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First published online 2 January 2007
doi: 10.1242/jcs.03343

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Connexin43 increases the sensitivity of prostate cancer cells to TNF-induced apoptosis

Department of Pediatrics, Section of Hematology/Oncology and Stem Cell Transplantation, University of Chicago, Chicago, IL 60637, USA

View larger version (47K): [in this window] [in a new window]   Fig. 1. Cx43 induces the formation of functional gap junction channels in LNCaP but not PC3 cells. (A,B) Photomicrographs show the distribution of Cx43 immunoreactivity in PC3 cells that were uninfected (A) or infected with Ad-Cx43 (B). (C-F) Photomicrographs show the distribution of connexin immunoreactivity in LNCaP cells that were uninfected (C) or infected with Ad-Cx43 (D), Ad-Cx37 (E), or Ad-Cx40 (F). (G) Immunoblot detection of Cx43 in cell lysates from PC3 cells that were uninfected (lane 1) or infected with Ad-Cx43 (lane 2) and in lysates of uninfected LNCaP cells (lane 3) or LNCaP cells infected with Ad-Cx43 (lane 4) or Ad-Cx43DN (lane 5). The migration positions of the molecular mass markers are indicated on the right. (H-K) Representative fluorescence images of Lucifer Yellow transfer after microinjection of the tracer into a single cell within a cluster of uninfected LNCaP cells (H) or LNCaP cells infected with Ad-Cx43 (I), Ad-Cx43DN (J), or both Ad-Cx43 and Ad-Cx43DN (K). (L) Histogram showing the extent of intercellular transfer of Lucifer Yellow in uninfected LNCaP cells and LNCaP cells infected with Ad-Cx43, Ad-Cx43DN, or both adenoviruses. Values represent the number of recipient cells containing Lucifer Yellow (mean ± s.e.m., n=8). (M-O) Photomicrographs show the localization of Cx43DN (M) and Golgi 58K protein (N) in LNCaP cells infected with Ad-Cx43DN after double-labeling immunofluorescence using anti-Cx43 and anti-Golgi 58K antibodies. The overlap of the two signals appears yellow in the merged image (O). (P-R) Photomicrographs show the localization of Cx43 and Cx43DN in LNCaP cells co-infected with Ad-Cx43 and Ad-Cx43DN after double-labeling immunofluorescence using anti-Cx43IL (P) and anti-HA (Q) antibodies. The overlap of anti-Cx43IL and anti-HA immunoreactivities appears yellow (R). Bar, 20 µm for A-D and P-R, 8 µm for E, 12 µm for F, 66 µm for H-K and 17 µm for M-O.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Cx43 increases the susceptibility of LNCaP cells to TNF-induced cell death. (A) PC3 cells were left uninfected () or infected with Ad-Control () or Ad-Cx43 () for 12 hours and then treated with 0.01-100 ng/ml TNF for 48 hours. (B) LNCaP cells were left uninfected () or infected with Ad-Control () or Ad-Cx43 () for 12 hours and then treated with 0.01-100 ng/ml TNF for 48 hours. Cell viability of untreated and Ad-Control infected cells showed a slight, but insignificant decline with increasing doses of TNF. These curves did not differ significantly from each other. Ad-Cx43-infected cells showed significantly less viability at 0.1, 1, 10, and 100 ng/ml TNF (P<0.0001). (C) LNCaP cells were infected with Ad-Control (), Ad-Cx43 (), or both Ad-Cx43 and Ad-Cx43DN () for 12 hours and then treated with 0.01-100 ng/ml TNF for 48 hours. The viability of Ad-Control- or Ad-Cx43 + Ad-Cx43DN-infected cells did not differ from each other. The viability of cells infected with Ad-Cx43 was significantly lower than that of cells infected with both viruses at 0.1-100 ng/ml TNF (P=0.0264 for 0.1 ng/ml; P<0.0001 for 1.0, 10, and 100 ng/ml). The results are presented as mean ± s.e.m. although in many cases the error bars are so small that they are obscured by the symbols.

View larger version (17K): [in this window] [in a new window]   Fig. 3. TNF-induced cell death in Cx43-expressing LNCaP cells is greater at higher cell densities. LNCaP cells were plated at different densities, 1250 (, ), 2500 (, ), 5000 (, ) or 10,000 (, ) initial cells/well. After 12 hours, cells were left uninfected (open symbols) or infected with Ad-Cx43 (filled symbols), incubated for an additional 12 hours and then treated with different concentrations of TNF for 48 hours, and cell viability was measured using the MTS assay. At the highest density (10000 cells/well), viability of Ad-Cx43-infected LNCaP cells was significantly reduced by all concentrations of TNF (P=0.0015 for 0.01 ng/ml, P<0.0001 for concentrations 0.1 ng/ml) as compared to untreated cells. When the initial cell number was decreased to 5000 cells/well, only TNF concentrations 1 ng/ml induced a significant decrease in cell viability as compared to untreated cells (P<0.0001). At the highest concentrations of TNF, slight decreases in cell viability were observed for cells plated at 1250 or 2500 cells per well, but these changes were not statistically significant. In uninfected LNCaP cells, TNF only reduced cell viability significantly when used at 10 ng/ml in cells cultured at the highest initial density (viability=85.3±2.2%, P=0.0052).

View larger version (13K): [in this window] [in a new window]   Fig. 4. Cx43 increases the susceptibility of LNCaP cells to cell death induced by anti-Fas antibodies or TRAIL. (A,B) LNCaP cells were left uninfected () or they were infected with Ad-Cx43 () for 12 hours and then treated with 1-1000 ng/ml anti-Fas antibodies (A) or 0.1-1000 ng/ml TRAIL (B) for 48 hours. Cell viability after treatment with anti-Fas antibodies (10 ng/ml) was significantly less in Ad-Cx43 infected LNCaP cells as compared to untreated, infected cells (P<0.0001). Moreover, the viability of Ad-Cx43-infected LNCaP cells treated with 10 ng/ml anti-Fas antibodies was significantly reduced as compared to non-infected cells treated with the same doses of antibodies (P<0.0001). High concentrations of anti-Fas antibodies (100 ng/ml) moderately, but significantly reduced the viability of uninfected LNCaP cells (to 82.0±1.7% for 100 ng/ml and 81.9±2.3% for 1000 ng/ml, respectively; P<0.0001 as compared to untreated cells). Similarly, in Ad-Cx43-infected LNCaP cells, TRAIL concentrations as low as 10 ng/ml induced a significant decrease in cell viability (P<0.0001), but only 1000 ng/ml TRAIL induced a significant decrease in cell viability of uninfected LNCaP cells (viability=86.0±2.2%; P=0.017 as compared to untreated cells).

View larger version (23K): [in this window] [in a new window]   Fig. 5. TNF induces the death of Cx43-expressing LNCaP cells through TNFR1. (A) Immunoblots showing levels of TNFR1, TNFR2 and -actin in uninfected LNCaP cells (Control), LNCaP cells 12 hours after infection with Ad-Cx43 (Ad-Cx43), uninfected LNCaP cells after 12 hours of treatment with 10 ng/ml TNF (TNF), and LNCaP cells 12 hours after Ad-Cx43 infection and TNF treatment (Ad-Cx43 + TNF). (B) Bar graph showing viability of Cx43-expressing LNCaP cells in the presence or absence of neutralizing anti-TNFR1 (Ab-R1) or –TNFR2 (Ab-R2) antibodies (concentrations indicated in µg/ml). Cell viability determined by MTS assay is expressed as a percentage of control values. Statistical analysis of the raw absorbance data showed that treatment with 1 or 10 µg/ml of anti-TNFR1 antibodies or the combination of 10 µg/ml anti-TNFR1 and 10 µg/ml anti-TNFR2 antibodies significantly antagonized the effects of TNF (P<0.0001 when compared to the viability of cells treated with TNF alone).

View larger version (49K): [in this window] [in a new window]   Fig. 6. TNF induces apoptotic cell death in Cx43-expressing LNCaP cells. Graphs show the distribution of propidium iodide and/or annexin V staining in uninfected or infected LNCaP cells under control conditions or after treatment with 10 ng/ml TNF for 12 hours. The percentage of cells positive for only annexin V is indicated in the lower right quadrants and that of cells positive for both annexin V and PI is indicated in the top right quadrants.

View larger version (29K): [in this window] [in a new window]   Fig. 7. TNF treatment leads to an increase in the percentage of cells in the sub-G1 fraction. Graphs show the distribution of propidium iodide staining intensities in uninfected or infected LNCaP cells under control conditions or after treatment with 10 ng/ml TNF for 48 hours. The percentage of cells containing a hypodiploid amount of DNA (sub-G1) is indicated.

View larger version (13K): [in this window] [in a new window]   Fig. 8. TNF induces activation of caspase 8. (A) Time course of activation of caspase 8 in LNCaP cells infected with Ad-Control () or Ad-Cx43 () after treatment with 10 ng/ml TNF for variable lengths of time. Caspase 8 activity was significantly greater in the Ad-Cx43-infected cells as compared to those infected with control virus at 30 minutes, 1 hour and 3 hours (P<0.01). (B) Bar graph showing the effects of pre-treatment with TNFR neutralizing antibodies (10 µg/ml) upon the TNF-induced caspase 8 activation in LNCaP cells infected with Ad-Control (black bars) or Ad-Cx43 (gray bars). Ab-R1 and Ab-R2 indicate antibodies directed against TNFR1 and TNFR2, respectively. Ad-Control-infected LNCaP cells showed a small increase in caspase 8 activity that was abolished by pretreatment with anti-TNFR1 antibodies (P=0.0183 for Ad-Control + TNF vs Ad-Control alone; P=0.0381 for Ad-Control + TNF vs Ad-Control + TNF + Ab-R1; and not significant for Ad-Control + TNF + Ab-R1 vs Ad-Control alone). Ad-Cx43-infected cells showed a much larger increase in caspase 8 activity that was abolished by pretreatment with anti-TNFR1 antibodies or anti-TNFR1 plus anti-TNFR2 antibodies (P<0.0001 for Ad-Cx43 + TNF vs Ad-Cx43 alone or Ad-Cx43 + TNF +Ab-R1 or Ad-Cx43 + TNF +Ab-R1 +Ab-R2; Ad-Cx43 + TNF +Ab-R1 or Ad-Cx43 + TNF +Ab-R1 +Ab-R2 did not significantly differ from Ad-Cx43 alone). Pretreatment of Ad-Cx43-infected cells with anti-TNFR2 antibodies partially antagonized the TNF-induced activation of caspase 8, since it significantly reduced the caspase 8 activity as compared to Ad-Cx43-infected cells treated with TNF (P=0.0009), but levels were still much higher than those in untreated Cx43-expressing cells (P<0.0001).