Preclinical and clinical estimates of the basal apoptotic rate of a cancer predict the amount of apoptosis induced by subsequent proapoptotic stimuli.
OBJECTIVE
We hypothesized that the basal apoptotic rate (BAR) of a cancer would predict sensitivity to subsequent proapoptotic stimuli. To explore this, preclinical and clinical BAR assays were developed measuring cumulative apoptotic events through ELISAs for soluble caspase-cleaved cytokeratin 18 (M30) normalized to either cell number increase or total tumor volume, respectively.


METHODS
The BARs of A549, HCC44, and SW1573 non-small cell lung carcinoma cell lines were measured following different pro/antiapoptotic manipulations. In isogenic wild-type and stable knockdown (KD) series, pretreatment BARs were correlated with response to proapoptotic stimuli and compared with established apoptosis assays. Pretreatment and posttreatment serum was available from stereotactic body radiation therapy patients.


RESULTS
Caspase inhibition and p53 KDs reduced the BAR, whereas serum deprivation, XIAP, or Bcl2 KDs increased the BAR. The nontreated BAR rank ordering of the XIAP series recapitulated that with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase-3/7 activity assays, and predicted each line's sensitivity to TRAIL or irradiation. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, however, underestimated basal apoptosis during increased apoptotic stress, and caspase-3/7 activity detected minimal death in the media. P53 KDs with lower nontreated BARs were less sensitive to TRAIL and cisplatinum than wild-type. Stereotactic body radiation therapy increased serum M30 values, and the pretreatment clinical BAR strongly correlated with fold change in M30 on treatment (r = 0.93).


CONCLUSIONS
M30-based BAR assays reflect apoptosis accurately and are more amenable to clinical application than existing apoptosis assays. The pretreatment BAR correlates with cell and/or tumor sensitivity to extrinsic and intrinsic apoptotic pathway stimulation. Prospective clinical exploration is warranted.
Zhang
Lian
L
Department of Pharmacology, University of Colorado, School of Medicine, Aurora, CO, USA.
Kavanagh
Brian D
BD
Thorburn
Andrew M
AM
Camidge
D Ross
DR
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3 / Q000378:metabolism*; D061945:Caspase Inhibitors; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D015151:Immunoblotting; D020287:In Situ Nick-End Labeling; D053538:Keratin-18 / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D009369:Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D011379:Prognosis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction / Q000187:drug effects; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D053538:Keratin-18; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D053148:Caspase 3