Roles of GalNAc-disialyl Lactotetraosyl Antigens in Renal Cancer Cells

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Details are described in Supplementary Methods.


Cell lines

Human renal cancer cell lines (SK-RC 1, SK-RC 6, SK-RC 7, SK-RC 17, SK-RC 29, SK-RC 35, SK-RC 39, SK-RC 44, SK-RC 45, SK-RC 99 and Moroff) were established at Memorial Sloan-Kettering Cancer Center (New York, NY, U.S.A). Human renal cancer cell lines (Caki-1, VMRC-RCW, VMRC-RCZ, TUHR10TKB and TUHR14TKB) were obtained from the Human Science Research Resources Bank (Osaka, Japan). Human renal cancer cell lines (OS-RC-2, RCC10RGB, TUHR4TKB) were obtained from the Riken Cell Bank (Wako, Japan). A human renal cancer cell line, ACHN, was purchased from Dainihonseiyaku Co. Normal HRPTE were purchased from Sanko Junyaku Co. All human renal cancer cell lines were grown in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% (v/v) FCS. HRPTE cells were cultured in renal epithelial cell growth medium Bullet KitTM (Sanko Junyaku Co.). Mouse fibroblast L cells was grown in D-MEM supplemented with 7.5% FCS.


Antibodies

Monoclonal antibodies (mAbs) used were as follows: anti-MSGG (RM1), anti-DSGG (5F3) and anti-GalNAc-DSLc4 (RM2) were established as described previously12; anti-DSLc4 mAb (FH9) was kindly provided by R. Kannagi at Aichi Medical University, Nagakute. FITC-labeled anti-mouse IgG antibody and anti-mouse IgM antibody were purchased from ICN/Cappel (Durham, NC). Anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) was purchased from Cell Signaling Technology (Beverly, MA). Anti-mouse IgG (H + L) conjugated with HRP was from BETHYL Laboratories, Inc. (Montgomery, TX). Anti-mouse IgG conjugated with HRP and anti-goat IgG conjugated with HRP were from Santa cruz biotechnology, Inc. (Dallas, TX). Anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-Akt, anti-phospho-EGFR (Tyr1068), anti-EGFR, anti-phospho-cMet (Tyr1234/1235), anti-phospho-cMet (Tyr1349), anti-cMet, anti-phospho-ERK p44/42 (Thr202/Tyr204), anti-ERK p44/42, anti-Caveolin-1, and anti-ILK, were purchased from Cell Signaling Technology (Beverly, MA). Goat polyclonal anti-integrin β1 antibody and mouse monoclonal anti-integrin α3 were purchased from Santa cruz biotechnology, Inc. Mouse anti-CD29 mAb was purchased from BD Transduction Laboratories (San Jose, CA). Monoclonal mouse anti-human CD29 (integrin β1)/biotin antibody was from Ancell (Bayport, MN). Anti-β-actin mAb conjugated with horseradish peroxidase and anti-GAPDH mAb conjugated with horseradish peroxidase were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan).


Construction of expression vectors

Plasmid DNA construction is described in Supplementary Methods.


Flow cytometric analysis

Cell surface expression of glycolipids on 20 renal cancer cell lines and HRPTE cells was analyzed using mAbs by a FACScaliburTM with CellQuestTM version 3.1 f software (Becton Dickinson). Cells were incubated with mAbs for 1 h on ice and stained with FITC-conjugated second antibodies. Control cells for flow cytometry were prepared using the second antibody alone.


Preparation of membrane fraction

L cells (3 × 106) were plated in 10-cm dishes and transiently transfected with an expression plasmid (4 μg) by LipofectamineTM 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The cells were harvested after 48 h, and the membrane fraction was prepared using a nitrogen cavitation apparatus (Parr Instrument Co., Moline, IL) as described11.


N-acetylgalactosaminyltransferase assay

The N-acetylgalactosaminyltransferase assay was performed as previously described11.


Transfection for flow cytometric analysis

The THUR14TKB cells were transiently transfected by LipofectamineTM 2000 as described in Supplementary Methods.


D-PDMP treatment of transfectant cells

D-PDMP (an inhibitor of glucosylceramide synthase42) was purchased from Sigma, and dissolved in distilled water at concentration of 4 mM. This stock solution was directly added to culture medium. The transfectant cells were treated with 50 μM D-PDMP for 6 days.


Glycolipid extraction and TLC

Glycolipids were extracted as described previously43.


MTT assay

For cell proliferation assay, transfectant cells and control cells (2.5 × 103 cells/well) were prepared in 48-well plates in serum-containing medium and cultured for 6 days. MTT assay was performed by assessing the reduction of MTT to formazan based on the absorbance at 590 nm using Molecular Devices Vmax kinetic microplate readerTM with SOFT max Pro.3.1.1TM software (Molecular Devices). For cell growth inhibition assay, transfectant cells and control cells (2.5 × 103 cells/well) were seeded in 48-well plates in serum-containing medium and treated with mAb RM2 antibody diluted to the indicated concentrations for 5 days.


Cell viability assay

To determine cell viability, we quantify the amount of ATP present, which indicates the presence of metabolically active cells. Cells grown in 96-well plates for 4 days (7.5 × 102 cells/well) were prepared. Then CellTiter-Glo™ Reagent (Promega Corporation, Madison, MI) was added with equal volume to that of cell medium in each well. Plates were incubated at room temperature for 10 min on a shaker and luminescence was measured on a luminometer (TriStar2 LB942, Berthold Technology) according to the manufacturer’s instruction.


In vitro invasion assay

Invasion assay were performed using BioCoat™ Matrigel™ Invasion Chamber (Corning) as described previously16 with some modifications.


Cell adhesion assay using real time cell electronic sensing (RT-CES)

ACEA e-plates (ACEA Biosciences, San Diego, CA) were coated with FN from human plasma, LN from human placenta, CL type I, or CL type IV from human placenta for 1 h at 37 °C. FN, CL type I, and CL type III were purchased from Chemicon (Temecula, CA). LN was purchased from Sigma. The plates were washed with PBS and coated with 0.5% BSA in PBS for 30 min at 37 °C. The wells were washed with PBS before the addition of culture medium with or without serum, and cells (2 × 104) were added on ACEA e-plate coated with various extracellular matrix proteins. The adhesion of cells was continuously monitored using the RT-CES system (Wako Pure Chemical).

To examine the inhibition of cell adhesion by mAbs reactive with anti-integrin β1, anti-integrin α3, or mAb RM2, cells were pretreated with these mAbs for 30 min at room temperature before plating into wells.


Integrin-mediated adhesion to laminin

Cells were starved for 14–16 h in serum-free D-MEM, and harvested with 0.02% EDTA in PBS. To reduce basal phosphorylation of signaling molecules, cells were rotated for 30 min at 37 °C using a tube rotator (TR-118, AS ONE, Osaka, Japan). Cell suspensions (4 × 105) were added to 6-cm dishes pre-coated with LN from human placenta (Sigma-Aldrich, Germany). Cells were lysed after incubation at 37 °C, and lysates were used for Western immunoblotting.


Western immunoblotting

Cells were lysed with cell lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 μg/ml of leupeptin) (Cell Signaling), Protease Inhibitor MixtureTM (Calbiochem, SanDiego, CA), and 1 mM PMSF. Insoluble materials were removed by centrifugation at 15,000 × g for 10 min at 4 °C. Cell lysates were separated by SDS-PAGE using 7.5% or 10% gels. The separated proteins were transferred onto a Fluoro Trans™ W Membrane (PALL, Dreieich, Germany). Blots were blocked with 3% BSA in PBS. The membrane was first probed with primary antibodies. After being washed with PBS containing 0.05% Tween 20, the blots were incubated with respective secondary antibodies conjugated with horseradish peroxidase. After the membrane was washed, bound conjugates were visualized with an enhanced chemiluminescence (ECLTM) detection system (PerkinElmer Life Sciences) and imaged using a Biorad ChemiDoc XRS™ (BioRad).


Knockdown of ILK

Suppression of ILK with 3 kinds of siRNA was examined by immunoblotting using an anti-ILK antibody (Cell signaling) after 72 h of transfection into GalNAc-DSLc4-expressing cells as previously described28. GL2, siRNA for a firefly luciferase (B-Bridge International, Inc. CA) was used as a negative control. Si-GAPDH was also used as a control. si-ILK3 (5′AAGUUAAGCUGUUUGAAGUCAAUGC-3′) (Invitrogen) was the most effective (knockdown rate was about 80–90%).


Preparation of GEM/raft fractions

GEM/rafts were isolated using a detergent extraction method as described previously17,44.


Immunofluorescence staining

Cells were rotated under serum-free conditions for 30 min, and stimulated with 10% FCS followed by incubation for 0–60 min. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After being washed with PBS, nonspecific binding was blocked with 2.5% normal donkey serum and 5% normal goat serum in PBS for 30 min at room temperature. Cells were incubated with biotin-conjugated anti-human CD29 antibody and anti-caveolin-1 antibody in PBS for 45 min at room temperature, then with streptavidin-Alexa 488 and anti-rabbit IgG-Alexa 594 in PBS for 30 min at room temperature. The resulting staining patterns were imaged using a confocal microscope (Fluoview FV10i-DOC, Olympus, Tokyo, Japan).


PI3K inhibitor treatment

Cells were cultured with serum-free D-MEM overnight, and then treated with 50 μM or 150 μM LY294002 for 30 min. After treatement with serum-containing D-MEM for 5 min, the cell lysates were prepared and used for Western immunoblotting with anti-EGFR (Tyr1068) or anti-phospho Akt (Thr308) antibodies. In the same manner, 0–0.5 nM of LY294002-treated cells were harvested with 0.02% EDTA in PBS, and used for cell proliferation assay, 0–150 μM of LY294002-treated cells were used for invasiveness assay and 50 μM of LY294002-treated cells were used for adhesion assay.


mAb RM2 treatment

Cells were cultured with serum-free D-MEM overnight, and mAb RM2-treated cells were harvested with 0.02% EDTA in PBS. The cells were treated with 1/200 conc. of stock solution (1 mg/ml) of mAb RM2 in serum-free D-MEM for 30 min, and were used for cell proliferation assay, invasiveness assay and adhesion assay.


Statistical analysis

The statistical significance was examined by the Student’s t-test.



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