1. Volume 114, Issue 5, Pages 930-939 (May 1998)
Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells 
William E. Karnes, Shaun G. Weller*, Philip N. Adjei*, Timothy J. Kottke‡, Kahlil S. Glenn*, Gregory J. Gores*, Scott H. Kaufmann‡ 
Gastroenterology 
Volume 114, Issue 5, Pages (May 1998)
DOI: /S (98)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Concentration-dependent inhibition of EGF-induced EGFR tyrosine phosphorylation in ligand-dependent and ligand-independent colon cancer cell lines by PD (A) Antiphosphotyrosine immunoblots were prepared after exposure of cells to EGF and varying concentrations of PD and resolution of total cell lysates by 7.5% SDS–polyacrylamide gel electrophoresis (PAGE). Treatments are shown on top. Immunoblots were cropped to include only the 180-kilodalton EGFR. (B) Gray-scale densitometry of the autoradiographs. Y axis reflects relative phosphorylation expressed as fraction of unstimulated (no EGF, no PD ) value for each cell line. Ligand-dependent cell lines: SNU-C1 (◊) and SNU-C4 (○); ligand-independent cell lines: SNU-C5 (▴), HCT116 (●), and SW480 ().
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Concentration-dependent effects of 72-hour treatment with (A) PD or (B) tyrphostin 51 on viable cell mass of ligand-dependent and ligand-independent cells. MTS assay was used to assess viable cell mass. Data are from representative experiments repeated a minimum of three times for each cell line. Ligand-dependent cells are shown in open symbols (SNU-C1 [□] and SNU-C4 [○]), and ligand-independent cells are shown in closed symbols (SNU-C5 [●]), SW480 [■], and HCT116 [(▴]). Ligand-dependent cells treated with EGF 10 ng/mL EGF are shown by dotted lines. Y axes are expressed as absorbance values at 72 hours as a percentage of absorbance values of identically plated cells at time 0 hour.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Nuclear and DNA fragmentation (Hoescht and TUNEL images; original magnification 400×) after 18-hour incubation with diluent or 10 μmol/L PD Arrows indicate representative cells with nuclear fragmentation. All visible cells in the TUNEL column are TUNEL positive and were only seen in EGFR ligand-dependent cells after treatment with PD
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

5. Fig. 4
PD induces apoptosis in SNU-C4 cells in a concentration- and time-dependent manner. (A) Cells were treated for 16 hours with the indicated concentration of PD , then stained with Hoescht so that the percentage of cells with fragmented nuclei could be quantified (% apoptosis). Values represent means ± SD of a minimum of 3 separate experiments for each PD concentration. (B) Cells were treated with 10 μmol/L PD for the indicated length of time before the percentage of cells with fragmented nuclei (% apoptosis) was determined. Error bars represent SDs of the mean from a representative experiment repeated three times.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

6. Fig. 4
PD induces apoptosis in SNU-C4 cells in a concentration- and time-dependent manner. (A) Cells were treated for 16 hours with the indicated concentration of PD , then stained with Hoescht so that the percentage of cells with fragmented nuclei could be quantified (% apoptosis). Values represent means ± SD of a minimum of 3 separate experiments for each PD concentration. (B) Cells were treated with 10 μmol/L PD for the indicated length of time before the percentage of cells with fragmented nuclei (% apoptosis) was determined. Error bars represent SDs of the mean from a representative experiment repeated three times.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

7. Fig. 5
Caspase activation in PD –treated ligand-dependent SNU-C4 cells. (A) Cytosol was prepared from SNU-C4 cells after various periods of exposure to 10 μmol/L PD Activities that cleave DEVD-AFC (caspase 3–like activity) and YVAD-AFC (caspase 1–like activity) were assayed as described in Materials and Methods. ○, Caspase 3; ◊, caspase 1. (B) Immunoblots showing proteolysis of PARP (top panel) and lamin B1 (bottom panel) after exposure of ligand-dependent and ligand-independent cell lines to 20 μmol/L PD for 24 hours. Arrows indicate caspase cleavage products.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

8. Fig. 5
Caspase activation in PD –treated ligand-dependent SNU-C4 cells. (A) Cytosol was prepared from SNU-C4 cells after various periods of exposure to 10 μmol/L PD Activities that cleave DEVD-AFC (caspase 3–like activity) and YVAD-AFC (caspase 1–like activity) were assayed as described in Materials and Methods. ○, Caspase 3; ◊, caspase 1. (B) Immunoblots showing proteolysis of PARP (top panel) and lamin B1 (bottom panel) after exposure of ligand-dependent and ligand-independent cell lines to 20 μmol/L PD for 24 hours. Arrows indicate caspase cleavage products.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

9. Fig. 6
PD induces Bcl-2 cleavage but no change in p53 levels. (A) Effects of PD on levels of p53 and Bcl-2 polypeptides in ligand-dependent and ligand-independent cell lines. Immunoblots are shown after 24-hour incubation with 20 μmol/L PD or diluent. Arrow indicates a 22–23-kilodalton Bcl-2 immunoreactive peptide that appears in response to PD treatment exclusively in ligand-dependent cell lines. Asterisks indicate cells bearing mutant p53. (B) Effects of DEVD-fmk on appearance of 22–23-kilodalton Bcl-2 immunoreactive band after 24 hours exposure to 20 μmol/L PD The left panel shows immunoblot using MAb (Ab-1) and the right panel shows immunoblot using polyclonal (Ab-2). Treatments are labeled on top: −, diluent control; +, DEVD-fmk (100 μmol/L). Arrowhead indicates a 22–23-kilodalton Bcl-2 cleavage product.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

10. Fig. 6
PD induces Bcl-2 cleavage but no change in p53 levels. (A) Effects of PD on levels of p53 and Bcl-2 polypeptides in ligand-dependent and ligand-independent cell lines. Immunoblots are shown after 24-hour incubation with 20 μmol/L PD or diluent. Arrow indicates a 22–23-kilodalton Bcl-2 immunoreactive peptide that appears in response to PD treatment exclusively in ligand-dependent cell lines. Asterisks indicate cells bearing mutant p53. (B) Effects of DEVD-fmk on appearance of 22–23-kilodalton Bcl-2 immunoreactive band after 24 hours exposure to 20 μmol/L PD The left panel shows immunoblot using MAb (Ab-1) and the right panel shows immunoblot using polyclonal (Ab-2). Treatments are labeled on top: −, diluent control; +, DEVD-fmk (100 μmol/L). Arrowhead indicates a 22–23-kilodalton Bcl-2 cleavage product.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

11. Fig. 7
Effects of DEVD-fmk on PD –induced apoptosis, viable cell mass, and [3H]thymidine incorporation. Treatments are shown at the bottom (0, 1, or 10 μmol/L PD ) with DEVD-fmk (0 or 100 μmol/L). (A) Percent fragmented nuclei by Hoescht staining (% apoptosis) after 18-hour treatment. (B) MTS assay after 48-hour treatment. (C) [3H]Thymidine incorporation after 18-hour treatment. Data show representative experiments repeated three times. Error bars represent SD.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

12. Fig. 8
Protection by DEVD-fmk is temporary. (A) Time course of protective effects of DEVD-fmk. Medium with PD , DEVD-fmk, or diluent was replaced every 24 hours. The percentage of cells displaying fragmented nuclei (% apoptosis) was assessed by Hoescht staining after 0-, 6-, 24-, or 48-hour treatment with 20 μmol/L PD in the presence of DEVD-fmk (100 μmol/L) or diluent. □, Control; ■, DEVD-fmk. Error bars represent SDs among 10 fields for each treatment in duplicate. (B) Representative images of Hoescht-stained cells treated with PD μmol/L for 24 or 48 hours with and without DEVD-fmk (100 μmol/L).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

13. Fig. 8
Protection by DEVD-fmk is temporary. (A) Time course of protective effects of DEVD-fmk. Medium with PD , DEVD-fmk, or diluent was replaced every 24 hours. The percentage of cells displaying fragmented nuclei (% apoptosis) was assessed by Hoescht staining after 0-, 6-, 24-, or 48-hour treatment with 20 μmol/L PD in the presence of DEVD-fmk (100 μmol/L) or diluent. □, Control; ■, DEVD-fmk. Error bars represent SDs among 10 fields for each treatment in duplicate. (B) Representative images of Hoescht-stained cells treated with PD μmol/L for 24 or 48 hours with and without DEVD-fmk (100 μmol/L).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions