examine annexin-V staining.

 
Association of PTEN with the Cellular Localization of MDM2.
It has been demonstrated that activation (phosphorylation) of Akt increased the translocation of MDM2 from the cytoplasm to the nucleus. We anticipated that high levels of PTEN expression would restrict MDM2 to the cytoplasm, and that loss of PTEN expression would result in higher levels of MDM2 in the nucleus. We selected two lines, EU-1 (PTEN-/doxorubicin-resistant), and EU-3 (PTEN+/doxorubicin-sensitive), to evaluate MDM2 cellular localization. EU-1 cells transfected with PTEN (EU-1/PTEN) were also examined. Western blot assay showed high levels of MDM2 in the cytoplasm of EU-3 and EU-1/PTEN cells. In contrast, EU-1 parental cells expressed enhanced levels of nuclear MDM2 (Fig. 4)Citation . Because PTEN restricted MDM2 to the cytoplasm, the interaction of MDM2 with p53 in the cytoplasm resulted in degradation of p53. Although the unstimulated ALL cells expressed low levels of p53, we were able to detect p53 in the cytoplasm and nucleus of EU-1 cells, but in the nucleus of EU-3 and EU-1/PTEN cells (Fig. 4)Citation . As also shown in Fig. 4Citation , transfected PTEN was localized in the cytoplasm, whereas endogenous PTEN can be detected in both cytoplasm and nucleus.



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  		Fig. 4. Localization of MDM2, p53 and PTEN proteins in nuclear (N) and cytoplasmic (C) cell fractions from EU-3, EU-1, and PTEN-transfected EU-1 cells was examined by Western blot assay. {alpha}-Tubulin and nucleolin served as positive controls for nuclear and cytoplsmic fractions.

 
Physical Interactions between PTEN and p53.
As described above, transfection of the PTEN phosphatase-inactive mutant did not completely block the effect of PTEN on cell growth and sensitivity to doxorubicin. We hypothesized that PTEN may regulate cell growth and apoptosis through an alternative, phosphatase-independent pathway. Therefore, we studied the direct interactions among PTEN, p53, and MDM2 by coimmunoprecipitation assay. As anticipated, endogenous MDM2 binds p53 in both EU-1 and EU-3 cells. An interesting finding is that endogenous PTEN can bind p53 but not MDM2 in PTEN-expressing EU-3 cells (Fig. 5A)Citation .



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  		Fig. 5. Physical interaction of PTEN and p53 examined by immunoprecipitation (IP)-Western blot assays. A, binding of endogenous PTEN to p53 in ALL cells. Cell lysates from EU-1 and EU-3 treated with IR (10 Gy) for 3 h were precipitated with antibodies as indicated. Normal mouse or rabbit antibodies served as control (Con). Proteins in immune complexes were separated on denaturing gels, transferred to filters, and detected by Western blotting with anti-MDM2, anti-p53, and anti-PTEN antibodies; antibodies for Western blotting were from different species than those used in IP. B, interaction of ectopic PTEN, p53, and MDM2 proteins expressed in EU-4 cells. EU-4 cells without endogenous MDM2, p53, and with low level of PTEN expression were transiently cotransfected with MDM2 and either wild-type (w) or mutant (m) -p53 and -PTEN plasmids, as indicated below each panel. Transfection was performed by electroporation at 300 V and 950 µF. At 48 h post-transfection, cell protein was prepared, and IP-Western blot was conducted as described in A.

 
To additionally confirm the p53 binding activity of PTEN, we transfected PTEN, p53, and MDM2 into EU-4, a p53-/MDM2- ALL line with low levels of PTEN expression (Fig. 6Citation , inset). Similar results showing physical interaction among ectopic PTEN, p53, and MDM2 were obtained by coimmunoprecipitation assay. Additionally, we found that the codon-248 p53 mutant bound MDM2 but not PTEN, whereas the PTEN mutant was able to bind p53 (Fig. 5B)Citation .



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  		Fig. 6. Effect of PTEN on MDM2-mediated p53 transcription in transient transfection assays. EU-4 cells were cotransfected with 5 µg p21-promoter plasmid and 5 µg p53 expression plasmid alone (Lane 2), either with increasing amounts (1, 3, and 5 µg) of MDM2 plasmid (Lanes 3–5) or with increasing amounts (1, 3, and 5 µg) of PTEN plasmid in the presence of a fixed amount (5 µg) of MDM2 (Lanes 6–8). Controls (Lanes 9–13) consisted of cotransfected plasmids (5 µg), including the PTENm as indicated. Total amount of plasmid was adjusted to 20 µg/transfection with an empty plasmid. Electroporation was performed at 300 V and 950 µF. At 48 h after transfection, cell extracts were analyzed for luciferase activity. Data represent mean of three independent experiments normalized to ß-galactosidase activity; bars, ±SD. Fold induction was determined from the transfection with p21-promoter plasmid alone. Titrations of the cellular extracts from plasmid-transfected cells were analyzed for p53, MDM2 and PTEN by Western blot analysis (inset).

 
Functional Interactions of PTEN, p53, and MDM2.
Because PTEN can bind p53, we next wanted to know whether binding of PTEN to p53 regulates p53 function. We performed cotransfection and gene reporter assays to evaluate the effect of PTEN on p53-mediated transcriptional activity. From the data presented in Fig. 6Citation , it can be seen that p53 induced p21 promoter activity (Fig. 6Citation , Lanes 2). This inducing effect was inhibited by MDM2 in a dose-dependent manner (Fig. 6Citation , Lanes 3–5). Addition of PTEN reversed the p53-inhibitory activity of MDM2 (Fig. 6Citation , Lanes 6–8), but did not increase the p53 activity in the absence of MDM2 (Fig. 6Citation , Lane 10). Similarly, addition of PTEN mutant reversed the p53-inhibitory activity of MDM2 (Fig. 6Citation , Lane 9) and failed to increase p53 activity directly (Fig. 6Citation , Lane 11). No effect of PTEN and PTEN mutant on p21 promoter activity was observed (Fig. 6Citation , Lanes 12 and 13, respectively). Titrations of transfected p53, MDM2, and PTEN in cellular extracts were determined by Western blot assay (Fig. 6Citation , inset).

Furthermore, we performed a similar experiment to detect the effect of PTEN on p53-mediated transcriptional-inhibitory activity regulated by MDM2. As shown in Fig. 7Citation , p53 suppressed p65-promoter (pKBCAT) activity, and this inhibitory effect was abolished by MDM2. Addition of PTEN reversed the p53-inhibitory effect of MDM2. Consistent with our previous finding that MDM2 transcriptionally activated the p65 promoter in a p53-independent manner (5) , MDM2 increased pKBCAT activity in the absence of p53 (Fig. 7Citation , Lane 10). In this experiment, addition of PTEN did not show any effect on MDM2-mediated pKBCAT activity (Fig. 7Citation , Lane 11).



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  		Fig. 7. Effect of PTEN on p53- and MDM2-mediated p65 promoter activity. EU-4 cells were cotransfected with 5 µg of pKBCAT (containing the p65 promoter) and 5 µg p53 expression plasmid alone (Lane 2), either with increasing amounts (1, 3, and 5 µg) of MDM2 plasmid (Lanes 3–5) or with increasing amounts (1, 3, and 5 µg) of PTEN plasmid in the presence of a fixed amount (5 µg) of MDM2 (Lanes 6–8). Controls (Lanes 9–11) included cotransfected plasmids (5 µg). Total amount of plasmid was adjusted to 20 µg/transfection, using an empty plasmid. Electroporation was performed as described in Fig. 6Citation . At 48 h post-transfection, cell extracts were analyzed for CAT protein expression with a CAT ELISA kit. Data represent mean of three independent experiments normalized to ß-galactosidase activity; bars, ±SD. The value of one is assigned to CAT expression from the transfection with pKBCAT plasmid alone.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We reported previously that the MDM2 oncogene is overexpressed in a significant percentage of childhood ALL (3 , 27) . Although MDM2-overexpressing leukemic cells are typically resistant to the chemotherapeutic drug doxorubicin, a subset of cases overexpress MDM2 and are sensitive to doxorubicin (27) . On the basis of studies of pediatric ALL cell lines, we additionally evaluated the regulation of MDM2 and its role in chemotherapy-induced apoptosis. These studies demonstrated that the tumor suppressor gene PTEN regulated the cellular localization of MDM2 and directly bound p53, resulting in abrogation of MDM2-mediated inhibition of p53. Moreover, overexpression of MDM2 in the absence of PTEN expression conferred resistance to apoptosis induced by chemotherapeutic drugs.

PTEN is a tumor suppressor gene inactivated in many common malignancies, including acute myeloid leukemia (28) . The status of PTEN expression in pediatric ALL has not been reported previously. By Western blot assay to screen the expression of PTEN protein in 36 primary leukemic bone marrow samples from children with ALL and 27 ALL cell lines, we found that 12 of the 36 (33%) primary bone marrow specimens and 11 of the 27 (41%) ALL lines have lost PTEN expression.4 To evaluate the interaction of PTEN with MDM2 and p53, we selected 9 B-cell precursor-ALL lines with high levels of MDM2 expression and a wt-P53 phenotype to evaluate the relationship between PTEN expression and sensitivity to doxorubicin. We found that lines with high levels of endogenous or enforced PTEN expression were sensitive to doxorubicin, whereas lines lacking PTEN expression were generally resistant. Because all of the lines studied overexpressed MDM2 and expressed wt-p53, it appears that PTEN may play a more important role than MDM2 in determining sensitivity to doxorubicin.

It has been shown previously that PTEN regulates cell-survival signaling through the PI3k/Akt pathway. Loss of PTEN expression results in hyperactivation of Akt. This is consistent with our observation that PTEN-negative lines had constitutively activated Akt. Previous studies have demonstrated that activation of Akt phosphorylates MDM2 (24) , which is required for translocation from the cytoplasm to the nucleus (25) ; furthermore, PTEN inhibits PI3k/Akt, resulting in accumulation of MDM2 in the cytoplasm (10) . In agreement with these results, we found that the majority of MDM2 is localized in the nucleus in PTEN-negative, doxorubicin-sensitive ALL cells, whereas MDM2 is expressed predominantly in the cytoplasm in either PTEN-positive or PTEN-transfected cells. However, PTEN-expressing lines did not show significantly reduced PI3k and Akt-p, although 2 lines with high levels of PTEN expression displayed increased pro-Akt. In addition, transfection of PTEN into the PTEN-negative line EU-1 did not significantly inhibit Akt-p expression (data not shown). Similarly, transfection of phosphatase-inactive PTEN mutant resulted in decreased cell growth and increased sensitivity to doxorubicin. These results indicate that PTEN-mediated cell survival and apoptosis is partially independent of the PI3k/Akt pathway.

Consistent with a very recent study by Freeman et al. (29) showing that PTEN interacts directly with p53, we demonstrated that PTEN binds p53 in vitro and in vivo. In our study, both PTEN and PTEN mutant are able to bind p53. Although we do not have evidence to support whether PTEN binds p53 in competition with MDM2, binding of PTEN to p53 seems to diminish the binding of MDM2 to p53 as demonstrated in Fig. 5Citation A, in which coprecipitated p53 or MDM2 in PTEN+ EU-3 cells is less than that in PTEN- EU-1 cells. Furthermore, by cotransfection and gene reporter assays, we demonstrated that PTEN regulated the transcriptional activity of p53 via inhibition of MDM2, suggesting that binding of PTEN to p53 in vivo abrogate the binding of MDM2 to p53. In addition, we did not detect binding of PTEN to MDM2 by coimmunoprecipitation assay. Furthermore, cotransfection assay did not detect an effect of PTEN on MDM2-mediated activation of the p65 subunit of NF{kappa}B in the absence of p53, suggesting that PTEN does not directly regulate MDM2.

In conclusion, the present study has demonstrated that loss of PTEN and overexpression of MDM2 are two important events in regulating sensitivity to chemotherapy in childhood ALL. Although overexpression of MDM2 occurs frequently in childhood ALL and is associated a wt-p53 phenotype, loss of PTEN is required for the development of resistance to chemotherapy in MDM2-overexpressing ALL cells. Endogenous PTEN expression or PTEN-transfer into PTEN-null ALL cells induces a decrease of MDM2-mediated inhibition of p53. PTEN suppresses MDM2 function through both the PI3k/Akt-dependent pathway (by inhibiting translocation of MDM2 to the nucleus) and the PI3k/Akt-independent mechanism (by directly binding to p53). Thus, our results contribute to defining the molecular mechanisms for loss of MDM2 antiapoptotic function in childhood ALL and suggest that the PTEN gene may be a therapeutic target for treatment of refractory leukemia.


   ACKNOWLEDGMENTS
 
We thank Drs. Donald J. Tindall, Bert Vogelstein, Moshe Oren, and Klaus Ueberla in providing us with expression plasmids used in this work.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the National Cancer Institute, NIH (R01 CA82323), CURE Childhood Cancer, Inc., Children’s Healthcare of Atlanta. Back

2 To whom requests for reprints should be addressed, at Division of Pediatric Hematology/Oncology/BMT, Emory University School of Medicine, 2040 Ridgewood Drive, N.E., Atlanta, GA 30322. Phone: (404) 727-1426; Fax: (404) 727-4455; E-mail: mzhou{at}emory.edu Back

3 The abbreviations used are: PI3k, phosphatidylinositol 3'-kinase; NF{kappa}B, nuclear factor {kappa}B; CAT, chloramphenicol acetyltransferase; XTT, 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt; wt, wild-type; IR, ionizing radiation; PTENm, mutant PTEN. Back

4 Unpublished observations. Back

Received 4/ 9/03. Revised 6/16/03. Accepted 7/22/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Oliner J. D., Kinzler K. W., Meltzer P. S., George D. L., Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature (Lond.), 358: 80-83, 1992.[Medline]
  2. Bueso-Ramos C. E., Yang Y., deLeon E., McCown P., Stass S. A., Albitar M. The human MDM-2 oncogene is overexpressed in leukemia. Blood, 82: 2617-2623, 1993.[Abstract/Free Full Text]
  3. Zhou M., Yeager A. M., Smith S. D., Findley H. W. Overexpression of the MDM2 gene by childhood acute lymphoblastic leukemia cells expressing the wild-type p53 gene. Blood, 85: 1608-1614, 1995.[Abstract/Free Full Text]
  4. Fakharzadeh S. S., Trusko S. P., George D. L. Tumorigenic potential associated with enhanced expression of a gene that is amplified in a mouse tumor cell line. EMBO J., 10: 1565-1569, 1991.[Medline]
  5. Gu L., Findley H. W., Zhou M. MDM2 induces NF-kB/p65 expression transcriptionally through Sp1-binding sites: A novel, p53-independent role of MDM2 in adriamycin resistance in acute lymphoblastic leukemia. Blood, 99: 3367-3375, 2002.[Abstract/Free Full Text]
  6. Momand J., Zambetti G. P., Olson D., George D., Levine A. J. The MDM-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69: 1237-1245, 1992.[Medline]
  7. Chen J., Wu X., Lin J., Levine A. J. mdm-2 inhibits the G1 arrest and apoptosis functions of the p53 tumor suppressor protein. Mol. Cell. Biol., 16: 2445-2452, 1996.[Abstract]
  8. Tao W., Levine A. J. Nucleo-cytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc. Natl. Acad. Sci. USA, 96: 3077-3080, 1999.[Abstract/Free Full Text]
  9. Prives C. Signaling to p53: breaking the MDM2–p53 circuit. Cell, 95: 5-8, 1998.[Medline]
  10. Mayo L. D., Dixon J. E., Durden D. L., Tonks N. K., Donner D. B. PTEN protects p53 from Mdm2 and sensitizes cancer cells to chemotherapy. J. Bio. Chem., 277: 5484-5489, 2002.[Abstract/Free Full Text]
  11. Li J., Yen C., Liaw D., Podsypanina K., Bose S., Wang S. I., Puc J., Miliaresis C., Rodgers L., McCombie R., Bigner S. H., Giovanella B. C., Ittmann M., Tycko B., Hibshoosh H., Wigler M. H., Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science (Wash. DC), 275: 1943-1947, 1997.[Abstract/Free Full Text]
  12. Steck P. A., Pershouse M. A., Jasser S. A., Yung W. K., Lin H., Ligon A. H., Langford L. A., Baumgard M. L., Hattier T., Davis T., Frye C., Hu R., Swedlund B., Teng D. H., Tavtigian S. V. ldentification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat. Genet., 15: 356-362, 1997.[Medline]
  13. Di Cristofano A., Pandolfi P. P. The multiple roles of PTEN in tumor suppression. Cell, 100: 387-390, 2000.[Medline]
  14. Huang H., Cheville J. C., Pan Y., Roche P. C., Schmidt L. J., Tindall D. J. PTEN induces chemosensitivity in PTEN-mutated prostate cancer cells by suppression of Bcl-2 expression. J. Bio. Chem., 276: 38830-38836, 2001.[Abstract/Free Full Text]
  15. Depowski P. L., Rosenthal S. I., Ross J. S. Loss of expression of the PTEN gene proein product is associated with poor outcome in breast cancer. Mod. Pathol., 14: 672-676, 2001.[Medline]
  16. Hyun T., Yam A., Pece S., Xie X., Zhang J., Mili T., Gutkind J. S., Li W. Loss of PTEN expression leading to high Akt activation in human multiple myelomas. Blood, 96: 3560-3568, 2000.[Abstract/Free Full Text]
  17. Sakai A., Thieblemont C., Wellmann A., Jaffe E. S., Raffeld M. PTEN gene alteration in lymphoid neoplasms. Blood, 92: 3410-3415, 1998.[Abstract/Free Full Text]
  18. Cantley L. C., Neel B. G. New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway. Proc. Natl. Acad. Sci. USA, 96: 4240-4245, 1999.[Abstract/Free Full Text]
  19. Datta S. R., Dudek H., Tao X., Masters S., Fu H., Gotoh Y., Greenberg M. E. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell, 91: 231-241, 1997.[Medline]
  20. del Peso L., Gonzalez-Garcia M., Page C., Herrera R., Nunez G. Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science (Wash. DC), 278: 687-689, 1997.[Abstract/Free Full Text]
  21. Cardone M. H., Roy N., Stennicke H. R., Salvesen G. S., Franke T. F., Stanbrldge E., Frlsch S., Reed J. C. Regulation of cell death protease caspase-9 by phosphorylation. Science (Wash. DC), 282: 1318-1321, 1998.[Abstract/Free Full Text]
  22. Ozes O. N., Mayo L. D., Gustin J. A., Pfeffer S. R., Pfeffer L. M., Donner D. B. NF-KB activation by tumour necrosis factor requires the Akt serine-threonine kinase. Nature (Lond.), 401: 82-85, 1999.[Medline]
  23. Romashkova J. A., Makarov S. S. NF-KB is a target of AKT in anti-apoptotic PDGF signalling. Nature (Lond.), 401: 86-90, 1999.[Medline]
  24. Ashcroft M., Ludwig R., Woods D. B., Copeland T. D., Weber H. O., MacRae E. J., Vousden K. H. Phosphorylation of HDM2 by Akt. Oncogene, 21: 1955-1962, 2002.[Medline]
  25. Mayo L. D., Donner D. B. A. phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl. Acad. Sci. USA, 98: 11598-11603, 2001.[Abstract/Free Full Text]
  26. Findley W. H., Gu L., Yeager A. M., Zhou M. Expression and regulation of Bcl-2, Bcl-xl and Bax correlate with p53 status and sensitivity to apoptosis in childhood acute lymphoblastic leukemia. Blood, 89: 2986-2993, 1997.[Abstract/Free Full Text]
  27. Zhou M., Gu L., Abshire T. C., Homans A., Billett A. L., Yeager A. M., Findley H. W. Incidence and prognostic significance of MDM2 oncoprotein overexpression in relapsed childhood acute lymphoblastic leukemia. Leukemia (Baltimore), 14: 61-67, 2002.
  28. Liu T., Lin P., Chang J., Lee J., Chen T., Lin S. Mutation analysis of PTEN/MMAC1 in acute myeloid leukemia. Am. J. Hem., 63: 170-175, 2000.
  29. Freeman D. J., Li A. G., Wei G., Li H., Kertesz N., Lesche R., Whale A. D., Martinez-Diaz H., Rozengurt N., Cardiff R. D., Liu X. PTEN tumor suppressor regulates p53 protein levels and activity through phosphatase-dependent and -independent mechanisms. , 3: 117-130, 2002.



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