Protein Kinase C-β Gene Variants, Pathway Activation, and Enzastaurin Activity in Lung Cancer

Sang-Haak Lee; Tingan Chen; Jun Zhou; Jennifer Hofmann; Gerold Bepler

Disclosures

Clin Lung Cancer. 2010;11(3) 

In This Article

Patients and Methods

Cell Culture

Sixteen NSCLC cell lines and 12 small-cell lung cancer (SCLC) lines were obtained from their original sources or the American Type Culture Collection (Bethesda, MD). They were maintained in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and antibiotics, and their authenticity was confirmed by DNA fingerprinting or isoenzyme patterns. They were free of mycoplasma contamination.

Gene Sequencing

For sequencing of the human PRKCB1 gene in cell lines, genomic DNA was extracted using resin-exchange chromatography. The complete genomic sequence, as reported in GenBank accession number NC_000016, was used as a reference. Exons 2–18 were amplified using intronic primer pairs and sequenced in both directions. Exon 1 required two primer pairs for a complete sequence analysis. The primer sequences are listed in Table 1. Sequencing was performed on an ABI Prism 377 DNA Sequencer, using BigDye Terminator Cycle Sequencing (Applied Biosystems, Inc; Carlsbad, CA). Results were compared with the reference sequence, using Sequencher software (version 3.0, Gene Codes Corp; Ann Arbor, MI) and BLAST software.

Proliferation Assays

Cell proliferation was assessed using a luminescent cell-viability assay (CellTiter-Glo; Promega; Madison, WI). This method is based on the quantification of adenosine triphosphate (ATP) levels, which correlate with the number of viable cells in culture. Cell lines H23, H125, H322, A549, H69, H211, DMS76, and SW210.5 were plated at the appropriate density (3000 cells per well for NSCLC; 50,000 cells per well for SCLC) in 96-well opaque white plates (Matrix Technologies, Hudson, NH) for 24 hours. The next day, growth medium was replaced with RPMI-1640 containing 1% FBS, at 90 μL per well. Enzastaurin was dissolved in phosphate-buffered saline (PBS) containing 0.02% dimethyl sulfoxide (DMSO), and we added 10 μL to each well. The final enzastaurin concentrations were 0.0 μM, 0.001 μM, 0.01 μM, 0.05 μM, 0.1 μM, 1.0 μM, and 5.0 μM. Cells were incubated for 72 hours at 37°C. We then added 100 μL of CellTiter-Glo reagent to each well. Cells and reagents were mixed with an orbital shaker for 2 minutes to induce cell lysis and were incubated at room temperature for 10 minutes to stabilize the luminescent signal. Luminescence was measured in a Veritas Microplate Luminometer (Turner Biosystems; Sunnyvale, CA). Cell proliferation was calculated as percentage of relative luminescence units of treated vs. untreated cells. Error bars represent the standard error, which was calculated using statistical tools in Microsoft Excel. At least three independent experiments were performed.

Immunoblotting

The NSCLC (H23, H125, H322, and A549) and SCLC (H69, H211, DMS76, and SW210.5) cell lines were incubated with 1 μM enzastaurin in RPMI-1640 supplemented with 1% FBS for up to 4 hours in six-well plates at approximately 1,000,000 cells/well. They were harvested in protein lysis buffer (20 mmol/L Tris-HCl at pH 7.6, 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetate, 0.5% NP40, 1 mmol/L dithiothreitol, 5 μmol/L trichostatin A, 1 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L NaF, and complete protease inhibitors; Roche Applied Science; Penzberg, Germany). Lysates were centrifuged at 4°C for 15 minutes at 13,000 × g, and the supernatants were recovered. Protein extracts (50 μg) were fractionated through 10% Novex tris-glycine gels (InVitrogen; Carlsbad, CA), blotted onto pure nitrocellulose membranes (Bio-Rad, Hercules, CA), and probed for the targets PKCβ2, phospho-PKCβ2 (ser660), GSK3β, phosphor-GSK3β (Ser9), S6RP, phosphor-S6RP (Ser240/244), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with commercially available antisera (Cell Signaling, Beverly, MA; Santa Cruz, Inc, Santa Cruz, CA). For final protein detection, a goat anti-mouse or anti-rabbit immunoglobulin G (IgG) or rabbit anti-goat IgG horseradish peroxidase secondary antibody was used (Santa Cruz, Inc), together with SuperSignal West Pico chemiluminescent substrate (Pierce; Rockford, IL).

In-cell Western Assay

For in-cell Western blot analysis, NSCLC (H23, H125, H322, and A549) and SCLC (H69, H211, DMS76, and SW210.5) cell lines were seeded in RPMI-1640 with 10% FBS at 20,000 cells/well in 96-well clear-bottom plates (B.D. Falcon; Franklin, NJ). After 24 hours, the medium was changed to RPMI-1640 with 1% FBS, at 180 μL per well. Enzastaurin was dissolved in PBS containing 0.02% DMSO, and we added 20 μL to each well. The final enzastaurin concentrations were 0.0 μM, 0.001 μM, 0.01 μM, 0.1 μM, 1.0 μM, and 5.0 μM. After 4 hours of incubation, the medium was completely aspirated, cells were fixed with 3.7% formaldehyde in PBS for 20 minutes, and cells were permeabilized with five 5-minute washes in PBS + 0.1% Triton-X 100. Li- Cor Odyssey blocking buffer (Li-Cor Biosciences; Lincoln, NB) was added to each well (150 μL) for 90 minutes at room temperature with moderate shaking on a rotator, followed by overnight incubation with primary antibodies phospho-PKCβ2 (Ser660) and PKCβ2, 1:50; phospho-GSK3β (Ser9) and GSK3β, 1:100; S6RP and phospho-S6RP (Ser240/244), 1:500; phospho-Akt (Thr308), phospho-Akt (Ser473), and Akt, 1:100; and phospho-forkhead transcription factor (FKHR) (Ser256) and FKHR, 1:100, in Li-Cor Odyssey blocking buffer. After five 5-minute washes in PBS + 0.1% Tween-20, detection was performed using two species-specific infrared fluorescent dye-conjugated secondary antibodies (IRDye 800CW-conjugated goat anti-rabbit IgG 1:800, and IRDye 680CW-conjugated goat anti-mouse IgG 1:200 in Li-Cor Odyssey blocking buffer + 0.2% Tween-20). After 1 hour of incubation and washing, targets were simultaneously visualized using the Odyssey Infrared Imaging Scanner (Li-Cor), with the 700-nm fluorophore emitting a red color and the 800-nm fluorophore emitting a green color. Relative fluorescent units for enzastaurin-treated samples were divided by vehicle controls to determine percent change in expression. Error bars represent standard error, which was calculated using statistical tools in Microsoft Excel. At least three independent experiments were performed.

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