Coordinate expression of apoptosis-associated proteins in human breast cancer before and during chemotherapy.
OBJECTIVE
Induction of apoptosis is a key factor in the response of tumors to chemotherapy. Laboratory studies have established many of the factors that regulate and execute apoptosis, but the significance of these in human tumors is poorly understood. Therefore, the relationship between key components of this machinery was examined in primary human breast carcinomas before and 24 h after the initiation of chemotherapy.


METHODS
Apoptosis was measured using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay, and proliferation was assessed using the anti-Ki67 antibody MIB-1. Monospecific polyclonal antibodies were used for immunohistochemical detection of Bcl-2, Bax, XIAP, activated (cleaved) caspase 3 and 6, and cleaved DNA Fragmentation Factor-40 (DFF40) using paraffin-embedded tissues.


RESULTS
Before treatment, a significant correlation was found between apoptosis and proliferation (r = 0.64, P < 0.0001), between caspases 3 and 6 (r = 0.49, P = 0.004) and between cleaved DFF40 and active caspases 3 (r = 0.66, P < 0.0001) or 6 (r = 0.47, P = 0.006). Before treatment, expression of inhibitor of apoptosis protein, XIAP, also correlated positively with cleaved caspase 3 (r = 0.64, P < 0.0001), caspase 6 (r = 0.36, P = 0.04), and DFF40 (r = 0.61, P = 0.0001). At 24 h after chemotherapy, significant increases in apoptosis and decreases in proliferation were observed, with the degree of increase in apoptosis inversely associated with decrease in proliferation. Chemotherapy-induced increases in Bax were correlated with increases in cleaved DFF40 (r = 0.54, P = 0.0008), but no other variables showed significant change at 24 h after initiation of chemotherapy.


CONCLUSIONS
The pretreatment biomarker relationships suggest parallel cleavage and activation of these executioner proteins in breast cancer and that XIAP may maintain cell survival in the face of caspase activation. The findings provide in vivo evidence in human breast cancer that chemotherapy induces an apoptotic program characterized by up-regulation of Bax and cleavage of caspase substrate DFF40.
Breast Unit, Royal Marsden NHS Trust and Institute of Cancer Research, London SW3 6JJ, United Kingdom.
Marina
M
Parton
Stanislaw
S
Krajewski
Ian
I
Smith
Maryla
M
Krajewska
Caroline
C
Archer
Mihikito
M
Naito
Roger
R
Ahern
John
J
Reed
Mitchell
M
Dowsett
D000328:Adult; D000368:Aged; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D017209:Apoptosis; D014408:Biomarkers, Tumor / Q000378:metabolism*; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D018275:Carcinoma, Lobular / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism; D002945:Cisplatin / Q000627:therapeutic use; D003520:Cyclophosphamide / Q000627:therapeutic use; D003851:Deoxyribonucleases / Q000378:metabolism; D004317:Doxorubicin / Q000627:therapeutic use; D015251:Epirubicin / Q000627:therapeutic use; D005260:Female; D005472:Fluorouracil / Q000627:therapeutic use; D006801:Humans; D007124:Immunoenzyme Techniques; D019394:Ki-67 Antigen / Q000378:metabolism; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism*; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein
D014408:Biomarkers, Tumor; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; D002945:Cisplatin; D003520:Cyclophosphamide; D003851:Deoxyribonucleases; D004317:Doxorubicin; D015251:Epirubicin; D005472:Fluorouracil; D019394:Ki-67 Antigen; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D011980:Receptors, Progesterone; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C113696:DFFB protein, human; C505407:CASP3 protein, human; C505514:CASP6 protein, human