Figure 1.
Effect of pH on contractolation of IM-26 cells in monolayer culture.
(A) Cells grown in complete medium in a 12 well plate were photographed immediately after removal from the CO2 incubator (pH 7.4) and after exposure to atmospheric conditions for 10 and 30 min (as the pH increased to 8.3). (B) Area occupied by the cells as quantified with Adobe Photoshop CS4 Measuring Tool. Histobars represent means ± SEM of at least 3 separate fields of at least 5 independent determinations. Asterisks denote significant difference from the pH 7.4 with *p = 0.01, and **p = 0.005. (C) Snapshots from time lapse photography of IM-26 cells undergoing pH-induced contractolation. The initial cell (labeled ‘X’) changes completely over 39 min and moves away from its neighboring cell. (D) Snapshots for cells changing back to normal shape after pH change back from 8.3 to 7.4 (recovery). Elapsed time in min is indicated in each snapshot. 20 x magnification.
Figure 2.
Effect of pH on contractolation of breast and non-breast cell lines.
Cells were photographed immediately after removal from the CO2 incubator (0 min, pH 7.4) and after 30 min of exposure to normal atmospheric conditions (as the pH increased to 8.3). The following cell lines are shown: (A) HBL-100, (B) YS1.2, (C) MDA-MB-231, (D) pII, (E) YS2.5, (F) HEK 293, and (G) HEK 499. Panels (H) and (I) represent the combined phase contrast and fluorescence micrographs for the co-culture of EII cells (which express a transfected green fluorescent protein) with either pII or YS2.5 cells respectively.
Figure 3.
Effect of substratum on pH-induced contractolation of IM-26 cells.
Cells were seeded into 24-well microwell plates coated with either fibronectin or collagen, left to grow for 48h in a 5% CO2 atmosphere (pH 7.4; solid bars) and then exposed to normal atmospheric conditions for 10 and 30 min (as pH increased to 8.3; hatched bars). Panel A and B show quantitative analysis of at least 3 separate fields measured with Adobe Photoshop CS4 Measuring Tool (means ± SEM) for cells coated with fibronectin and collagen respectively. Asterisks denote significant difference from pH 7.4 (set as 100%) with *p = 0.03, and **p = 0.02.
Figure 4.
Effect of ion pump inhibitors on contractolation.
Quantitative analysis of pH induced contractolation of pII cells exposed to pH 7.4 (solid bars) or after exposure to alkaline pH for 1h (open bars). The following inhibitors were used: (A) amiloride, (B) zoniporide, (C) ouabain, (D) 3, 4,5, 6-tetrahydroxyxanthone, (E) nigericin, (F) bafilomycin A1 and (G) phloretin. Histobars represent means ± SEM of at least 3 independent determinations. Asterisk denotes significant difference from the pH 7.4 condition (set as 100%) with *p ≤ 0.05.
Figure 5.
Association of pH induced contractolation with activation of intracellular signaling molecules.
Cells were lysed immediately after removal from the incubator (0 min, pH 7.4), after 60 min exposure to normal atmosphere (as the pH increased to 8.3), or after 60 min exposure to normal atmosphere and returning back to pH 7.4 for 120 min (recovery). The pH of the medium was measured at the time of cell lysis and is indicated under each test condition. (A) pII and YS2.5 and (B) MDA-MB-231, MCF-7 and HBL-100 cell lysate proteins (8 µg) were electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to p-ERK 1/2, p-p38 MAPK, p-Akt, total-Akt and actin as described in Methods.
Figure 6.
Temporal association of pH induced contractolation with activation of intracellular signaling molecules and other proteins necessary for cell morphology/function.
Cells were lysed immediately after removal from the CO2 incubator (0 min, pH 7.4), after various time points of exposure to normal atmosphere (5-60 min), or after 60 min exposure to normal atmosphere and returning back to pH 7.4 for 60 or 120 min (recovery). The pH of the medium was measured at the time of cell lysis and is indicated under each test condition. (A) pII and (B) YS1.2 cell lysate proteins (8 µg) were electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to p-ERK 1/2, p-p38 MAPK, p-Akt, JAM-1 and -2, integrin α2, FAK, BIP and actin as described in Methods.
Figure 7.
Association of pH induced contractolation with expression of heat shock proteins.
Cells were lysed immediately after removal from the CO2 incubator (0 min, pH 7.4),or after 10-60 min exposure to normal atmosphere. The pH of the medium was measured at the time of cell lysis and is indicated under each test condition. (A) pII and (B) YS1.2 cell lysate proteins (8 µg) were electrophoresed on 10% SDS polyacrylamide gel, blotted onto nitrocellulose membrane and probed with antisera to HSPs 40, 90, 60, 70, HSF1 and actin as described in Methods.
Figure 8.
Behavior of contractolated cells in motility and invasion assays, and association with matrix metalloproteinase activity.
(A) The mean distance moved (in pixels), as a measurement of cell motility of pII cells after an overnight incubation in either a gassed incubator (pH 7.4, solid bar) or after a brief (1h) culture in un-gassed incubator at 37°C and subsequent overnight incubation in a gassed incubator (open bar). (B) The total number of pII cells showing random invasion (as defined in Methods) after an overnight incubation in a gassed incubator (pH 7.4, solid bar, set as 100%), after culture in un-gassed incubator at 37°C for 1-2h and subsequent overnight incubation in a gassed incubator, or after an overnight incubation in un-gassed incubator at 37°C (hatched bars). Asterisks denote significant difference from pH 7.4 with * p≤0.0001, and # p= 0.0004. (C) The total number of pII cells showing random invasion in response to EGF stimulation (50ng/ml) after either an overnight incubation in a gassed incubator (pH 7.4, set as 100%), or after a brief (1h) culture in un-gassed incubator at 37°C and subsequent overnight incubation in a gassed incubator (open bar). Asterisks denote significant difference from pH 7.4 with * p=0.0024 (D) The effect of amiloride or zoniporide (10µM) treatment of the total number of pII cells showing random invasion after an overnight incubation in a gassed incubator (pH 7.4, open bars), or after a brief (1h) culture in un-gassed incubator at 37°C and subsequent overnight incubation in a gassed incubator (solid bars). Asterisks denote significant difference from pH 7.4 (no drug) with * p=0.01 (E) Effect of pH and EGF stimulation on general MMP activity in pII cells. Cells were either cultured in a gassed incubator (pH 7.4) or in un-gassed incubator for 1h and either left untreated or stimulated with EGF (50ng/ml) for 30 min and metalloproteinase activity was determined using a fluorogenic substrate as described in Methods. Asterisks denote significant difference from no EGF treatment groups with * p≤ 0.05. (F) Effect of pH, EGF, amiloride, and zoniporide treatment of MMP2/9 activity in pII (open bars) and YS1.2 (solid bars) cells. Cells were either cultured in a gassed incubator (pH 7.4) or in un-gassed incubator for 1h and either left untreated, treated with amiloride or zoniporide (10µM, 60 min), or stimulated with EGF (50ng/ml, 30 min) and MMP2/9 activity was determined using a fluorogenic substrate as described in Methods. Asterisks denote significant difference from pH 7.4 with * p≤0.001, from pH7.4 +EGF with # p≤0.0001, and from alkaline pH (1h) with ** p ≤0.001. Bars represent means ± SEM for at least 3 independent determinations.
Figure 9.
Preliminary model for the contractolation phenomenon.
Brief exposure of endocrine resistant ER silenced breast cancer cells (that have undergone EMT) to extracellular alkaline pH environment modulates the activity of Na+/H+ and Na+/K+ pumps, resulting in reduced activity of the intracellular signaling molecules p38 MAPK, Akt, and ERK1/2, enhanced expression level of FAK, JAM-1 and 2, and integrin alpha 2, and dramatic morphological changes including cell shrinkage, rounding, and separation from neighboring cells (possibly due to reduced level of E-cadherin and catenins as a result of the EMT process). This leads to enhanced secretion of MMP2/9 in the extracellular environment and enhances cell invasion, particularly that responsive to EGF and serum factors. Conditioned medium from cells at pH 8.3 can confer enhancement of invasion on cells that had been maintained at pH 7.4. The entire contractolation process with accompanying changes can all be reversed by returning cells to pH 7.4.