Colorectal cancer cell adhesion attenuates Ad-E2F-1 mediated apoptosis.
BACKGROUND
The complex interplay of cell adhesion molecules, intracellular signaling, and tumor growth behavior have important implications for the failure of most conventional cancer therapies. Cell adhesion to the basement membrane has been shown to promote tumor cell survival. We hypothesize that the presence of matrix substrate contributes to chemoresistance through signaling via cell adhesion molecule.


METHODS
RKO colorectal cancer cells express integrin beta1 to adhere to substrate. We measured apoptosis of the cells after infection with adenovirus vector containing the transgene E2F-1 (Ad-E2F-1), a potent tumor suppressor gene, and Ad-LacZ (as control), both under the control of the cytomegalovirus promoter. Cells were plated on Matrigel, an extracellular substrate similar to basement membrane and compared to tissue culture plastic. Apoptosis was assessed by flow cytometry-based TUNEL assay and cell proliferation was assessed by WST-1 assay. E2F-1 expression was confirmed by Western blot analysis. A function-blocking anti-beta1 integrin antibody was used to assess the contribution of beta1 on cell survival.


RESULTS
At 120 h postinfection of RKO cells with 50 multiplicity of infection, cells plated on plastic underwent marked apoptosis in response to Ad-E2F-1 compared with Ad-LacZ control-treated cells (53% vs 1% apoptosis, respectively). However, when cells were plated on Matrigel, the same dose of E2F-1 was ineffective at inducing apoptosis (3% vs 1% apoptosis, comparing Ad-E2F-1 with Ad-LacZ control). The cell proliferation assay showed >3-fold cell survival in E2F-1-infected cells on Matrigel vs plastic (P < 0.004). By Western blot analysis, attenuation of apoptosis may be a result of reduction in transduction efficiency on Matrigel and function-blocking anti-beta1 integrin antibody does not abolish the decrease in apoptosis afforded by Matrigel.


CONCLUSIONS
These data suggest that escape from adenoviral E2F-1-mediated apoptosis, at least in part, is related to reduction of intracellular E2F-1 expression. Interactions involving cellular adhesion via beta1 integrin to matrix proteins does not seem to contribute toward gene therapy resistance. Further studies will investigate other specific receptor-ligand interactions after gene and/or chemotherapy.
Chao
Celia
C
Department of Surgery, James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky 40202, USA. celia.chao@nortonhealthcare.org
Jamshidi-Parsian
Azemat
A
Wang
Warner W
WW
McMasters
Kelly M
KM
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D018797:Cell Cycle Proteins; D003094:Collagen / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000235:genetics*; D004268:DNA-Binding Proteins; D004338:Drug Combinations; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D005109:Extracellular Matrix / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D039641:Integrin beta Chains / Q000378:metabolism; D007797:Laminin / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004338:Drug Combinations; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D039641:Integrin beta Chains; D007797:Laminin; D011509:Proteoglycans; D014157:Transcription Factors; C059485:matrigel; D003094:Collagen