Volume 120, Issue 7, Pages 1700-1712 (June 2001) PKC-δ inhibits anchorage-dependent and -independent growth, enhances differentiation, and increases apoptosis in CaCo-2 cells  Sonia R. Cerda, Marc Bissonnette, Beth Scaglione–Sewell, Matthew R. Lyons, Sharad Khare, Reba Mustafi, Thomas A. Brasitus  Gastroenterology  Volume 120, Issue 7, Pages 1700-1712 (June 2001) DOI: 10.1053/gast.2001.24843 Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 1 Western blot analysis of PKC-δ expression in CaCo-2 cells and in human colonic normal mucosa and colon tumor samples. Whole cell lysates were prepared from rat brain control, CaCo-2 cells, and human normal (N) and tumor (T) paired colon tissues. Proteins (20 μg/lane) were resolved by SDS-PAGE on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with antihuman PKC-δ and β-actin monoclonal antibodies. Upper panel: PKC-δ expression. PKC-δ protein comigrates with rat brain PKC-δ with MR = 78 kilodaltons. Lower panel: β-actin expression. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 2 Western blot analysis of PKC-δ–transfected CaCo-2 cells. Whole cell lysates were prepared from rat brain control and EV- and PKC-δ–transfected cells. Proteins (15 μg/lane) were resolved by SDS-PAGE on 10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunoblotted with PKC-δ monoclonal antibodies. (A) PKC-δ expression in PKC-δ1 and EV-1 stable transfectants. (B) PKC-δ expression in PKC-δ2 and EV-2 stable transfectants. Human PKC-δ comigrates with rat brain PKC-δ; MR = 78 kilodaltons. (B) PKC-δ may migrate as a doublet because of phosphorylation. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 3 PKC-δ overexpression increases PKC-δ–specific phosphorylating activity in situ. CaCo-2 cells, transfected with EV or full length human PKC-δ (PKC-δ1 and PKC-δ2), were plated in 24-multiwell plates. PKC-δ phosphorylating activity was measured in subconfluent cells in the presence of a permeabilizing phosphorylation reaction mixture containing digitonin, [γ32P] ATP, and a PKC-δ–specific substrate derived from eEF-1α as described in Materials and Methods. 32P-labeled eEF-1α peptide was then measured by scintillation counting. Values are expressed as fold increases in PKC-δ activity (mean ± SD) in transfected cells (PKC-δ1 and PKC-δ2), compared with the EV controls (n = 3 independent experiments in triplicate). *P < 0.05, compared with their respective EV control. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 4 PKC-δ overexpression inhibits the anchorage-dependent growth of CaCo-2 cells. EV- (●) and PKC-δ– (■) transfected cells (0.5 × 105 cells/well) were seeded in 6-well plates as described in Materials and Methods. At the indicated days after plating, cells were trypsinized, collected, and then resuspended and counted using a Coulter counter. Data represents the average ± SD of 2 independent experiments, each carried out in triplicate. Error bars not shown are contained within the data points. (A) PKC-δ1–transfected cells. (B) PKC-δ2–transfected cells. *P < 0.05, compared with their respective EV controls. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 5 PKC-δ overexpression causes a G1 arrest in CaCo-2 cells. Preconfluent CaCo-2 cells, transfected with EV (EV1 ≅ EV2; data pooled and expressed as EV, □; or full length human PKC-δ, PKC-δ2, ■), were assessed for their distribution in the cell cycle as described in Materials and Methods. Cells were fixed and analyzed using CellQuest software in a FacScan flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). The distribution of DNA in the cell cycle was determined using Modfit LT software. Values are reported as mean of 3 samples ± SD. *P < 0.05, compared with EV control. As noted in the text, cell cycle distributions for PKC-δ1 were similar to those of PKC-δ2. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 6 PKC-δ overexpression inhibits anchorage-independent growth of CaCo-2 cells. CaCo-2 cells (1 × 105), transfected with EV (EV1 ≅ EV2; data pooled and expressed as EV) or PKC-δ (PKC-δ1 or PKC-δ2), were plated in 0.35% agar and analyzed for their ability to form colonies in soft agar as described in Materials and Methods. Data are the mean ± SD of 2 independent experiments of 10 determinations each. *P < 0.05, compared with EV. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 7 Effect of PKC-δ overexpression on alkaline phosphatase–specific activity. EV- (●) and PKC-δ– (■) transfected cells (0.5 × 105) were plated in 6-well plates. Cells from triplicate wells were collected on the indicated days and assayed for alkaline phosphatase activity as described in Materials and Methods. Specific activity was expressed as units of alkaline phosphatase per milligrams of protein per minute. (A) PKC-δ–stable transfectants. (B) PKC-δ2–stable transfectants. Data are means ± SD of 2 separate experiments each carried out in triplicate. *P < 0.05, compared with their respective EV control. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions

Fig. 8 PKC-δ overexpression increases the apoptosis of CaCo-2 cells. Preconfluent CaCo-2 cells, transfected with EV or full length human PKC-δ cDNA (PKC-δ1 and PKC-δ2), were treated with either 10 nmol/L TPA (+TPA) or vehicle DMSO (−TPA) as described in Materials and Methods. Treated cells were fixed, and nuclei or fragmented DNA were stained by DAPI or TUNEL assay, respectively, and visualized by microscopy. Representative fields of view were photographed at 200× magnification. (A) DAPI-stained cells from the indicated clones. Examples of apoptotic cells are indicated with arrows. (B) Apoptotic rates. Rates of apoptosis, expressed as a percentage of apoptotic nuclei, were assessed in DAPI- (■) and TUNEL- (□) stained cells by counting 1000 random cells in duplicate platings per experiment. Results are the mean ± SD of 2 independent experiments each in duplicate. a,bP < 0.05, compared with EV control group. c,dP < 0.05, compared with their respective DMSO-treated clone (TPA control). Because the rates of apoptosis among parental- and EV-transfected cells (EV-1, EV-2) were similar, only EV-1 is shown for clarity. Gastroenterology 2001 120, 1700-1712DOI: (10.1053/gast.2001.24843) Copyright © 2001 American Gastroenterological Association Terms and Conditions