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Abstract
The Selective Protein Kinase C B Inhibitor Enzastaurin
Induces Apoptosis In Cutaneous T-Cell Lymphoma
Cell Lines Through The AKT Pathway
INTRODUCTION
Correspondence: Dr Christiane Querfeld, Robert H. Lurie Comprehensive Cancer Center, 3-250, 303 E Superior Avenue, Chicago, Illinois 60611, USA. E-Mail: C-Querfeld@Northwestern.Edu
RESULTS
DISCUSSION
MATERIALS AND METHODS
Cell Culture And Reagents
Cell Proliferation Assay
Immunoblotting Analysis
Cell Cycle Analysis
Statistical Analysis
CONFLICT OF INTEREST
ACKNOWLEDGMENTS
Abstract
Enzastaurin displays pro-apoptotic properties against a spectrum of malignancies and is currently being investigated in clinical trials. We have investigated the effects of enzastaurin on the viability of the cutaneous T-cell lymphoma cell lines HuT-78 and HH by using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium assay, cell cycle analysis, propidium iodide and annexin-V staining, and caspase3-mediated proteolytic activation. Enzastaurin-treatment decreased cell viability, increased annexin V-FITCpositive cells, and increased the proportion of sub-G1 populations in both cell lines that was not reversed by the T-cell growth stimulating cytokines IL-2, IL-7, IL-15. Enzastaurin-induced cell death involved caspase-3-activated cleavage of poly(ADP-ribose) polymerase that was inhibited by the pan-caspase inhibitor ZVAD-fmk, whereas the increase in sub-G1 population was only partially inhibited by ZVAD-fmk. Furthermore, enzastaurin downregulated AKT activity and its downstream effectors GSK3b and ribosomal protein S6. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the growth and survival of hematologic malignancies and inhibition of this pathway is considered as a therapeutic target. Protein kinase C activation contributes to PI3K/AKT activation, but it is unknown how enzastaurin may interfere with signaling through this pathway. These results demonstrate that enzastaurin, at clinically achievable concentrations, induces apoptosis and affects AKT signaling, and provide a rationale for further in vivo studies addressing the therapeutic efficacy in cutaneous T-cell lymphoma patients.
The Selective Protein Kinase C b Inhibitor Enzastaurin
Induces Apoptosis in Cutaneous T-Cell Lymphoma
Cell Lines through the AKT Pathway
Christiane Querfeld1,4, Mujahid A. Rizvi2, Timothy M. Kuzel2,4, Joan Guitart1,4, Alfred Rademaker3,4, Simran S. Sabharwal4, Nancy L. Krett4 and Steven T. Rosen2,4 Enzastaurin displays pro-apoptotic properties against a spectrum of malignancies and is currently being investigated in clinical trials. We have investigated the effects of enzastaurin on the viability of the cutaneous T-cell lymphoma cell lines HuT-78 and HH by using 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium assay, cell cycle analysis, propidium iodide and annexin-V staining, and caspase3-mediated proteolytic activation. Enzastaurin-treatment decreased cell viability, increased annexin V-FITCpositive cells, and increased the proportion of sub-G1 populations in both cell lines that was not reversed by the T-cell growth stimulating cytokines IL-2, IL-7, IL-15. Enzastaurin-induced cell death involved caspase-3-activated cleavage of poly(ADP-ribose) polymerase that was inhibited by the pan-caspase inhibitor ZVAD-fmk, whereas the increase in sub-G1 population was only partially inhibited by ZVAD-fmk. Furthermore, enzastaurin downregulated AKT activity and its downstream effectors GSK3b and ribosomal protein S6. The phosphatidylinositol 3-kinase (PI3K)/AKT pathway has been implicated in the growth and survival of hematologic malignancies and inhibition of this pathway is considered as a therapeutic target. Protein kinase C activation contributes to PI3K/AKT activation, but it is unknown how enzastaurin may interfere with signaling through this pathway. These results demonstrate that enzastaurin, at clinically achievable concentrations, induces apoptosis and affects AKT signaling, and provide a rationale for further in vivo studies addressing the therapeutic efficacy in cutaneous T-cell lymphoma patients. Journal of Investigative Dermatology (2006) 126, 1641–1647. doi:10.1038/sj.jid.5700322; published online 27 April 2006
INTRODUCTION
Primary cutaneous T-cell lymphomas (CTCLs) originate from skin-homing T lymphocytes. Mycosis fungoides (MF) with patch, plaque and/or tumor manifestations and the leukemic variant Sézary syndrome are the most common types and account for 50% of all primary cutaneous lymphomas (Querfeld et al., 2003). Mycosis fungoides has an indolent course with slow progression over years; however, patients ultimately progress into more aggressive and advanced disease with either cutaneous or extracutaneous tumor manifestations. At present, no curative therapy exists. Systemic single-agent or multi-agent chemotherapies are used to treat advanced and aggressive forms of CTCL to palliate patients. Despite moderate response rates, however, no treatment has been shown to prolong disease-free or overall survival. Protein kinase C (PKC) consists of a family of at least 11 serine-threonine protein kinases that are involved in signal transduction pathways that regulate growth factor response, proliferation, and apoptosis. T lymphocytes express the classic PKC a and b isoforms, the novel isoforms PKC d, e, Z, and y, and the atypical isoform PKC z (Mischak et al., 1991; Altman et al., 2000). Members of the PKC family have been shown to play an important role in T-cell activation and proliferation (Sun et al., 2000; Long et al., 2001; Lisowska et al., 2003) as well as to contribute to the growth and survival of leukemic T cells (Villalba et al., 1999; Gorelik et al., 2002; Felli et al., 2005). PKC b has been implicated in hematologic malignancies such as B-cell lymphomas (Hans et al., 2005), but its role in T-cell malignancies has not been determined yet. Recently, it was found that PKC b is critical for IL-2 secretion from the CTCL cell line HuT-78 (Long et al., 2001). Keratinocyte-derived interferon-g-inducible protein 10 (IP-10) expression, promoting the epidermotropism of CTCL, See related commentary on page 1431 & 2006 The Society for Investigative Dermatology www.jidonline.org 1641 ORIGINAL ARTICLE Received 8 November 2005; revised 10 February 2006; accepted 9 March 2006; published online 27 April 2006 1Department of Dermatology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA; 2Department of Medicine, Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA; 3Department of Preventive Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA and 4Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA
Correspondence: Dr Christiane Querfeld, Robert H. Lurie Comprehensive Cancer Center, 3-250, 303 E Superior Avenue, Chicago, Illinois 60611, USA. E-mail: c-querfeld@northwestern.edu
Abbreviations: CTCL, cutaneous T-cell lymphoma; GSK3b, glycogen synthetase kinase 3 beta; PKC, protein kinase C; PARP, poly(ADP-ribose) polymerase; PI, propidium iodide; PI3K, phosphatidylinositol 3-kinase has also been ascribed to PKC activation (Boorsma et al., 1998). Enzastaurin (LY317615), an acyclic bisindolylmaleimide, is a selective orally administered PKC b inhibitor. It is currently used in phase II trials for the treatment of refractory glioblastoma and diffuse large B-cell lymphoma. Tumorinduced angiogenesis requires the activation of PKC b and enzastaurin was originally evaluated in human tumor xenograft mice models for its antiangiogenic activity upon PKC b inhibition (Keyes et al., 2004). However, in addition to its antiangiogenic effects, enzastaurin, at concentrations reached in clinical trials, directly suppressed proliferation and induced apoptosis of tumor cells in culture and in human colon and glioblastoma xenografts through the suppression of phosphorylation of AKT and its downstream effectors glycogen synthetase kinase 3 beta (GSK3b) and ribosomal protein S6 (Fine et al., 2005; Graff et al., 2005). Based on these observations, we investigated the effect of this PKC b inhibitor on the survival of two well-characterized CTCL cell lines. In this study, we demonstrate that enzastaurin inhibits cell growth, induces partially caspase-dependent apoptosis via specific inhibition of the AKT signaling pathway in the CTCL cell lines HH and HuT-78.
RESULTS
Enzastaurin treatment suppresses tumor cell proliferation and induces apoptosis in CTCL cells To determine the effect of PKC bII inhibition to the survival of HH and HuT-78 cells, we cultured these cell lines in the presence of enzastaurin. HuT-78 and HH cells were treated with 1, 2, 3, 4, and 5 mM enzastaurin and DMSO (vehicle control) for 24, 48, 72, 120 hours and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. There was no significant cell growth inhibition at 24 and 48 hours (data not shown). At 72 hours (Figure 1), treatment with enzastaurin resulted in a dose-dependent inhibition of cell proliferation (P¼0.002 for HuT-78; P¼ 0.005 for HH). However, HH cells treated with 4 and 5 mM enzastaurin showed a significantly higher number of viable cells compared to cells treated with 1 to 3 mM doses suggesting a possible saturation concentration for PKC inhibition in this cell line. The growth inhibition in both cell lines was about 50%. At 120 hours a 60% growth inhibition was noted (data not shown). To determine whether enzastaurin induces apoptosis as evidenced by an increase in cells with a sub-G1 DNA content and annexin V-binding, CTCL cell lines were treated with 1 and 3mM enzastaurin for 72 h. Using cell cycle analysis the number of cells with sub-G1 populations treated with 3 mM enzastaurin was significantly increased compared with untreated control (P¼0.004 for HH; P¼0.003 for HuT-78), indicating cells were undergoing apoptosis (Figure 2a and b). The increase in sub-G1 populations was accompanied by a loss of cells in the G1, S, and G2–M phases. In all, 1 mM concentration of enzastaurin had no effects. The sub-G1 DNA content was not reversed by the T-cell growth-stimulating cytokines IL-2, IL-7, or IL-15 (data for IL-7 and IL-15 are not shown). In parallel to cell cycle analysis, the effect of enzastaurin on annexin V-FITC-binding was investigated. The number of annexin V-FITC-positive cells was significantly increased compared with untreated control (Po0.0001) and is consistent with the results of sub-G1 populations from propidium iodide (PI) staining for cell cycle (Figure 2c and d). As a positive control for annexin V binding, cells were treated with 10 mM 8-amino-adenosine (Krett et al., 2004). Effect of enzastaurin on caspase activation and poly(ADP-ribose) polymerase cleavage Enzastaurin treatment for 72 hours increases the proportion of sub-G1 populations in both cell lines. To determine whether enzastaurin-induced cell death was caspase dependent, HH and HuT-78 cells were treated with 40 mM of the pan-specific caspase inhibitor ZVAD-fmk for 1 hour prior to exposure to enzastaurin. Enzastaurin-induced cell death was partially reversed after pretreatment with ZVAD-fmk (Figure 3a). Furthermore, immunoblotting analysis showed caspase-3 activation, revealing a specific proteolytic cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP) from 116 to 85 kDa (Figure 3b). Cleavage occurred at 24 hours of drug treatment. Equal amounts of total protein from enzastaurin-treated HuT-78 cells were analyzed separately and showed similar results (data not shown). Cleavage of PARP was prevented if cells were preincubated with ZVADfmk (Figure 3c) indicating that caspase activity is at least partially required for enzastaurin-induced cell death. Enzastaurin decreases GSK3b protein, phosphorylation of GSK3b and ribosomal protein S6 PKC activation has been linked to the AKT pathway that plays a critical role in cell survival and apoptosis. Enzastaurin was reported to block AKT activity through inhibition of PKC b. By immunoblotting, there was no enzastaurin-stimulated change 1642 Journal of Investigative Dermatology (2006), Volume 126 in phosphorylated PKC b levels (Figure 4a). We investigated the changes in AKT phosphorylation after exposure to 3 mM enzastaurin for 0.5, 1 hour, 2, 4, and 24 hours. Enzastaurin decreased AKT phosphorylation at 0.5 hours persisting through 24 hours (Figure 4b). To confirm the inhibition of AKT kinase, we examined the phosphorylation status of 2 downstream AKT substrates, GSK3b and ribosomal protein S6. In general, phosphorylated/activated AKT maintains a www.jidonline.org 1643 pro-survival pathway by preventing apoptosis through phosphorylation and inactivation of pro-apoptotic proteins such as GSK3b. Enzastaurin treatment suppressed active phosphorylated GSK3b levels (Figure 4c). Immunoblot analysis showed a reproducible reduction at 30 minutes, persisting through 72 hours after treatment with enzastaurin. Ribosomal protein S6 is another downstream effector of AKT signaling, critical for protein synthesis. Enzastaurin also decreased the phosphorylation of ribosomal protein S6 in a time-dependent fashion (Figure 4d).
DISCUSSION
Enzastaurin has been shown to inhibit cell growth and induce apoptosis in various cultured human tumor cell lines and tumor xenografts (Teicher et al., 2001a, b, 2002; Keyes et al., 2004; Graff et al., 2005). In this study, enzastaurin effectively inhibited growth and induced apoptosis in two well-characterized human CTCL cell lines, HH cells, and HuT-78 cells, with evidence of a time- and dose-dependent manner. Apoptosis was measured by increased annexin V-binding and cleavage of cellular components such as PARP and DNA. Exposure of these cell lines to ZVAD-fmk did only partially reduce apoptosis upon treatment with enzastaurin, but did prevent PARP cleavage. The data further indicate that enzastaurin-induced apoptosis appears partially caspase mediated. Both the serine/threonine PKC and AKT subfamilies have been implicated in signaling pathways leading to activation, differentiation, and cell survival of T lymphocytes. AKT is mainly activated by the upstream phosphatidylinositol 3-kinase (PI3K) and deregulated PI3K/AKT signaling in oncogenic transformation and cancer progression has been reported (Vivanco and Sawyers, 2002). The PI3K/AKT pathway was reported to be particularly important for the growth and survival of acute lymphoblastic T-cell leukemia cell lines deficient for the PI3K-antagonistic enzyme PTEN (Uddin et al., 2005). Apoptosis induced by PI3K inhibition correlated with AKT dephosphorylation in contrast to the PTEN-expressing HuT-78 CTCL cell line. Various models have also shown that enhanced PI3K/AKT-signaling pathway in T cells triggers lymphoid hyperplasia and autoimmunity (Di Cristofano et al., 1999). The proto-oncogene human T-cell prolymphocytic leukemia 1 was found to be an AKT kinase coactivator (Laine et al., 2000). AKT activation requires the recruitment of the enzyme to the plasma membrane by interacting with membrane-bound lipid products of PI3K. Phosphorylation of both threonine (Thr308) and serine (Ser473) residues fully activates AKT, which maintains a survival signal that protects cells from apoptosis by phosphorylating pro-apoptotic proteins such as caspase-9, Bad, and the Forkhead family of transcription factors, the cell regulatory protein GSK3b, and indirectly by 1644 Journal of Investigative Dermatology (2006), Volume 126 cell proliferative modulating p53 and NF-kB (Cardone et al., 1998; Pap and Cooper, 1998; Khwaja, 1999; Shankar et al., 2004) and mediates growth factor-induced cell proliferation (Chang et al., 2003). Recent studies done by Graff et al. (2005) emphasized the importance of the PI3K/AKT signaling pathway in enzastaurin-induced apoptosis in human cancer cell lines. In our study, expression of phosphorylated AKT was also decreased by enzastaurin treatment. Downregulation was apparent at 30 minutes. We further analyzed the enzastaurin effect on AKT downstream targets. Both cell lines showed a marked decrease in phosphorylated GSK3b and RP S6 levels at 30 minutes of drug treatment. AKT was suppressed in CTCL cell lines within 30 minutes and further indicates that enzastaurin inhibits GSK3b and RP S6 activity by AKT downregulation. Several PKC isoforms are known to interfere with PI3K/ AKT activity in T lymphocytes. In the myeloid HL-60 leukemia cell line, a potential role of PKC b in tumor necrosis factor-a-induced apoptosis has been reported (Laouar et al., 1999). PKC y was identified to protect T lymphocytes from Fas-induced apoptosis (Villalba et al., 1999). PKC-dependent p90 ribosomal S6 kinase phosphorylation promoted cell survival through phosphorylation and inactivation of the proapoptotic Bcl-2 family member Bad (Bertolotto et al., 2000). Enzastaurin impairs PI3K/AKT signaling, but the pathway remains to be elucidated. It is thought to selectively inhibit PKC b at low concentrations, but inhibits other PKC isozymes at concentrations reached in clinical trials (1–4 mM). Therefore, it is feasible that inhibition of the AKT-signaling pathway may be influenced by enzastaurin-induced effects on other PKC isoforms. CTCL is a slowly progressive malignancy for which there is no cure and patients ultimately develop advanced or relapsed disease that is refractory to standard treatment options. No treatment has been shown to prolong disease-free or overall survival and therefore, there is a great need for the development of novel emerging therapies. It appears that enzastaurininduced AKT suppression affects both caspase-mediated apoptosis and cell cycle regulatory pathways, but may involve various biochemical mechanisms. The results of this study provide a rationale for further evaluation of enzastaurin as a potential therapeutic agent for CTCL. Inhibition of the PI3K/ AKT pathway prevents uncontrolled proliferation and enzastaurin has shown to downregulate this pathway, however, the overall role of PKC in CTCL needs to be elucidated.
MATERIALS AND METHODS
Cell culture and reagents
The CTCL cell lines HuT-78 and HH were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown in RPMI 1640 (Invitrogen, Baltimore, MD) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 U/ml penicillin, 100mg/ml streptomycin, and 2.5mg/ml amphotericin B. Cells were maintained at 371C in a humidified atmosphere containing 5% CO2. HuT-78 phenotype (CD2 , CD3þ dim, CD4þ , CD5þ , CD7þ dim, CD8 , CD25 , CD26 , CD52 ) and HH phenotype (CD2 , CD3þ dim, CD4þ , CD5þ , CD7þ partial dim, CD8 , CD25 , CD26 , CD52 ) was established by flow cytometry. Enzastaurin was a gift from Eli Lilly & Co (Indianapolis, IN). Stock solutions of 10 mM were prepared in DMSO and stored at 201C. IL-2, IL-7, IL-15, and the pan-specific caspase inhibitor ZVAD- fluoromethylketone (ZVAD-fmk) were purchased from R&D Systems (Minneapolis, MN). Enzastaurin has a high protein-binding affinity and to achieve clinically relevant concentrations in each experiment RPMI 1640 containing 1% fetal bovine serum was used. The final DMSO concentration in all experiments was o0.001%. Polyclonal antibodies against the phosphorylated/activated form of PKC-bII, GSK3b (pS9), and ribosomal protein S6 (pS240/244) and against total S6 ribosomal protein, and AKT were purchased from Cell Signaling Technologies (Beverly, MA). Antibody directed against phosphorylated AKT (pS473) was obtained from R&D Systems (Minneapolis, MN). Antibody to total PKC-bII was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Total GSK3b antibody and PARP monoclonal antibody were obtained from BD Pharmingen (San Diego, CA).
Cell proliferation assay
Cells were seeded at a density of 2.5 104 cells per well in a 96-well tissue plate and were treated with enzastaurin, DMSO (vehicle), IL-2, IL-7, or IL-15 for 72 hours in a total volume of 100 ml RPMI with 1% fetal bovine serum. Cell viability and growth was determined using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium Cell Titer Assay (Promega, Madison, WI) that measured the conversion of a tetrazolium compound into formazan spectrophotometrically. The quantity of formazan product as measured by the amount of 490 nm absorbance is directly proportional to the number of living cells in culture. The data are expressed as percentage of untreated control cells. Each condition was performed in quadruplicate and all experiments were performed at least three times. Each data point represents the mean value (percentage)7SE.
Immunoblotting analysis
Cells were seeded in 25 cm2 cell culture flasks at a density of 5 106 cells/flask and allowed to grow overnight in RPMI containing 1% fetal bovine serum. After 24 hours cells were treated with 3 mM enzastaurin for the indicated times, harvested, washed with ice-cold phosphate-buffered saline (PBS), and lysed in protein lysis buffer (50 mM N-[2-hydroxyethyl]piperazine-N0-[2-ethanesulphonic acid] (HEPES), 10% glycerol, 0.5% Triton X-100, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EDTA, pH 8.0, 100 mM NaF, 10 mM sodium pyrophosphate, 500mM phenylmethylsulfonyl fluoride, 200 mM sodium orthovanadate, and 10 mg/ml aprotinin) at 41C for 1 hour. Lysates were centrifuged at 10,000 g at 41C for 1 minute and the supernatants were collected in 1.5-ml microcentrifuge tubes and stored at 201C. Protein concentration was determined by BioRad protein assay (BioRad Laboratories, Hercules, CA). Protein at a concentration of 30mg was fractionated on a precast 8–16% Tris-glycine gel (Invitrogen, Carlsbad, CA) and subsequently transferred to a polyvinylidene fluoride membrane. The membranes were incubated with 5% nonfat milk diluted in Tris-buffered saline (10 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween 20) for 1 hour to block nonspecific binding and were then incubated with the appropriate primary antibodies overnight at 41C. The blots were then washed with Trisbuffered saline for 15 minutes and incubated with horseradish peroxidase–conjugated secondary antibody (Amersham, Arlington www.jidonline.org 1645 Heights, IL) for 1 hour. Antibody binding was detected using Enhanced Chemiluminescence Plus Western Blotting Detection Reagent (Amersham) and the signal was visualized with X-ray film (Hyperfilm, Amersham).
Cell cycle analysis
Cells were grown in 25 cm2 flasks at a concentration of 1 106 cells/ml and treated with 3 mM enzastaurin and indicated cytokines. To determine the distribution of cells within the cell cycle, cells were collected after 72 hours, centrifuged (500 g for 5 minutes at 41C), washed twice in ice-cold PBS, fixed in ice-cold 40% ethanol, and stored at 41C until analyzed. Before flow cytometry analysis, the fixed cells were washed in PBS, treated with 50 mg/ml DNase-free RNase in PBS, and incubated for 30 minutes at 371C. The samples were then resuspended in 25 mg/ml PI in 38 mM sodium citrate buffer. Flow cytometry was performed on a Coulter EPICs XL instrument, and data were analyzed using the System II software package. To determine whether enzastaurin-induced apoptosis was caspase dependent, cells were pretreated with the pan-specific caspase inhibitor ZVAD-fmk for 1 hour prior to enzastaurin treatment at a final concentration of 40 mM. Annexin V staining Cells (1 106/ml) were treated with 3mM enzastaurin and harvested after 72 h. As cells undergo apoptosis, the integrity of the cell membrane is disrupted and phosphatidylserine is exposed. Detec- tion of phosphatidylserine on the outer leaflet of apoptotic cells was performed by using annexin V binding according to the manufac- turer’s instructions (BD Pharmingen, San Diego, CA). Briefly, cells were incubated with 5mg/ml FITC-conjugated annexin V in the presence of 5 mg/ml PI and further screened by flow cytometry (Coulter EPICs XL). Annexin V-FITC-positive PI-negative cells were scored as early apoptotic. Annexin V-positive PI-positive cells correspond to late apoptotic cells. As a positive control for apoptosis, cells were treated with 10 mM of the purine nucleoside analogue, 8-amino-adenosine.
Statistical analysis
Data were analyzed using one-way analysis of variance with post hoc t-tests adjusted for multiple comparisons at Po0.05 using Tukey’s method (Figures 1 and 2a), and the two-factor analysis of variance test for interaction (Figure 2b). Statistical analyses were carried out using the SAS statistical software (SAS Institute Inc., SAS OnlineDocs, Version 9.1, Cary, NC: SAS Institute Inc., 2003).
CONFLICT OF INTEREST
Dr Steven T Rosen is a consultant for Eli Lilly & Co.
ACKNOWLEDGMENTS
Dr Christiane Querfeld is a Young Investigator Award Recipient and supported by the Northwestern Medical Foundation.
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