Progressive resistance to apoptosis in a cell lineage model of human proliferative breast disease.
BACKGROUND
Proliferative breast disease (PBD) may increase a woman's risk of developing breast cancer, perhaps by decreasing cellular sensitivity to apoptosis. To determine whether resistance to apoptosis develops during PBD, we investigated apoptosis initiated through the Fas pathway in a series of cell lines that recapitulates the morphologic changes of PBD in nude/beige mice.


METHODS
The series of cell lines used was MCF-10A cells (parental preneoplastic human breast epithelial cells), MCF-10AT cells (transformed with T(24) Ha-ras), and MCF-10ATG3B cells (derivative cells that progress to carcinoma). Fas-mediated apoptosis, induced when a Fas monoclonal antibody bound to and activated the Fas receptor on these cells, was assessed morphologically and by flow cytometry. Levels of proteins involved in Fas-mediated apoptosis and cleavage of poly(adenosine diphosphate-ribose) polymerase (PARP), an end product of caspase activation, were determined by immunoblotting. Bcl-2 and Bax heterodimerization was examined by coimmunoprecipitation. All statistical tests were two-sided.


RESULTS
Sensitivity to Fas-mediated apoptosis decreased with the tumorigenic potential of cells: MCF-10A cells were extremely susceptible, MCF-10AT cells were less susceptible, and MCF-10ATG3B cells were resistant. The percentage of apoptotic cells declined, from 24% to 8% to 6%, respectively. All lines produced Fas ligand (FasL) and had comparable levels of Fas receptor, FasL, Fas-associated death-domain protein, and caspases 3 and 6. Levels of caspase 8 were similar in MCF-10A and MCF-10AT cells but about 30% lower in MCF-10ATG3B cells (P>.01 but <.05). Levels of caspase 10 were about 20% lower in MCF-10AT cells (P>.005 but <.01) and about 59% lower in MCF-10ATG3B cells than in MCF-10A cells (P>.01 but <.05). PARP cleavage was detected in MCF-10A and MCF-10AT cells but not in MCF-10ATG3B cells. Levels of Bax, Bid, and Bak proteins were similar in all lines, but levels of Bcl-2 were lower in MCF-10AT and MCF-10ATG3B cells than in MCF-A cells, and Bcl-2-Bax heterodimerization progressively declined in the series.


CONCLUSIONS
Resistance to Fas-mediated apoptosis appears to develop progressively in the MCF-10AT cell series.
Starcevic
S L
SL
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI, USA.
Elferink
C
C
Novak
R F
RF
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology*; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism; D053455:Caspase 10; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis; D002461:Cell Line, Transformed; D019070:Cell Lineage; D019281:Dimerization; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008819:Mice, Nude; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; C494772:BAK1 protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; C494773:Bak1 protein, mouse; C494827:Bax protein, mouse; C494815:Bid protein, mouse; D002352:Carrier Proteins; C505533:FADD protein, human; C505661:FASLG protein, human; C505534:Fadd protein, mouse; D053222:Fas Ligand Protein; D053200:Fas-Associated Death Domain Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014404:Tubulin; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D019014:fas Receptor; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; C505409:Casp3 protein, mouse; C505516:Casp6 protein, mouse; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053455:Caspase 10; D053148:Caspase 3; D053178:Caspase 6; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C506438:CASP10 protein, human