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Signaling and Regulation

Pim Family Kinases Enhance Tumor Growth of Prostate Cancer Cells

¹ Hollings Cancer Center and ² Department of Pathology, Medical University of South Carolina, Charleston, South Carolina; ³ Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, Colorado; and ⁴ Center for Molecular Biology and Gene Therapy, Loma Linda University School of Medicine, Loma Linda, California

Requests for reprints: Andrew S. Kraft, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425. Phone: 843-792-8284; Fax: 843-792-9456. E-mail: Kraft{at}musc.edu

	Abstract

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
Recent analyses indicate that the expression of the Pim-1 protein kinase is elevated in biopsies of prostate tumors. To identify the mechanism by which the Pim kinases may affect the growth of prostate tumors, we expressed Pim-1, Pim-2, or a kinase-dead Pim-2 protein in human PC3 prostate cancer cells. On implantation of the transfectants in nude mice, the growth of the cells expressing Pim-1 or Pim-2 was significantly faster than the growth of the control cells transfected with the neomycin-resistant gene or the kinase-dead Pim-2 protein. When grown in medium, the doubling time of the Pim-1 and Pim-2 transfectants was faster (0.75 days) than that of the control cells (1.28 days). We, therefore, examined the ability of Pim to control the phosphorylation of proteins that regulate protein synthesis. On growth factor starvation or rapamycin treatment, the Pim-1 and Pim-2 transfectants maintained their ability to phosphorylate 4E-BP1 and S6 kinase, although this phosphorylation did not occur in the control-transfected PC3 cells. We have found that the cellular levels of c-Myc were elevated in the Pim-1 and Pim-2 transfectants under these conditions. The Pim-1 and Pim-2 transfectants have lower levels of serine/threonine protein phosphatase 2A (PP2A) activity and the - and ß-subunit B56 of the PP2A phosphatase do not coimmunoprecipitate in these cells. Thus, the effects of Pim on PP2A activity may mediate the levels of c-Myc and the phosphorylation of proteins needed for increased protein synthesis. Both of these changes could have a significant impact on tumor growth.

	Introduction

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
Prostate cancer is the most common malignancy diagnosed in men in the United States. Recent evidence suggests that the Pim family of protein kinases may play a role in the development or progression of this cancer. The report of elevated levels of pim-1 in human prostate tumors on cDNA and microarray analysis implicates the Pim family of serine/threonine kinases in the progression of human prostate tumors (1). In transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene (2). Although the Pim kinases have been identified as oncogenes in transgenic models, by themselves they are only weakly transforming. They have, however, been shown to greatly enhance the ability of c-myc gene to induce lymphomas (3, 4). The regulation of the expression of the Pim kinases and their function has been analyzed extensively in hematopoietic cells. It has been shown that the levels of Pim-1 and Pim-2 protein can be regulated by the addition of granulocyte-macrophage colony stimulating factor, interleukin-3, and interleukin-7 to normal hematopoietic cells (5) through activation of the Janus-activated kinase/signal transducers and activators of transcription pathway (6). In addition, tumor necrosis factor and Toll-like receptor ligands have been shown to induce the expression of Pim-1 (7-9). Although the overexpression of either Pim-1 or Pim-2 factor–dependent hematopoietic cells makes them resistant to apoptosis induced by interleukin-3 withdrawal (10-12), kinase-dead mutants of pim-1 do not protect against apoptosis (13, 14). It also has been shown that the BH3 protein BAD can be phosphorylated by Pim-1 and Pim-2 and protect against apoptosis (12, 15).

Other previously described phosphorylation targets include HP-1 (16), cdc 25A phosphatase (17), and SOCS-1 (18). More recently, Pim-2 has been shown to phosphorylate the ribosomal protein 4E-BP1, causing it to dissociate from eIF-4E, which may affect protein synthesis (11). These data indicated that the ability of these protein kinases to inhibit cell death and regulate tumorigenesis is likely controlled by phosphorylation of specific target proteins. To explore the role of Pim in human prostate cancer, we have established overexpressor cell lines in PC3 cells.

	Results and Discussion

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
To investigate the role of the Pim protein kinase family in controlling prostate cancer growth, we have created PC3 human prostate cancer cell lines that overexpress either murine Pim-1 or Pim-2. At the amino acid level, human and mouse Pim-1 are 89.9% identical, whereas the Pim-2s are 86.4% identical. We have created two control cell lines by transfecting PC3 cells with either a kinase-inactive form of Pim-2 that was generated by mutating Lys¹²⁰ to alanine using the Quik Change Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) or the neomycin-resistance gene (Fig. 1A). These are called PC3/Pim-1, PC3/Pim-2, PC3/Pim-2 K/A, and PC3/neo, respectively. To evaluate the effect of increasing Pim levels on tumor growth, the cell lines were injected subcutaneously into BALB/c nu/nu mice. Both PC3/Pim-1– and PC3/Pim-2–derived tumors (overlapping growth curves in Fig. 1B) grew significantly faster than PC3/neo–derived tumors when comparing tumor size on day 33 (paired Student's t test PC3/neo compared with PC3/Pim-1, P < 1.36 x 10^–6; PC3/neo compared with PC3/Pim-2, P < 3.33 x 10^–6). Interestingly, the kinase-dead Pim-2–transfected cells did not grow well as tumors (Fig. 1B). It is possible that kinase-dead Pim acts as a dominant-negative mutation inhibiting protein synthesis or phosphorylation of specific substrates and blocking tumor growth.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Pim kinases enhance the growth PC3 prostate tumor cells implanted in BALB/c nu/nu mice. A. Cell lines were established from PC3 cells that had been transfected with Pim-1, Pim-2, a kinase-dead mutation of Pim-2 (Pim-2 K/A), or the neomycin resistance gene. The expression of Pim-1 and Pim-2 in the cell lines is equivalent as indicated by Western blot analysis of cell lysates on 10% SDS-PAGE using monoclonal antibodies (12) to probe the blotted polyvinylidene difluoride membrane. The PC3/neo transfectant does not express levels of Pim-1 or Pim-2 that are detectable using this technique. B. PC3/neo, PC3/Pim-1, PC3/Pim-2, and PC3/Pim-2 K/A cells (2 x 106 per xenograft) were injected subcutaneously into the flanks of BALB/c nu/nu mice with five mice in each group with four tumors injected into each mouse. The tumor volume was calculated from caliper measurements in two dimensions at the indicated time points. Points, mean of 20 measurements (four tumors per mouse and five mice per group); bars, SD. The growth of Pim-1– and Pim-2–containing tumors are shown as overlapping lines.

View larger version (26K): [in this window] [in a new window]   FIGURE 2. Expression of Pim-1 or Pim-2 enhances the doubling time of PC3 prostate tumor cells in culture. A. The PC3 cells were plated at low density in triplicate wells and the cells harvested and counted at the indicated time points. Points, average of triplicate determinations; bars, SD. B. PC3 cell lines were starved of serum (–S) for 24 hours then either treated with rapamycin (80 nmol/L) for an additional 24 hours (+Ra) or maintained under serum-free conditions. The cells were then trypsinized, fixed in 70% ethanol, stained with propidium iodide, and subjected to DNA histogram analysis by fluorescence-activated cell sorting. The results presented are the average of triplicate determinations. However, cell cycle histograms representing individual experiments are shown.

4E-BP1 is highly phosphorylated on Thr³⁷, Thr⁴⁶, Thr⁷⁰, Ser⁶⁵, Ser⁸³, Ser¹⁰¹, and Ser¹¹². A two-stage mechanism of phosphorylation has been proposed in which Thr³⁷ and Thr⁴⁶ are phosphorylated and then allow phosphorylations to occur on Ser⁶⁵ and Thr⁷⁰ (21). The latter two phosphorylations are essential for inhibiting the binding of 4E-BP-1 to eIF4E (22). A number of the phosphorylations may be minor, including Ser⁸³, or constitutive, e.g., Ser¹⁰¹ and Ser¹¹² (23). More highly phosphorylated 4E-BP1 has been associated with the malignant phenotype in human mammary epithelial cells (24). On SDS polyacrylamide gels, the more highly phosphorylated 4E-BP1 has been labeled , whereas the least phosphorylated form is denoted (Fig. 3A; ref. 24). Multiple protein kinase pathways are thought to play a role in regulating phosphorylation of 4E-BP1. TOR activity may be required as a priming event stimulating the phosphorylation of Thr³⁷ and Thr⁴⁶. The protein kinases that phosphorylate Ser⁶⁵ and Thr⁷⁰ have not been fully identified. It has been suggested that these phosphorylations may be downstream of the extracellular signal-regulated kinase or protein kinase B and directly regulated by insulin and phorbol esters (25, 26). We show that the addition of rapamycin, and to a lesser extent serum starvation, to wild-type PC3 cells markedly diminishes the phosphorylation of Thr³⁷, Thr⁴⁶, and Thr⁷⁰ (Fig. 3A). In Pim-containing cells growing in serum, the presence of either Pim-1 or Pim-2 seems to enhance the phosphorylation of 4E-BP1. In Pim-2–containing cells, neither serum starvation alone nor in combination with rapamycin decreases this phosphorylation. In Pim-1–containing PC3 cells, there is a clear decrease in phosphorylation with these treatments although there is no decrease to baseline. It is possible that Pim-1 may not function identically to Pim-2. We have found that transfected Pim-1 is largely located in the nucleus (27), whereas Pim-2 is thought to be cytoplasmic. Pim-1 could function in the nucleus to effect the transcription of proteins that modify the function of TOR, e.g., rictor and raptor, or specific phosphatases that regulate these proteins. Pim-2 could work directly in the cytoplasm to phosphorylate 4E-BP1 or regulate the activity of specific phosphatases.

View larger version (51K): [in this window] [in a new window]   FIGURE 3. Expression of Pim-1 or Pim-2 kinase maintains phosphorylation of key enzymes on serum starvation or treatment with rapamycin. A. PC3 cell lines containing either the neomycin-resistance gene, Pim-1, Pim-2, or Pim-2 K/A, were grown in serum, or after serum starvation for 24 hours, fresh serum-free medium was added followed by an additional 24 hours incubation with or without rapamycin (80 nmol/L). To examine the ability of serum to overcome inhibition by rapamycin (80 nmol/L), a portion of the serum-starved rapamycin (80 nmol/L)–treated cells was incubated with 20% FCS for 30 minutes before homogenization. Cellular extracts were electrophoresed on a 12% SDS-PAGE gel and then transferred to polyvinylidene difluoride membranes. Additional extracts were run to probe for S6 kinase isoforms. Arrow, phosphorylated p70 and p85 S6K protein; arrows, multiple forms of 4E-BP1doublet points at the multiple phosphorylated forms of 4E-BP1. B. RNA interference was used to validate the role of Pim-2 in controlling 4E-BP1 phosphorylation. PC3 cells expressing Pim-2 were transfected with an RNA interference that knocks down Pim-2 protein levels. Cells were then starved of serum for 24 hours followed by treatment with rapamycin (80 nmol/L) and wortmannin (20 nmol/L) for an additional 24 hours. Extracts were run on SDS-PAGE gels and immunoblotted with antibodies to the phosphorylated form of 4E-BP1, 4E-BP1, Pim-2, and GAPDH.

Using RNA interference directed at Pim-2 that has been validated in other cell lines, it is shown that the knockdown of Pim-2 protein levels in PC3 Pim-2 cells markedly inhibits the phosphorylation of 4E-BP1 (Fig. 3B; ref. 11). However, it had no effect on the total cellular levels of 4E-BP1. This knockdown occurs in the presence of rapamycin and wortmannin, where Pim seems to be regulating 4E-BP1 phosphorylation. In contrast, this small interfering RNA treatment had no effect on the levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in these cells. These data confirm that Pim-2 can modulate the phosphorylation of proteins that play a crucial role in controlling protein synthesis in prostate cancer. We also show the ability of overexpressed Pim-1 compared with Pim-2 to modulate the phosphorylation in a similar if not identical fashion. This regulation of protein synthesis may contribute significantly to the ability of Pim protein kinases to enhance tumor growth.

Cap-dependent translation controls the level of multiple proteins, including c-Myc, a protein that seems to collaborate with Pim (32, 33). Under the conditions described above, we evaluated the cellular levels of c-Myc and found that the level of this protein is higher in the Pim-1 and Pim-2 transfectants than in control PC3/neo cells (Fig. 4A). Growth factor starvation or rapamycin and wortmannin treatment decreased the level of c-Myc in control cells. Likewise, these treatments inactivated the endogenous AKT phosphorylation that is elevated in PC3 cells. These manipulations had little effect on the level of c-Myc in the Pim-containing cells. Phosphorylation of c-Myc on Ser⁶² by the action of the extracellular signal-regulated kinase pathway stabilizes the protein, whereas GSK-3ß phosphorylation of Thr⁵⁸ targets the protein to ubiquitin-mediated degradation (34, 35). Serum starvation of PC3/neo cells or treatment with wortmannin and rapamycin increased phosphorylation of Thr⁵⁸ while decreasing phosphorylation of Ser⁶². In the Pim-1 and Pim-2 transfectants, these treatments had no significant effect and Ser⁶² remained phosphorylated. The combined treatment with rapamycin and wortmannin clearly inhibited the activity of the AKT protein kinase in all cell lines (Fig. 4A). It has been shown previously that c-Myc in which Ser⁶² is phosphorylated, but not unphosphorylated c-Myc, functions to transform normal human fibroblasts transfected with human telomerase and Ras^G12V and is responsible for activation of the E2F promoter (36). Activated c-Myc can increase the transcription of eIF-4E, further enhancing protein synthesis (37-39). Thus, it is possible that modulation of the phosphorylation and, hence, the levels of c-Myc by Pim may be important in its mechanism of action and the regulation of tumor cell growth.

View larger version (40K): [in this window] [in a new window]   FIGURE 4. Regulation of c-Myc levels in Pim-containing PC3 cells. A. To evaluate c-Myc levels, PC3/neo, PC3/Pim-1, and PC3/Pim-2 were grown in RPMI, including 10% fetal bovine serum, then serum-starved for 24 hours and treated with rapamycin (80 nmol/L) and wortmannin (20 nmol/L) for an additional 24 hours before harvesting. Extracts were run on SDS-PAGE gels. The membrane was stripped and probed with antibodies specific for c-Myc, phospho-Ser62, phospho-Thr58, phospho-Ser473 AKT, AKT, and GAPDH, a loading control. B. To measure the level of c-Myc mRNA in PC3 cells, mRNA was extracted as described in Materials and Methods and subjected to quantitative reverse transcription-PCR. The level of c-myc mRNA was compared with GAPDH and expressed as a ratio for each sample. The experiment was repeated with three individual RNA samples and was done in triplicate. Columns, mean; bars, SD.

The phosphorylation of serine PP2A results in the dephosphorylation of c-Myc on Ser⁶², thereby allowing its Thr⁵⁸-directed protein ubiquination and degradation (36). The degradation of c-Myc blocks its cell cycle– and growth-promoting activity, inhibiting its ability to regulate transcription. The serine phosphatases also have been shown to play an important role in regulating protein synthesis in yeast (41). Pim has been shown to coimmunoprecipitate with the catalytic subunit of PP2A (42), suggesting that Pim might regulate the activity of this phosphatase. Using a colorimetric assay for PP2A that was validated by demonstrating its inhibition with okadaic acid and sodium fluoride (Fig. 5A), we measured PP2A activity in the four PC3 cell lines (Fig. 5A). This experiment showed that the Pim-1 and Pim-2 transfectants exhibited a significantly lower level of PP2A activity than the PC3/neo control cells. Interestingly, the PC3/Pim-2 K/A cells had a slightly lower level of phosphatase activity than PC3/neo controls, possibly suggesting that this protein could interact with PP2A in the absence of kinase activity. Recent evidence (43) has shown that polyoma small-T antigen inhibits PP2A activity by causing the dissociation of the catalytic and regulatory subunits of this phosphatase. To investigate this possibility, we immunoprecipitated the PP2A -subunit and then Western blotted with an antibody to the ß-component of the complex, B56. Our results show that the overexpression of Pim-1 or Pim-2 compared with the PC3/neo cells caused apparent disassociation of these two subunits. However, the total levels of B56 do not change in these cells (Fig. 5B). This dissociation could function to inhibit PP2A activity, and thus indirectly regulate the phosphorylation of c-Myc as seen in Fig. 4A. By modulating c-Myc phosphorylation and thus its degradation, Pim protein kinases can function to enhance the activity of c-Myc and potentially to stimulate transit through the cell cycle.

View larger version (27K): [in this window] [in a new window]   FIGURE 5. Expression of Pim-1 or Pim-2 regulates PP2A activity in PC3 prostate tumor cells. A. Cells (2.5 x 106) were harvested in Tris-HCl–buffered saline, centrifuged, and then sonicated in 500 µL buffer [10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, and 0.02% sodium azide]. The phosphatase assay was carried out as described in Materials and Methods. As controls, either sodium fluoride (50 mmol/L) or okadaic acid (5 µmol/L) was added to cell extracts before beginning the assay. Columns, mean of triplicate determinations; bars, SD. 1, PC3/neo; 2, PC3/Pim-2 K/A; 3, PC3/Pim-2; 4, PC3/Pim-1; 5, PP2A catalytic subunit. B. 1 x 106 cells were lysed in 50 mmol/L Tris-HCl (pH 7.4); 150 mmol/L NaCl; 1 mmol/L EDTA; 1 mmol/L phenylmethylsulfonyl fluoride; and 1 µg/mL each of aprotinin, leupeptin, and pepstatin. The extracts were immunoprecipitated with an antibody to the -subunit of PP2A. The immunoprecipitates were run on a 10% SDS-PAGE gel, transferred to polyvinylidene difluoride membranes, and probed with antibodies to the -subunit of PP2A or an antibody to the ß-subunit B56. Arrows, - and ß-subunits and the IgG light and heavy chains.

There has been recent interest in the development of rapamycin analogues as treatment regimens for tumors in which PTEN is deleted (52-56) or as drugs that could sensitize tumors to other chemotherapeutic agents (20). Clearly, tumors that overexpress Pim would be resistant to both of these approaches. Knowledge of the level of overexpression of Pim in tumors may help in planning chemotherapy regimens. Additionally, targeting the Pim kinases with small molecule inhibitors may play a critical role in cancer therapy.

	Materials and Methods

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
Materials
Rapamycin, wortmannin, and antibodies to wild-type and phosphorylated forms of 4E-BP1, phospho-Ser⁴⁷³ AKT, AKT, and p70S6K were purchased from Cell Signaling Technologies (Beverly, MA). Okadaic acid was obtained from Invitrogen (Carlsbad, CA). Monoclonal Pim-2 and Pim-1 antibody were generated by immunization with a 20–amino acid peptide corresponding to the carboxyl terminus of the Pim-2 and Pim-1 protein. Antibodies were purified by affinity chromatography using peptides covalently liked to 4B Sepharose (Amersham, Piscataway, NJ). Anti-flag M2 was purchased from Eastman Kodak Company (New Haven, CT). Additional antibodies used include c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Ser⁶² (Abcam, Cambridgeshire, United Kingdom), phospho-Thr⁵⁸ (Cell Signaling), -subunit of PP2A (6F9; Covance, Berkeley, CA), an antibody to the ß-subunit B56 a gift of Dr. William Hahn (Harvard Medical School, Boston, MA), and GAPDH (Chemicon, Temecula, CA), a loading control.

Plasmids
The murine pim-1 and pim-2 cDNAs were PCR amplified using primers with EcoRI and XhoI restriction site at their ends and cloned into the PCDNA.3.1 expression vector (Invitrogen). A kinase-dead mutant form of pim-2 expression vector (pimK/A) was generated by mutating Lys¹²⁰ to alanine using the Quik Change Site-Directed Mutagenesis kit (Stratagene).

Cell Culture and Transfection
PC3 cell lines were maintained in RPMI 1640 containing 10% FCS penicillin/streptomycin. PC3 cells were transfected using Effectene (Qiagen, Valencia, CA) according to the instructions of the manufacturer. For derivation of cell lines after 48 hours, the culture medium was switched to the same medium supplemented with 1,000 µg/mL Geneticin. For rapamycin treatment of stable and transiently transfected cells, all cell lines were first placed in serum-free medium for 24 hours and then fresh serum-free medium was added before addition of 80 nmol/L rapamycin.

Western Blot Analysis
Cell extracts were prepared by lysing cells in a buffer containing 20 mmol/L Tris (pH 7.5),150 mmol/L NaCl, 1 mmol/L EDTA,1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium PPi,1 mmol/L ß-glycerolphosphate, 0.5% NP40, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, and protease inhibitor cocktail (Sigma Chemical, St. Louis, MO). The lysates were incubated on ice for 15 minutes followed by centrifugation for 10 minutes at 4°C. To the supernatant was added with 5x Ficoll and the mixture was heated at 100°C for 5 minutes. The cell extracts were resolved by 12% SDS-PAGE and then transferred to Immobilon-P membrane filters (Millipore, Bedford, MA).

Cell Cycle Analysis
Twenty-four hours after culturing prostate cancer cell lines in normal medium, they were transferred to serum-free medium and cultured for 24 additional hours. The tumor cells were then treated with 80 nmol/L rapamycin or DMSO for 18 hours and subjected to propidium iodide staining. The percentage of cells in different phases of the cycle was determined by fluorescence-activated cell sorting analysis.

Tumor Growth Curve
Subcutaneous tumor xenografts were established in nude mice by injecting tumor cells (2 x 10⁺⁶) subcutaneously in four locations on each mouse. Seven days after transplantation, tumor measurements were begun in a blinded fashion with five mice per group or 20 total tumors.

Phosphatase Assay
Cells (2.5 x 10⁶) were harvested in Tris-HCl–buffered saline, centrifuged, and then sonicated in 500 µL buffer [10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, and 0.02% sodium azide]. A 250 µL aliquot of the homogenate was applied to a G25 column to decrease the levels of free phosphate, and the flow through subjected to a PP2A colorimetric assay following the instructions of the manufacturer (Promega, Madison WI) and using the PP2A-specific buffer. As controls, either sodium fluoride (50 mmol/L) or okadaic acid (5 µmol/L) was added to cell extracts before beginning the assay.

RNA Interference
A short interfering RNA for Pim-2 (5'-GGGATAGATGGACATCTGTTGAA-3') that had been previously validated (11) was purchased from Ambion (Austin, TX) and transfected into PC3 cells at a concentration of 100 pmol using LipofectAMINE 2000 (Invitrogen). Thirty-six hours later, the serum was withdrawn for 24 hours and then rapamycin (80 nmol/L) and wortmannin (20 nmol/L) were added for an additional 48 hours.

RNA Isolation and Quantitative Reverse Transcription-PCR
Total RNA (0.5 µg per reaction) was reverse transcribed into cDNA utilizing random primers (Promega). AMV Reverse Transcriptase II enzyme (500 units per reaction; Promega) was used for first-strand synthesis and Tfl DNA Polymerase for second-strand synthesis (500 units per reaction; Promega) as per the protocol of the manufacturer. In each case, 50 pg of cDNA was used per ensuing PCR reaction. Two-step quantitative reverse transcription-PCR was done on cDNA generated using the MultiScribe Reverse Transcriptase from the TaqMan Reverse Transcription System and the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA).

The primer pair for c-Myc was generated from the published c-Myc sequence (accession no. NM_002467). Forty cycles of PCR were done under standard conditions using an annealing temperature of 56°C. In addition, GAPDH was amplified as a housekeeping gene to normalize the initial content of total cDNA. Here, c-Myc expression was calculated as the relative expression ratio between c-Myc and GAPDH and was compared for each condition. As a negative control, quantitative reverse transcription-PCR reactions without cDNA template were also done. All reactions were run thrice in triplicate.

	Acknowledgements

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
We thank William C. Hahn for the gift of the PP2A ß-subunit antibodies.

	Notes

Top Notes Abstract Introduction Results and Discussion Materials and Methods Acknowledgements References

 
Grant support: Prostate Cancer Research Foundation (A.S. Kraft and W.W. Chen), NIH and Leukemia and Lymphoma Society of America (M.B. Lilly), and NIH Specialized Programs of Research Excellence in Lung Cancer (D.C. Chan).

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.

Received 1/26/05; revised 6/29/05; accepted 7/11/05.

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