This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Green, L. J. Articles by Thornton, D. Search for Related Content PubMed PubMed Citation Articles by Green, L. J. Articles by Thornton, D.

Development and Validation of a Drug Activity Biomarker that Shows Target Inhibition in Cancer Patients Receiving Enzastaurin, a Novel Protein Kinase C-ß Inhibitor

Authors' Affiliations: ¹ Lilly Research Laboratories, Indianapolis, Indiana; ² The University of Texas M.D. Anderson Cancer Center, Houston, Texas; ³ Johns Hopkins Medical Institutions, Baltimore, Maryland; ⁴ University of California at Los Angeles Medical Center, Los Angeles, California; and ⁵ University of Colorado Cancer Center, Aurora, Colorado

Requests for reprints: Lisa J. Green, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis IN, 46285. Phone: 317-276-6048; Fax: 317-277-2934; E-mail: lisa.green{at}lilly.com.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Purpose: To evaluate the effects of the novel protein kinase C (PKC) inhibitor enzastaurin on intracellular phosphoprotein signaling using flow cytometry and to use this approach to measure enzastaurin effects on surrogate target cells taken from cancer patients that were orally dosed with this agent.

Experimental Design: The activity of PKC was assayed in intact cells using a modification of published techniques. The U937 cell line and peripheral blood mononuclear cells were stimulated with phorbol ester, fixed, permeabilized, and reacted with an antibody specific for the phosphorylated forms of PKC substrates. The processed samples were quantitatively analyzed using flow cytometry. The assay was validated for selectivity, sensitivity, and reproducibility. Finally, blood was obtained from volunteer cancer patients before and after receiving once daily oral doses of enzastaurin. These samples were stimulated ex vivo with phorbol ester and were assayed for PKC activity using this approach.

Results: Assay of U937 cells confirmed the selectivity of the antibody reagent and enzastaurin for PKC. Multiparametric analysis of peripheral blood mononuclear cells showed monocytes to be the preferred surrogate target cell. Day-to-day PKC activity in normal donors was reproducible. Initial results showed that five of six cancer patients had decreased PKC activity following enzastaurin administration. In a following study, a group of nine patients displayed a significant decrease in PKC activity after receiving once daily oral doses of enzastaurin.

Conclusion: An inhibition of surrogate target cell PKC activity was observed both in vitro and ex vivo after exposure to the novel kinase inhibitor, enzastaurin.

Enzastaurin, formerly LY317615 HCl, a potent, novel macrocyclic bisindolylmaleimide, disrupts the intrinsic phosphotransferase activity of PKC-ß. Administration of enzastaurin decreases vascular endothelial growth factor levels in tumor-bearing mice and suppresses growth of human glioblastoma and colon carcinoma xenografts in mice (5). Enzastaurin has been well tolerated in early human testing (6, 7) and has shown early promising phase II results in treatment of high-grade glioma (8). In this report, we describe the development and clinical validation of a biomarker assay to assess intracellular phosphoprotein signaling in human peripheral blood leukocytes. Our study showed decreased PKC activity in peripheral blood monocytes taken from cancer patients treated with enzastaurin.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Patients. Patients had advanced nonhematologic malignancies and participated in a phase I single-agent dose escalation trial at The University of Texas M.D. Anderson Cancer Center (Houston, TX) or at Johns Hopkins Medical Center (Baltimore, MD; ref. 6) or in a phase I study of enzastaurin in combination with capecitabine at University of California at Los Angeles Medical Center or at the University of Colorado Cancer Center (Denver, CO). Written informed consent was obtained from all patients and healthy donors according to institutional, state, and federal guidelines. For flow cytometric assays, patients were identified by numerals in sequence according to sample receipt (single agent study) or letters (combination study) and normal donors were assigned numbers in sequence.

Collection of peripheral blood mononuclear cells. Venous blood was collected from healthy normal donors or clinical trial patients into Becton Dickinson Vacutainer CPT tubes containing sodium heparin anticoagulant (Becton Dickinson, Franklin Lakes, NJ). Tubes were centrifuged for 20 minutes at 1,500 x g at room temperature. Processed CPT tubes were inspected for presence of mononuclear cell layer, then mononuclear cells were resuspended in the plasma layer by inverting the tube three to four times. The processed CPT tubes were stored or shipped overnight at 4°C. CPT tubes are important because they permit isolation of a relatively pure population of peripheral blood mononuclear cell (PBMC) preserved in autologous plasma sample. The PBMCs are remixed into the supernatant plasma for shipment but remain isolated from red cells and neutrophils by a gel barrier. Before beginning the assay, cells/plasma were mixed well, passed through a 70 µmol/L cell strainer, and warmed to room temperature.

Cell line and reagents. Cell culture medium and buffers were purchased from Invitrogen (Carlsbad, CA). For in vitro PKC inhibition studies, enzastaurin analogue (Eli Lilly & Co., Indianapolis, IN) was used. The human histiocytic lymphoma cell line U937 (9) was purchased from the American Type Culture Collection (Manassas, VA) and maintained in RPMI 1640 with 10% fetal bovine serum. The cell staining and wash buffer (assay buffer) was Dulbecco's PBS with addition of fetal bovine serum to 5% v/v. Methanol (anhydrous), DMSO, anisomycin, forskolin, 3-isobutyl-1-methylxanthine, and phorbol myristate acetate (PMA) were purchased from Sigma (St. Louis, MO). Stock solutions of agonists were prepared in DMSO and aliquots stored frozen at –70°C. Cytofix buffer and FITC-labeled surface antigen antibodies were purchased from BD Biosciences (San Diego, CA). Anti-phospho-(Ser)-PKC substrate antibody (rabbit polyclonal) was purchased from Cell Signaling Technology (Beverly, MA). Phycoerythrin-labeled goat anti-rabbit IgG was purchased from Biosource International (Camarillo, CA).

Stimulation and staining of phospho-PKC substrates in U937 cells. Aliquots of cells in log phase growth were washed with PBS and resuspended at 2 million/mL in RPMI with 10% fetal bovine serum. An aliquot of cells was pretreated with enzastaurin for 1 hour at 37°C. Cell stimulation and phosphoprotein staining was done using a modification of the method of Chow et al. (10). Briefly, 90 µL cells were dispensed into 12 x 75 mm polypropylene tubes followed by 10 µL of 10x working dilutions of agonist. Cells were stimulated for 20 minutes at 37°C, followed by addition of 100 µL of Cytofix buffer (diluted 1:1 in PBS) and incubation at 37°C for 10 minutes. Samples were then transferred to an ice bath, 1 mL ice-cold methanol added, and tubes were vortexed and held at 4°C for at least 30 minutes. Samples were then washed (2 mL wash buffer was added, tubes were centrifuged at 650 x g for 5 minutes, and supernatant was aspirated) and resuspended in 100 µL anti-phospho (p)-PKC substrate antibody (diluted 1/100 in assay buffer) or rabbit IgG (2 µg/mL) for control. Samples were incubated with primary antibody for 30 to 120 minutes, washed, and then incubated with phycoerythrin-labeled anti-rabbit IgG at 2 µg/mL for 15 to 30 minutes. Samples were washed and cells resuspended in 300 µL Cytofix buffer diluted 1:4 in standard PBS.

Stimulation and staining of p-PKC substrates in human leukocytes. Aliquots of cells in plasma were pretreated with enzastaurin (as appropriate) for 30 minutes at 37°C. PBMC stimulation and phosphoprotein staining was similar to that of U937 cells with some modifications. First, surface marker antibody (10 µL/test) was dispensed into tubes, followed by cells/plasma (sample volumes ranging from 200 to 400 µL were used). PMA was added using either 10x or 100x working solutions and cells were stimulated for 20 minutes at 37°C. This was followed by a rapid hypotonic lysis step to remove contaminating RBC (2 mL ice-cold distilled water was added to tubes, tubes were vortexed, and 250 µL of 10x PBS were added). Samples were centrifuged at 650 x g for 3 minutes, supernatant was aspirated, and cells were resuspended in 100 µL Cytofix buffer diluted 1:1 with standard PBS. Cells were fixed at 37°C for 10 minutes, permeabilized with methanol, and stained for intracellular p-PKC substrates as described above for U937 cells. Note that PMA treatment was found to cause monocyte adherence and clumping in some donors, leading to reduced monocyte numbers for analysis. This can be overcome, without affecting phosphoprotein results, by adding a small amount of EDTA solution (10 mmol/L) to tubes and vortexing well just before fixation. PKC activation in some patient samples was assayed by single-concentration (400 nmol/L PMA) stimulation of PBMC as described above. For others, a PMA titration curve was done. In addition, to provide a reference inhibitory control, an aliquot of patient PBMC in plasma was spiked in vitro with enzastaurin. Both aliquots were incubated at 37°C for 1 hour before proceeding with stimulation and measurement of PKC activation.

Flow cytometry analysis. Light scatter (forward and 90°), FITC, and phycoerythrin fluorescence signals were collected in listmode format on a Coulter XL or Beckman-Coulter FC500 flow cytometer (Beckman-Coulter, Miami, FL). For monocyte analysis, 3,000 CD14⁺ events were routinely analyzed (with a minimum of 500 cells). The collected listmode files were deconvoluted using Winlist software (Verity Software House, Topsham ME). Single-color controls were used for color compensation in the Winlist program. The mean phycoerythrin fluorescence intensity (MFI) was computed for each sample using a CD14-FITC versus 90° scatter gate.

Enzastaurin pharmacokinetic assay. Heparinized blood samples (5 mL) were collected from patients for pharmacokinetic assessment at steady state (on D7 and D15 of enzastaurin therapy). High-performance liquid chromatography with mass spectrometry was used to detect enzastaurin and its metabolites in plasma (Advion BioSciences, Inc., Ithaca, NY). The lower limit of quantification of this assay was 0.50 ng/mL. Pharmacokinetic variables, such as C_max and AUC_0-24, were calculated using noncompartmental methods from the plasma concentration-time profiles of enzastaurin and its metabolites with WinNonlin Pro 3.1 (Pharsight, Mountain View, CA).

Data analysis. Percentage fluorescence inhibition was calculated using the following formula:

where MFI_T refers to MFI of an enzastaurin-treated sample, MFI_B refers to unstimulated (basal) sample, and MFI_M refers to maximal stimulation MFI (PMA but no in vitro enzastaurin spike). When PMA titration curves were done on normal donor and patient PBMC, the area under the titration curve was calculated according to the linear trapezoidal rule. This value was designated as the integrated PMA response (IPR). Enzastaurin inhibition of IPR was calculated according to the following formula: [(IPR_max – IPR_{enzastaurin-treated}) / (IPR_max)] x 100, where IPR_max refers to the IPR of the PMA-only titration curve and IPR_{enzastaurin-treated} refers to the IPR of the PMA titration curve in the presence of enzastaurin. Day-to-day reproducibility of normal donor results was tested using the intercept term from a random-effects model of the logarithm of the ratio of the two IPR values using JMP software (SAS Institute, Inc., Cary, NC) with REML option. Statistical significance of PKC inhibition by enzastaurin in the capecitabine combination study was determined by log transformation of data and analysis by a linear mixed effect model (SAS Institute).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Flow cytometric detection of PKC signaling in U937 cells. We characterized our p-PKC substrate antibody using U937 cells, a cell line that has been shown by other techniques to express the PKC ßI and II isoforms, among others (11–13). Fluorescence histograms from a representative experiment (Fig. 1 ) show that basal p-PKC substrate signal measured was 15-fold greater than nonspecific binding to rabbit IgG (MFI = 62.0 for p-PKC substrate antibody versus 4.3 for rabbit IgG control). A 20-minute treatment with PMA at 500 nmol/L resulted in a 3-fold increase in p-PKC substrate signal; however, treatment with the mitogen-activated protein kinase agonist anigsomycin (1 µg/mL) did not increase signal. Likewise, treatment conditions used to stimulate PKA activity (20-minute incubation with 10 µmol/L forskolin and 500 µmol/L 3-isobutyl-1-methylxanthine) did not affect the p-PKC substrate signal. A 1-hour pretreatment with 2 µmol/L enzastaurin blocked PMA-induced PKC activation by 73%.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Flow cytometric detection of PKC signaling in U937 cells. U937 cells were cultured, treated, and stained for reactivity with p-PKC substrate antibody as described in Materials and Methods. Phycoerythrin fluorescence histograms from the flow cytometric analysis of representative test samples are overlaid and labeled with both the treatment and the mean fluorescence intensity value for each condition.

View larger version (39K): [in this window] [in a new window]   Fig. 2. Leukocyte subset analysis. Enzastaurin (ENZ; or 0.05% DMSO for controls) at final concentration of 1 µmol/L was added to whole blood samples in CPT tubes. Tubes were incubated with gentle rocking at room temperature for 1 hour. CPT tubes were processed and aliquots of the resultant PBMC were treated with 400 nmol/L PMA (or PBS for controls) and surface marker antibodies, and then processed for detection of intracellular p-PKC substrates as described in Materials and Methods. Fluorescence data were acquired and plotted as shown with FITC fluorescence (CD19 or CD14; X axis) and phycoerythrin fluorescence (p-PKC substrates; Y axis).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Human monocyte p-PKC substrate fluorescence data from patients before and after receiving enzastaurin (single-agent study). Blood samples were collected predose and after 14 and 28 days (except patient 3 who discontinued trial before day 28) of once daily oral dosing. Samples were stained for expression of CD14 (monocytes), stimulated ex vivo with PMA, and assayed of p-PKC substrate as described in Materials and Methods. An aliquot from each specimen was added to enzastaurin (1 µmol/L) to serve as a positive control for inhibition. Columns, mean of duplicate measurements; bars, SD.

View larger version (9K): [in this window] [in a new window]   Fig. 4. In vitro enzastaurin effect on PMA-induced PKC activity in human monocytes. Dilutions of enzastaurin were added to normal donor whole blood in CPT tubes and incubated for 1 hour, tubes were centrifuged, and then PBMC/plasma was collected. Each PBMC sample was aliquoted, stimulated with PMA concentrations from 100 to 3,200 nmol/L, and then assayed for monocyte p-PKC substrates as described in Materials and Methods. The PMA concentration responses for each of the enzastaurin-treated samples was determined using the area under each curve (as described in Materials and Methods) to derive the IPR value. Inset, representative IPR curves for varying enzastaurin treatments of a representative normal donor. Inhibition curve summarizes three separate whole blood experiments. Bars, SD.

View larger version (7K): [in this window] [in a new window]   Fig. 5. PMA titration using predose and postdose patient PBMC. Patient PBMC samples were aliquoted, stimulated with PMA concentrations from 25 to 1,600 nmol/L, and then assayed for monocyte p-PKC substrates as described in Materials and Methods. Points, MFI (after subtraction of background fluorescence) for each PMA concentration tested.

View larger version (11K): [in this window] [in a new window]   Fig. 6. Intracellular p-PKC substrate reactivity in monocytes from patients orally dosed with enzastaurin (capecitabine combination study). Patient blood samples were collected at predose, 4-hour postdose, and after 14 days of oral dosing. PBMC were prepared, stimulated with PMA concentrations from 100 to 3,200 nmol/L, and assayed for monocyte p-PKC substrates as described in Materials and Methods. Points, averaged results for nine patients; bars, SE. *, P < 0.001.

	Discussion

Top Abstract Materials and Methods Results Discussion References

Flow cytometric measurement of intracellular signaling using phosphospecific antibodies was first reported by Chow et al. (10). The authors showed strong activation of extracellular signal-regulated kinase in CD3⁺ lymphocytes following PMA or anti-CD3 stimulation and dose-dependent inhibition of phospho–extracellular signal-regulated kinase following in vitro treatment with an investigational Raf kinase inhibitor. Analysis of phosphoproteins by flow cytometry provides several advantages over Western blots, the standard technique used to detect phosphoepitopes. Chief among these is single cell analysis. Using flow cytometry, responses in minor cell subpopulations can be measured easily. Western blotting provides a single average value obtained from millions of lysed cells, thus hiding robust responses from small subpopulations. In addition, multivariable acquisition makes it possible to correlate multiple intracellular and extracellular markers simultaneously. Jacoberger et al., for example, examined cell cycle related distribution of phospho-STAT-5 in chronic myelogenous leukemia cell lines using probes for cellular DNA content and antibody to the mitotic marker MPM2 (14). Success of the flow cytometry technique is highly dependent on availability of quality reagent-grade antibodies that bind specifically to the phosphorylated (active) epitopes and do so in fixed, intact cells. Although this technique is in its infancy, vendors are now responding by developing and testing phosphospecific antibodies designed specifically for flow cytometry. Krutzik et al. (15) recently published an excellent review of the techniques and challenges involved in measuring protein phosphorylation at the single cell level. Detection of phosphoproteins by flow cytometry has been pushed to the current analytic limits by Perez and Nolan (16). Using a flow cytometer modified to collect signals from up to 11 different fluorochromes, the authors evaluated activity and kinetics of multiple kinase families in naïve and memory T lymphocytes.

In our studies, we first tested phospho–extracellular signal-regulated kinase and phospho–mitogen-activated protein/extracellular signal-regulated kinase kinase antibodies (data not shown). A number of commercially available anti-phospho-extracellular signal-regulated kinase antibodies can be used for robust signal by flow cytometry; however, we found that the best sensitivity to enzastaurin action was revealed by using an antibody to phosphorylated PKC substrates. The antibody recognizes phosphorylated serine residues in specific motifs (serine residues surrounded by lysine or arginine in positions –2 and +2 and a hydrophobic residue in position +1) of substrates of the classic PKC isoforms , ßI, ßII, and (17). The antibody has been used for PKC studies with Western blot and ELISA readout (18–20). Although not specific for PKC ßII, we were able to use this antibody to show enzastaurin inhibition of the PKC signaling cascade. Our studies revealed that monocytes, a minor population of human PBMC, are potent responders to PMA-induced PKC activation. Both monocytes and B-lymphocytes have been shown to have abundant PKC activity, particularly the ß isoforms (21, 22). Although all cells responded to PMA to varying degrees, monocytes generated a distinctly better signal window. Flow cytometry permitted us to focus on the appropriate subset and improved the assay signal window and thus assay sensitivity.

Intracellular phosphoprotein assays at the single cell level should continue to make important contributions to our understanding of the complex signaling circuitry of cells. The early reports of this novel technique have focused on signaling in cell lines or PBMC treated in vitro. Our study is one of few to use ex vivo stimulation to glean pharmacodynamic information from patient samples. Our initial results suggest that the administered enzastaurin dose is active against its intended molecular target in humans, albeit in surrogate cells. The monocyte serves as a conveniently obtained specimen that is robustly stimulated and dramatically inhibited. Desplat et al. recently reported flow cytometry analysis of leukemic blasts for expression of intracellular phosphotyrosine. The assay was used to monitor patients on imitinab therapy. Interestingly, bone marrow cells from chronic myelogenous leukemia patients who progressed to blast crisis despite continuing imitinab therapy exhibited high constitutive phosphotyrosine signal that could not be reduced by ex vivo culture with imitinab (23). These results linked hyperphosphorylation in leukemic blasts to failed inhibition of the molecular target and to clinical relapse.

Our drug activity biomarker has provided one of the first glimpses of intended target inhibition by enzastaurin in humans. Two factors that contributed to our success were selection of an appropriate antibody and using monocytes as surrogate target cells. At the outset of our studies, we did not know how monocytes from cancer patients would perform in our PKC activity assay compared with normal donor monocytes. We increased our chances of detecting PMA and drug effects by testing a 32-fold concentration range of PMA, with this result being conveniently summarized using an area under curve calculation (IPR). Clinical use of molecularly targeted therapies, such as enzastaurin, may possibly be maximized by codevelopment of specific assays, similar to those described in this report, to assist clinicians in identifying appropriate dosage and monitoring efficacy, toxicity, and resistance to these novel agents. Despite intersubject variability in the magnitude of monocyte responses to PMA, our intrasubject study using normal donors showed excellent week-to-week reproducibility. This is an important feature because drug activity biomarkers are most informative when done on sequentially obtained patient samples taken before and during the course of drug administration. Our assay of monocyte PKC activity has provided valuable information for further development of enzastaurin, yet use of functional assays in the clinical trial setting presents certain unique challenges. Many clinical laboratory markers are measured on patient serum or plasma samples that may be frozen for later analysis. The logistics of transporting and maintaining viable leukocytes, together with the throughput of current methods, means that intracellular phosphoprotein assays are most suitable for small phase I trials.

Despite significant treatment advances in the last decade, patients with advanced or metastatic cancers still have poor prognoses. Enzastaurin is an exciting example of a molecularly targeted anticancer agent. Presumably, with such agents, physicians could use methods that evaluate specific target inhibition to determine effective dose. Enzastaurin blocks activity of PKC, a key enzyme in the vascular endothelial growth factor signaling cascade that can ultimately induce tumor angiogenesis. We have used a multiparameter, flow cytometry assay of peripheral blood monocytes to show PKC inhibition in these cells after patients received enzastaurin. Data obtained from these studies should be useful for selection of efficacy doses and design of future clinical evaluations.

	Acknowledgments

 
We thank Dr. Michael Lahn for critical review of the manuscript, Christelle Darstein for statistical analysis, Lilly associates Kim Gill and Jennifer Hatmacher for managing clinical specimen collection and shipment, Dr. David Hedley for fostering our initial interest in intracellular phosphoprotein assays, and Dr. Garry Nolan for his encouraging endorsement of 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.

Received 10/11/05; revised 2/ 9/06; accepted 3/30/06.

	References

Top Abstract Materials and Methods Results Discussion References

 

1. Spitaler M, Cantrell DA. Protein kinase C and beyond. Nat Immunol 2004;5:785–90.[CrossRef][Medline]
2. Koya D, King GL. Protein kinase C activation and the development of diabetic complications. Diabetes 1998;47:859–66.[Abstract]
3. Hofmann J. Protein kinase C isozymes as potential targets for anticancer therapy. Curr Cancer Drug Targets 2004;4:125–46.[CrossRef][Medline]
4. Yan Liu WS, Thompson A, Leitges M, Murray NR, Fields AP. Protein kinase C-ßII regulates its own expression in rat intestinal epithelial cells and the colonic epithelium in vivo. J Biol Chem 2004;279:45556–63.[Abstract/Free Full Text]
5. Graff JR, McNulty AM, Hanna KR, et al. The protein kinase Cß-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts. Cancer Res 2005;65:7462–9.[Abstract/Free Full Text]
6. Herbst RS, Thornton, DE, Kies MS, et al. Phase I study of LY317615, a protein kinase Cß inhibitor. Proc Am Soc Clin Oncol 2002;21:82a.
7. Rademaker-Lakhai JM, Beereport L, Witteveen EO, et al. Phase I and pharmacologic study of enzastaurin HCl, gemcitabine and cisplatin. J Clin Oncol 2004;22:3129.
8. Fine HA, Royce C, Draper D, et al. Results from phase II trial of Enzastaurin (LY317615) in patients with recurrent high grade glimomas. ASCO annual meeting. Abstract. No. 1504; 2005.
9. Sundstrom C, Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer 1976;17:565–77.[Medline]
10. Chow S, Patel H, Hedley DW. Measurement of MAP kinase activation by flow cytometry using phospho-specific antibodies to MEK and ERK: potential for pharmacodynamic monitoring of signal transduction inhibitors. Cytometry 2001;46:72–8.[CrossRef][Medline]
11. Kiley SC, Parker PJ. Differential localization of protein kinase C isozymes in U937 cells: evidence for distinct isozyme functions during monocyte differentiation. J Cell Sci 1995;108:1003–16.[Abstract]
12. Pongracz J, Deacon EM, Johnson GD, Burnett D, Lord JM. Doppa induces cell death but not differentiation of U937 cells: evidence for the involvement of PKC-ß1 in the regulation of apoptosis. Leuk Res 1996;20:319–26.[CrossRef][Medline]
13. Mahajna J, King P, Parker P, Haley J. Autoregulation of cloned human protein kinase Cß and gene promoters in U937 cells. DNA Cell Biol 1995;14:213–22.[Medline]
14. Jacobberger JW, Frisa PS, Peng Ye P, et al. Immunoreactivity of Stat5 phosphorylated on tyrosine as a cell-based measure of Bcr/Abl kinase activity. Cytometry 2003;54A:75–88.[Medline]
15. Krutzik PO, Nolan Garry P, Perez OD. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol 2004;110:206–21.[CrossRef][Medline]
16. Perez OD, Nolan GP. Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 2002;20:155–62.[Medline]
17. Nishikawa K, Toker A, Johannes FJ, Songyang Z, Cantley LC. Determination of the specific substrate sequence motifs of protein kinase C isozymes. J Biol Chem 1997;272:952–60.[Abstract/Free Full Text]
18. Pierchala BA, Ahrens RC, Paden AJ, Johnson EM, Jr. Nerve growth factor promotes the survival of sympathetic neurons through the cooperative function of the protein kinase C and phosphatidylinositol 3-kinase pathways. J Biol Chem 2004;279:27986–93.[Abstract/Free Full Text]
19. Iwabu A, Smith K, Allen FD, Lauffenburger DA, Wells A. Epidermal growth factor induces fibroblast contractility and motility via a protein kinase C -dependent pathway. J Biol Chem 2004;279:14551–60.[Abstract/Free Full Text]
20. Jones ML, Craik JD, Gibbins JM, Poole AW. Regulation of SHP-1 tyrosine phosphatase in human platelets by serine phosphorylation at its C terminus. J Biol Chem 2004;279:40475–83.[Abstract/Free Full Text]
21. Su TT, Guo B, Kawakami Y, et al. PKC-ß controls IB kinase lipid raft recruitment and activation in response to BCR signaling. Nat Immunol 2002;3:780–6.[Medline]
22. Chang ZL, Beezhold DH. Protein kinase C activation in human monocytes: regulation of PKC isoforms. Immunology 1993;80:360–6.[Medline]
23. Desplat VL, Belloc F, Chollet C, et al. Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells. Cytometry 2004;62A:35–45.[Medline]

This article has been cited by other articles:

				J. Kim, Y.-L. Choi, A. Vallentin, B. S. Hunrichs, M. K. Hellerstein, D. M. Peehl, and D. Mochly-Rosen Centrosomal PKC{beta}II and Pericentrin Are Critical for Human Prostate Cancer Growth and Angiogenesis Cancer Res., August 15, 2008; 68(16): 6831 - 6839. [Abstract] [Full Text] [PDF]

				D. W. Hedley, S. Chow, C. Goolsby, and T. V. Shankey Pharmacodynamic Monitoring of Molecular-Targeted Agents in the Peripheral Blood of Leukemia Patients Using Flow Cytometry Toxicol Pathol, January 1, 2008; 36(1): 133 - 139. [Abstract] [Full Text] [PDF]

				R. K. Bowers, P. Marder, L. J. Green, C. L. Horn, A. L. Faber, and J. E. Thomas A platelet biomarker for assessing phosphoinositide 3-kinase inhibition during cancer chemotherapy Mol. Cancer Ther., September 1, 2007; 6(9): 2600 - 2607. [Abstract] [Full Text] [PDF]

				R. S. Herbst, Y. Oh, A. Wagle, and M. Lahn Enzastaurin, a Protein Kinase C{beta} Selective Inhibitor, and Its Potential Application as an Anticancer Agent in Lung Cancer Clin. Cancer Res., August 1, 2007; 13(15): 4641s - 4646s. [Abstract] [Full Text] [PDF]

				K. Podar, M. S. Raab, J. Zhang, D. McMillin, I. Breitkreutz, Y.-T. Tai, B. K. Lin, N. Munshi, T. Hideshima, D. Chauhan, et al. Targeting PKC in multiple myeloma: in vitro and in vivo effects of the novel, orally available small-molecule inhibitor enzastaurin (LY317615.HCl) Blood, February 15, 2007; 109(4): 1669 - 1677. [Abstract] [Full Text] [PDF]

				B. A. Teicher Protein kinase C as a therapeutic target. Clin. Cancer Res., September 15, 2006; 12(18): 5336 - 5345. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Green, L. J. Articles by Thornton, D. Search for Related Content PubMed PubMed Citation Articles by Green, L. J. Articles by Thornton, D.