QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 16 September 2003
doi: 10.1242/jcs.00712

This Article Summary Full Text Full Text (PDF) Movie Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend 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 Joy, A. M. Articles by Berens, M. E. Search for Related Content PubMed PubMed Citation Articles by Joy, A. M. Articles by Berens, M. E.

Migrating glioma cells activate the PI3-K pathway and display decreased susceptibility to apoptosis

The Translational Genomics Research Institute, 400 North 5th Street, Suite 1600, Phoenix, AZ 85004, USA

View larger version (42K): [in a new window]   Fig. 1. Laminin stimulates glioma cell migration. (A) A typical migration assay for cells on laminin at the initial migration measurement (2 hours following plating, t=0) and after allowing cells to migrate out for 24 hours (t=24). The circle circumscribing cells used to calculate migration rate is shown. Also shown within the rectangle is a representative field of rim cells used to calculate apoptosis. Bar, 2 mm. (B) SF767 and T98G cells were plated onto 0.1% BSA- or 10 µg/ml laminin-coated slides and migration rate obtained after 24 hours. See movie online (http://jcs.biologists.org/supplemental).

View larger version (40K): [in a new window]   Fig. 2. Actively migrating cells suppress apoptosis induction by camptothecin and Trail. (A) Representative micrograph of Trail-treated, fixed and DAPI stained T98G cells. Arrows identify cells scored as apoptotic. (B) SF767 and T98G cells were plated onto laminin-(LAM, 10 µg/ml) or 0.1% BSA-coated slides in the migration assay format and allowed to migrate for 16 hours. Cells were treated with 1 µM camptothecin for 24 hours or 100 ng/ml Trail for 16 hours, fixed and stained with DAPI. Cells in rim and core were scored for total and apoptotic cells. Values represent the mean and standard deviation of five replicate measurements. Significance with unpaired Student's t test: *P<0.001 versus rim on laminin; **P<0.01 versus rim on laminin. (C) Differential sensitivity to apoptosis is not overcome at high concentrations of cytotoxic agent. SF767 and T98G cells were plated on laminin-(10 µg/ml) coated slides in the migration assay format and allowed to migrate for 24 hours. Cells were treated with the specified concentration of camptothecin or Trail as described in Materials and Methods, fixed and DAPI stained. Cells in the rim and core were scored for total and apoptotic cells. Values represent the mean and standard deviation of four replicate measurements. (D) Camptothecin treatment generates activated caspase 3. SF767 cells in the migration assay format were treated with 1 µM camptothecin as described in Materials and Methods, fixed, stained with DAPI and immunocytochemistry for activated caspase 3 performed. Arrows show cells with condensed fragmented chromatin and positive staining for caspase 3. Bar in A and D, 5 µm.

View larger version (15K): [in a new window]   Fig. 3. Camptothecin and Trail-induced apoptosis is inversely proportional to rate of migration. SF767 and T98G cells were plated onto slides coated with the specified concentration of laminin. Migration was measured 24 hours post seeding (), cells were then treated with 1 µM camptothecin for 24 hours (SF767) or 100 ng/ml Trail for 16 hours (T98G), stained with DAPI then % apoptotic cells in the rim quantitated as described in Materials and Methods (). Values represent the mean and standard deviation of three replicate measurements.

View larger version (26K): [in a new window]   Fig. 4. Migration inhibitors sensitize actively migrating but not migration-restricted cells to apoptosis. (A) Cells were seeded onto slides coated with 10 µg/ml laminin and allowed to adhere. The media was exchanged for media containing migration inhibitors, cells were incubated for 24 hours and the migration rate was determined as described in Materials and Methods. (B) Cells were plated onto slides coated with 10 µg/ml laminin, allowed to migrate for 24 hours then pretreated with migration inhibitors or solvent control for 2 hours in serum-free media. Media was replaced with fresh serum-free media containing migration inhibitors or solvent control plus 1 µM camptothecin (Cpt) for SF767 cells or 100 ng/ml Trail for T98G cells. Cells were then incubated with camptothecin plus inhibitors or solvent control for 24 hours or Trail plus inhibitors or solvent control for 16 hours, fixed, stained with DAPI and apoptotic and total nuclei quantitated. Gray bars represent cells treated with camptothecin or Trail alone and black bars represent cells treated with migration inhibitor plus camptothecin or Trail. Significance with unpaired Student's t test: *P<0.05 vs camptothecin only treatment of rim cells; **P<0.001 vs Trail only treatment of rim cells. (C) Migration rate vs % apoptosis was plotted (r2=0.79) using data from Fig. 3 () and Fig. 4A,B (). Data from anti-ß1 integrin treatment of SF767 cells was deleted from this analysis.

View larger version (93K): [in a new window]   Fig. 5. Phospho-Akt is detected only in migrating cells. T98G cells were plated in the migration format onto laminin-coated slides (10 µg/ml) and allowed to migrate overnight. Cells were then incubated in serum-free media for 4 hours, treated with 100 ng/ml EGF or solvent control for 15 minutes then fixed. For treatment with the PI3-K inhibitor LY294002 (20 µM) was added 2 hours before time of EGF addition. Slides were then processed for total Akt (a and b) or phospho-Akt Ser 473 (c-h) immunocytochemistry as described in Materials and Methods. Arrows in d indicate more intense staining for phospho-Akt at the leading edge type of structures. Arrows in g indicate a different pattern of staining for phospho-Akt following EGF treatment. Bar, 5 µm.

View larger version (45K): [in a new window]   Fig. 6. Cells plated sparsely on laminin have increased staining for phosphorylated Akt and suppress Trail-induced apoptosis. T98G cells were plated sparsely (10,000 cells/cm2) or densely (50,000 cells/cm2) onto slides coated with BSA or laminin as indicated and incubated overnight (A and B). (A) Cells were then incubated 4 hours in serum-free media and fixed, and immunocytochemistry for p-Ser-473 Akt was performed as described in Materials and Methods. Bar, 5 µm. (B) Some slides were then treated with Trail for 16 hours as described in the method section, fixed, stained with DAPI and % apoptotic cells determined.

View larger version (26K): [in a new window]   Fig. 7. Western analysis for phospho-Akt production in cells plated sparsely on laminin. T98G cells were plated onto laminin-coated dishes at 7000 (sparse) or 55,000 (dense) cells/cm2 and lysates collected. (A) Total Akt, Akt phosphorylated on Ser-473, Akt phosphorylated on Thr-308, total GSK-3ß, GSK-3 phosphorylated on Ser-21, GSK-3ß phosphorylated on Ser-9 and tubulin was evaluated by SDS-PAGE and western analysis as described in Materials and Methods. (B) Signals were quantified by densitometry using Gel Expert software by Nucleovision.

View larger version (13K): [in a new window]   Fig. 8. Pharmacological inhibition of PI3-K increases apoptosis sensitivity of migrating cells. SF767 cells were plated onto laminin coated (10 µg/ml) slides in the migration assay format and incubated overnight. Cells were treated with 1 µM camptothecin (Cpt) for 24 hours, or preincubated with LY294002 for 2 hours before camptothecin addition, fixed and stained with DAPI. Cells in rim and core were scored for total cells and cells with condensed, fragmented chromatin. Values represent the mean and standard deviation of five replicate measurements. Significance with unpaired Student's t test: *P<0.001 vs camptothecin alone.