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Blood, 1 October 2006, Vol. 108, No. 7, pp. 2407-2415. Prepublished online as a Blood First Edition Paper on June 8, 2006; DOI 10.1182/blood-2006-04-020305.
NEOPLASIA Autocrine release of interleukin-9 promotes Jak3-dependent survival of ALK+ anaplastic large-cell lymphoma cellsFrom the Departments of Hematopathology and Lymphoma/Myeloma, University of Texas M. D. Anderson Cancer Center, Houston, TX; Department of Laboratory Medicine and Pathology and Cross Cancer Institute, University of Alberta, Edmonton, AB, Canada; and Department of Biological Sciences, University of Texas at El Paso, El Paso, TX.
The aberrant fusion protein NPM-ALK plays an important pathogenetic role in ALK+ anaplastic large-cell lymphoma (ALCL). We previously demonstrated that Jak3 potentiates the activity of NPM-ALK. Jak3 activation is restricted to interleukins that recruit the common  chain (c) receptor, including IL-9. NPM-ALK was previously shown to promote widespread lymphomas in IL-9 transgenic mice by unknown mechanisms. We hypothesized that IL-9 plays an important role in ALK+ ALCL via Jak3 activation. Our studies demonstrate the expression of IL-9R and IL-9 in 3 ALK+ ALCL-cell lines and 75% and 83% of primary tumors, respectively. IL-9 was detected in serum-free culture medium harvested from ALK+ ALCL-cell lines, supporting autocrine release of IL-9. Treatment of these cells with an anti–IL-9–neutralizing antibody decreased pJak3 and its kinase activity, along with pStat3 and ALK kinase activity. These effects were associated with decreased cell proliferation and colony formation in soft agar and cell-cycle arrest. Evidence suggests that cell-cycle arrest can be attributed to up-regulation of p21 and down-regulation of Pim-1. Our results illustrate that IL-9/Jak3 signaling plays a significant role in the pathogenesis of ALK+ ALCL and that it represents a potential therapeutic target for treating patients with ALK+ ALCL.
Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is defined by the World Health Organization (WHO) classification of hematologic malignancies as a subtype of T/null-cell non-Hodgkin lymphoma that is characterized by the consistent expression of CD30.1 In approximately 80% of ALK+ ALCL tumors, the aberrant expression of ALK occurs as a result of a t(2;5)(p23;q35) translocation, which leads to the fusion of the nucleophosmin (NPM) gene on 5q35 and ALK gene on 2p23.2,3 Characteristically, ALK+ ALCL occurs more frequently in children and young adults with an initial 5-year overall survival rate of approximately 70% following conventional cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP)–based therapy.4 Nonetheless, the prognosis for 30% to 40% of the patients is relatively poor.5-7 The oncogenic potential of NPM-ALK has been demonstrated by its transforming ability in vitro and by its ability to induce different types of malignant lymphomas in vivo.8-11 Previous studies showed that NPM-ALK mediates tyrosine phosphorylation and activation of various SH- or PTB-containing signaling molecules, such as GRB-2, PLC- , PI3K/Akt, IRS-1, Ras, SHC, FOXO, and Stats, that are directly involved in the regulation of cell survival and growth.12-18 However, the exact mechanisms by which NPM-ALK induces its oncogenic effects are not completely understood. Janus kinase 3 (Jak3) is the final member identified of a family of protein tyrosine kinases that includes Jak1, Jak2, and tyrosine kinase 2 (Tyk2).19 Jaks reside in the cytoplasm; however, they can be recruited to certain cell-surface receptors on cytokine-induced receptor engagement. This process results in tyrosine phosphorylation and activation of Jaks (pJaks). Thereafter, pJaks phosphorylate receptor residues that act as docking sites for effector molecules including signal transducers and activators of transcription (Stats).20 pJaks subsequently tyrosine phosphorylate and activate Stats (pStats), which dissociate to the cytoplasm, dimerize, and translocate to the nucleus where they induce the transcription of a wide array of genes that can ultimately promote cell survival and proliferation. We and others have previously shown that Jak3 and ALK are physically associated in ALK+ ALCL cells and that selective pharmacologic inhibition of Jak3 reduces ALK tyrosine kinase activity and pStat3 levels in ALK+ ALCL cells.21,22
Jak3 activation is primarily restricted to interleukins (ILs) that possess the common
Cell lines, cell culture, and treatment with anti–human IL-9–neutralizing antibody Three previously characterized ALK+ ALCL-cell lines, Karpas 299, SU-DHL-1, and SUP-M2, were used in the present study. In some of the experiments, the Hodgkin lymphoma–cell line L1236 and the colon carcinoma-cell line HT29 were used as positive and negative controls, respectively. Cells were cultured in RPMI 1640 (DMEM/F-12 for HT29 cells) medium (Life Technologies, Grand Island, NY) supplemented with 10% (15% for L1236 cells) heat-inactivated (56°C for 30 minutes) fetal bovine serum (FBS; Sigma, St Louis, MO), penicillin (10 000 U/mL; Sigma), streptomycin (10 mg/mL; Sigma), and L-glutamine (200 mM, 29.2 mg/mL; Life Technologies). Cell cultures were maintained at 37°C in 95% oxygen, 5% carbon dioxide, and 98% humidity. To detect autocrine release of IL-9, the cell-culture supernatants were concentrated for 1 hour at 4°C by using Centricon YM-10 centrifugal filter devices, which can elute protein complexes of 10 kDa or larger (Millipore, Billerica, MA), and then frozen at –80°C until used in Western blot studies for the detection of IL-9 as explained in the following experiments. For treatment of the cells as denoted in some of the experiments, cells were maintained in FBS-free RPMI for 12 hours before being incubated with goat anti–human IL-9–neutralizing antibody (catalog no. AB-209-NA; lot number DV064030 [GenBank] ; R&D Systems, Minneapolis, MN). Goat IgG (R&D Systems) was used as a negative control. To demonstrate effective binding of anti–IL-9–neutralizing antibody with IL-9, the cell-culture supernatants were concentrated twice by passing through Centricon YM-10 filters as previously described. The second concentrate was then frozen at –80°C until used in Western blot studies to demonstrate lack of IL-9. [3H]-thymidine incorporation assay To detect changes in cell proliferation, [3H]-thymidine incorporation was performed using standard techniques. Briefly, cell samples (20 x 103) in triplicate were suspended in serum-free medium for 12 hours and then treated with anti–IL-9–neutralizing antibody or control goat IgG for 6 hours (24 hours for L1236). To demonstrate the specificity of the antibody, recombinant human IL-9 (rhIL-9, 209-IL; R&D Systems) was incubated at 5 ng/mL with different concentrations of the antibody for 1 hour at 37°C in a 96-well plate. The mixture in a total of 100 µL, containing the antibody, rhIL-9, and Karpas 299 cells (1 x 105 cells/mL) was incubated at 37°C for 6 hours in a humidified CO2 incubator. Thereafter, [3H]-thymidine (0.2 µCi/mL [0.0074 MBq]; Sigma) was incubated with the cells for 4 hours. Incorporation of [3H]-thymidine was measured using a liquid scintillation counter (Packard Biosciences, Meriden, CT). Colony formation in soft agar Soft agar (0.54% wt/vol) was prepared by autoclaving Bacto agar (Difco, Detroit, MI) in distilled water prior to use. Cells harvested in serum-fee medium were treated for 6 hours with anti–IL-9–neutralizing antibody (80 µg/mL) or IgG, then resuspended in cooled 0.33% agar in RPMI medium supplemented with 10% FBS at a density of 200 cells/35-mm plate, and seeded onto solidified 0.54% agar-containing culture medium; plates were kept at 4°C for 30 minutes and then 1 mL RPMI medium supplemented with 10% FBS was added to each well. Plates were cultured for 2 weeks as described. The colonies were stained with 0.5% crystal violet, counted, and photographed with FluorChem 8800 Imaging System (Alpha Innotech, San Leandro, CA). Triplicate samples were used in the experiment. Cell-cycle analysis For flow cytometric analysis of the cell cycle, cells (10 x 105) were fixed in 70% ice-cold ethanol and stored overnight at –20°C. Cells were then washed twice in PBS and incubated for 5 minutes with 100 U/mL ribonuclease A containing 0.1% Triton X in PBS. The cells were stained with propidium iodide (PI; Sigma) at a final concentration of 50 µg/mL for 20 minutes and analyzed using a flow cytometer after suspension in 7-AAD (Becton Dickinson, San Jose, CA). Tyrosine kinase activity assay Tyrosine kinase activity was measured using standard techniques and the Universal Tyrosine Kinase Assay Kit (Takara Bio, Otsu, Japan). Briefly, cells treated with anti–IL-9–neutralizing antibody or IgG were lysed in a buffer containing protease and phosphatase inhibitors. To limit nonspecific immunoglobulin binding, 20 µL/reaction of protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the cell lysates and then removed. Precleared cell lysates were then incubated with 10 µg anti-Jak3 (Santa Cruz Biotechnology) or anti-ALK antibody (DakoCytomation, Carpinteria, CA) overnight at 4°C. To capture the immune complex, 20 µL/reaction of protein A agarose was added to the lysates at room temperature for 20 minutes. The lysates were briefly centrifuged at 500g and the precipitate resuspended in 50 µL kinase-reacting buffer. Kinase reactions were initiated with 10 µL of 40 µM ATP-2Na in immobilized wells, incubated for 30 minutes at 37°C, and blocked by blocking solution according to manufacturer's recommendations. After the blocking solution was discarded, 50 µL horseradish peroxidase-conjugated antiphosphotyrosine (PY20) antibody (Takara Bio) was added to each well for 30 minutes and developed by addition of 100 µL horseradish peroxidase and substrate solution (TMBZ) for 15 minutes at 37°C. The reaction was stopped with 1 N H2SO4 and absorbance measured at 450 nm in a microplate reader (MRX II; Dynex, Frankfurt, Germany). The activity of Jak3 or ALK was normalized by an internal tyrosine kinase control. Immunoprecipitation and Western blotting
Briefly, cells were lysed in lysis buffer and centrifuged at 14 000g for 10 minutes at 4°C. The supernatant was collected and 50 to 80 µg protein was electrophoresed on a 6% to 12% SDS polyacrylamide gel or immunoprecipitated with a specific antibody. Cell lysates were incubated with anti- RT-PCR
Total cellular RNA was extracted from the ALK+ ALCL, L1236, and HT29-cell lines using RNeasy Mini Kit (Qiagen, Valencia, CA). Reverse transcription (RT) was performed by using 2 µg total RNA in a first-strand cDNA synthesis reaction with superscript reverse transcriptase as recommended by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). Polymerase chain reaction (PCR) was performed by adding 1 µL RT product into 50 µL total volume reaction containing 1 x buffer, 200 µMof each dNTPs, 20 pM of each oligonucleotide primer, and 0.2 U AmpliTaq polymerase. Oligonucleotides specific for IL-9R Immunofluorescence staining and confocal microscopy
To visualize the expression of IL-9R Patients, tissue microarray, and immunohistochemical staining
Archival tissue samples from 12 lymph node biopsies from patients with ALK+ ALCL were collected prior to therapeutic interventions following approval by the ethics research committee at Cross Cancer Institute (Edmonton, AB, Canada). All patients provided informed consent in accordance with the Declaration of Helsinki. The diagnosis of these cases was based on the criteria established by the WHO.1 Detailed clinical and follow-up data were available for all patients. The tumor samples were fixed in formalin, routinely processed, and embedded in paraffin. Representative cores were selected from each of the paraffin blocks and included in a tissue microarray. Cores from a reactive lymph node were also included in the tissue microarray as internal controls. The tissue microarray was constructed using a manual tissue arrayer (Beecher Instruments, Sun Prairie, WI). Immunohistochemical staining was performed on sections from the tissue microarray as well as on formalin-fixed and paraffin-embedded sections from cell blocks prepared from the ALK+ ALCL, L1236, and HT29-cell lines. Tissue sections were first deparaffinized in xylene and rehydrated using a graded series of ethanol. A 3-step streptavidin-biotin-horseradish peroxidase method was used after heat-induced epitope retrieval. Briefly, endogenous peroxidase activity was blocked for 30 minutes in 3% hydrogen peroxide and, subsequently, the slides were incubated with protein blocking solution (DakoCytomation) for 15 minutes. Thereafter, the slides were incubated overnight with the primary antibodies diluted in 0.1% bovine serum albumin, 50 mM Tris-HCl buffer, pH 7.6. The dilutions of the antibodies used in the study were 1:100 for IL-9 (R&D Systems) and 1:250 for IL-9R Statistical analysis Statistical analysis was performed using t test for paired data and Statview software (Abacus Concepts, Berkeley, CA).
Previous studies showed that enforced expression of NPM-ALK in IL-9 transgenic mice induces widespread malignant lymphomas.26 To determine whether a correlation exists within ALK+ ALCL-cell lines and patients' tumors, we initiated the following studies to probe the role of IL-9 in this disease.
Expression of IL-9R
RT-PCR studies demonstrated the presence of IL-9R
Because ALK+ ALCL-cell lines expressed IL-9R
We also used standard immunohistochemical staining techniques to evaluate the expression of IL-9R Taken together, these data demonstrate that ALK+ ALCL cells express IL-9 and its receptor suggesting a signaling cascade that may promote tumor progression in an autocrine fashion. To further validate that IL-9 is produced and secreted by ALK+ ALCL cells, we performed the following set of experiments.
Autocrine release of IL-9 by ALK+ ALCL cells IL-9 was detected in cell lysates from the 3 ALK+ ALCL-cell lines (Figure 4). Importantly, high levels of IL-9 also were readily detectable in the supernatants of FBS-free tissue culture medium from these cells (Figure 4). Similar studies showed no evidence of IL-9 in the cell lysate or supernatant from the negative control cells HT29 (Figure 4). As shown in Figure 4, treating the ALK+ ALCL cells with 80 µg/mL anti–IL-9–neutralizing antibody completely depleted IL-9 from the cell-culture supernatant after protein concentration and filtration. We previously showed that inhibition of Jak3 decreases pStat3 levels and ALK kinase activity in ALK+ ALCL cells.21 Because IL-9 signaling induces tyrosine phosphorylation and activation of Jak3, we sought to study the effects of blockade of IL-9 on Jak3, Stat3, and ALK in these cells via the following studies.
Specific blockade of IL-9 decreases pJak3 and its tyrosine kinase activity, along with pStat3 and ALK tyrosine kinase activity in ALK+ ALCL cells Using Western blotting, we measured protein levels of pJak3 and pStat3 after treatment of the ALK+ ALCL cells with anti–IL-9–neutralizing antibody. Indeed, specific blockade of IL-9 decreased pJak3 and pStat3 levels, without notable changes in the total levels of Jak3 or Stat3 (Figure 5A). To confirm the specificity of this antibody, we used HT29 cells that were previously shown to express Jak3, Stat3, and their phosphorylated forms.29 Significant changes in these proteins were not noted after treating HT29 cells with the anti–IL-9–neutralizing antibody using similar protocols (Figure 5B).
Because specific blockade of IL-9 signaling decreased tyrosine phosphorylated levels of Jak3 and Stat3, we next investigated whether this treatment inhibited the tyrosine kinase activity of Jak3 and ALK. Presumably, IL-9 engages its receptor resulting in generation of catalytically primed Jak3. To monitor their catalytic activity, lysates from ALK+ ALCL-cell lines were assayed for tyrosine kinase activity following pretreatment with the anti–IL-9–neutralizing antibody. Interestingly, specific blockade of IL-9 reduced both Jak3 and ALK tyrosine kinase activity to approximately 60% or less of the baseline levels (Figure 5C).
As mentioned, Jak3, ALK, and Stat3 promote cell growth and survival. To determine the effect of specific blockade of IL-9 on cell survival of ALK+ ALCL, we performed the following studies. Specific blockade of IL-9 decreases ALK+ ALCL-cell proliferation and colony formation in soft agar due to cell-cycle arrest The ALK+ ALCL cells were treated with the anti–IL-9–neutralizing antibody. Thereafter, cell proliferation was measured by [3H]-thymidine incorporation (Figure 6A). Anti–IL-9–neutralizing antibody at a concentration of 80 µg/mL decreased cell proliferation to approximately 40% of the baseline levels (P < .001). A similar effect on cell proliferation was not detected when the negative control cells HT29 were used in similar experiments (Figure 6A). In addition, preincubation of anti–IL-9–neutralizing antibody with rhIL-9 also abolished the decrease in proliferation of Karpas 299 cells (Figure 6A). The anti–IL-9–neutralizing antibody induced a gradual concentration-dependent decrease in the proliferation of the Hodgkin lymphoma cells L1236 to 69% of its baseline levels at a concentration of 80 µg/mL (P < .01; Figure 6A). Of note is that the decrease in L1236 cell proliferation was relatively less pronounced, albeit statistically significant from the baseline level, than the one observed in ALK+ ALCL cells. Most likely, this difference can be explained by the release of relatively higher levels of IL-9 by L1236 cells compared with ALK+ ALCL cells, as illustrated in the RT-PCR studies (Figure 2A).
To monitor the effect of blockade of IL-9 on cell growth in soft agar, Karpas 299 cells were treated as above with anti–IL-9–neutralizing antibody (80 µg/mL) and colonies were observed. The anti–IL-9–neutralizing antibody limited colony formation of ALK+ ALCL cells to only 40% of the control levels (Figure 6B). To explore possible explanations of the decrease in cell proliferation and growth after specific blockade of IL-9 in ALK+ ALCL, we studied changes in the cell cycle using flow cytometric analysis after staining with PI and 7-ADD. Anti–IL-9–neutralizing antibody induced G1 cell-cycle arrest as demonstrated by a significant decrease in ALK+ ALCL cells in the S phase (Figure 7A). Anti–IL-9–neutralizing antibody-induced cell-cycle arrest could be explained by a significant increase in p21 and decrease in Pim-1 kinase levels (Figure 7B). There were no significant changes in p27 or cyclin D3 levels (Figure 7B).
ALK+ ALCL is a unique type of non-Hodgkin lymphoma characterized by several chromosomal aberrations of which t(2;5)(p23;q35) is the most common. This translocation leads to the aberrant expression of the fusion protein NPM-ALK.2,3 It is believed that NPM-ALK plays a major role in the pathogenesis of ALCL. Previous in vitro studies showed that NPM-ALK possesses significant transformation potential.8,9 In addition, enforced expression of NPM-ALK gene induces malignant lymphoma in mice.10,11,30-32 Notably, a significant number of the NPM-ALK–induced tumors in the mice models, including tumors driven by T-cell–specific CD2 or CD4 promoters,11,32 demonstrated B-cell immunoblastic/plasmablastic morphologic and immunophenotypic features, were CD30–, and were confined to the mediastinum. Considering that the vast majority of ALK+ ALCL tumors in humans demonstrate T/null-cell immunophenotype, express CD30, and present as a widespread systemic disease, the findings from the animal models suggest that NPM-ALK is not the only factor that drives the biologic sequences that determine the characteristic features of ALK+ ALCL in humans. To further support this concept, previous studies demonstrated the presence of NPM-ALK in nonneoplastic cells.33-35 These observations implicate that additional events and signaling pathways are most likely required to drive the oncogenic events that lead to the characteristic histopathologic, immunophenotypic, and clinical features of ALK+ ALCL in humans.
The biologic processes that lead to lymphomagenesis are complex but likely differ among the cell lineages and different lymphoma histotypes. Numerous cytokines are thought to induce or support lymphomagenesis. IL-9 is a multifunctional cytokine secreted by TH2 lymphocytes.36 Despite the lack of significant effects on freshly isolated T cells,24,37 IL-9 induces significant proliferative effects on stimulated T cells.23,24 Nonetheless, Renauld et al showed that only 7% of IL-9 transgenic mice develop T-cell thymic lymphomas.38 Importantly, a subcarcinogenic dose of N-methyl-N-nitrosourea induced thymic lymphomas in all of the treated IL-9 transgenic mice.38 These findings demonstrate that dysregulated IL-9 requires additional factors for the induction and progression of malignant lymphomas. Recently, NPM-ALK was shown to induce massive and widespread lymphomas in IL-9 transgenic mice.26 Of these tumors, 46% demonstrated T-cell immunophenotype.
Jak3 is a protein tyrosine kinase whose activation/phosphorylation is limited to a small number of interleukins that recruit the IL-2 common
The aim of the present study was to test the hypothesis that IL-9 plays a significant role in the pathogenesis of ALK+ ALCL through a Jak3-dependent pathway. Using immunofluorescence and immunohistochemical staining and RT-PCR, we showed that IL-9R
To our knowledge, these findings are the first to demonstrate constitutive expression of IL-9R To examine the contribution of IL-9 to the pathogenesis of ALK+ ALCL cells, we used a commercially available IL-9–neutralizing antibody, which readily down-regulated pJak3 levels that corresponded to a marked decrease in Jak3 tyrosine kinase activity. Concomitantly, there was as a decrease in pStat3 levels. Total protein levels of Jak3 and Stat3 were not affected. Treating ALK+ ALCL cells with IL-9–neutralizing antibody also decreased ALK tyrosine kinase activity. These results are in agreement with our previous findings21 and provide further evidence supporting the concept that Jak3 plays an important role in the pathogenesis of ALK+ ALCL via interaction with ALK and Stat3, and further support the crosstalk between the 2 enzymes, Jak3 and ALK. Functionally, anti–IL-9–neutralizing antibody significantly abrogated the growth potential of ALK+ ALCL cells as demonstrated by a marked decrease in [3H]-thymidine incorporation and colony formation in soft agar. Anti–IL-9–neutralizing antibody also caused cell-cycle arrest in ALK+ ALCL cells. Previous studies showed that p21 plays a significant role in cell-cycle regulation in ALK+ ALCL via a CD30-dependent pathway.50 Indeed, we observed a concentration-dependent increase in p21 after treatment with anti–IL-9–neutralizing antibody. Pim-1 is a serine/threonine kinase involved in several important biologic functions including mitosis and cell-cycle progression.51-54 Previous studies showed that Pim-1 possesses a significant oncogenic potential in malignant neoplasms, including malignant lymphomas.55-58 A recent study demonstrated that Pim-1 kinase phosphorylates p21 and leads to its sequestration in the cytoplasm.59 The expression of Pim-1 appears to be regulated, at least in part, via IL-9/Jak/Stat signaling pathway.60,61 Our results demonstrated that anti–IL-9–neutralizing antibody reduces the expression of Pim-1 in ALK+ ALCL cells. This is the first report to demonstrate the expression of Pim-1 in ALK+ ALCL cells. These results also suggest that Pim-1 is a downstream target of the IL-9/Jak3/Stat3 signaling pathway in ALK+ ALCL and that it may have a role in the pathogenesis of this type of malignant lymphoma. We have previously demonstrated that the occurrence of cell-cycle arrest in ALK+ ALCL due to inhibition of Jak3 or other signaling pathways was associated with significant alterations in cyclin D3 and p27, 2 downstream targets of Jak/Stat signaling pathway.21,62 Therefore, we also sought to explore possible changes in these 2 proteins. The lack of significant changes in cyclin D3 and p27 levels after treatment with anti–IL-9–neutralizing antibody could be due to the effects of other signaling pathways known to affect the expression of these cell-cycle regulators, such as PI3K/Akt, FOXO, FAK, and PKC.18,62,63 Another possible explanation is that the experimental conditions in the present study were not sufficient to induce notable changes in cyclin D3 and p27 levels. In conclusion, the present study provides evidence that the IL-9/Jak3 signaling plays a major role in the pathogenesis of ALK+ ALCL via activation of ALK and Stat3. These findings have important clinical implications because they identify the IL-9/Jak3 signaling pathway as a potential therapeutic target to treat this type of malignant lymphoma. Considering the effects of IL-9/Jak3 on T-cell development and T-cell lymphomagenesis, our results suggest that, in addition to ALK, the constitutive activation of IL-9/Jak3 signaling may represent a secondary biologic event that leads to the development of the distinct features that characterize ALK+ ALCL.
Submitted December 17, 2005; accepted May 27, 2006.
Prepublished online as Blood First Edition Paper, June 8, 2006; DOI 10.1182/blood-2006-04-020305.
H.M.A. is supported by a K08CA114395 grant from the National Cancer Institute, the Physician Scientist Program Award at M. D. Anderson Cancer Center, and a Career Development Award from the National Institutes of Health (NIH) Leukemia Specialized Program of Research Excellence (SPORE) grant to M. D. Anderson Cancer Center. R.A.K. is supported by grants AI053566 and SG12RR008124 from NIH.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hesham M. Amin, Department of Hematopathology, Box 72, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail: hamin{at}mdanderson.org.
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Top
Abstract
Introduction
-actin (A5316; Sigma), other antibodies including Jak3 (sc-1080), pJak3 (sc-16567-R), Stat3 (sc-482), pStat3 (sc-8059), p21 (sc-6246), p27 (sc-1641), cyclin D3 (sc-6283), and Pim-1 (sc-13513) were from Santa Cruz Biotechnology. All blots were developed with a horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) and the enhanced chemiluminescence detection kit (Amersham Life Sciences, Arlington Heights, IL). 
