Kottke, Timothy

AbstractThis unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low‐molecular‐weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole‐cell lysates or subcellular fractions.

AbstractDT388–GM-CSF, a targeted fusion toxin constructed by conjugation of human granulocyte-macrophage colony-stimulating factor (GM-CSF) with the catalytic and translocation domains of diphtheria toxin, is presently in phase I trials for patients with resistant acute myeloid leukemia. HL-60/VCR, a multidrug-resistant human myeloid leukemia cell line, and wild-type HL-60 cells were used to study the impact of DT388–GM-CSF on metabolism of ceramide, a modulator of apoptosis. After 48 hours with DT388–GM-CSF (10 nM), ceramide levels in HL-60/VCR cells rose 6-fold and viability fell to 10%, whereas GM-CSF alone was without influence. Similar results were obtained in HL-60 cells. Examination of the time course revealed that protein synthesis decreased by about 50% and cellular ceramide levels increased by about 80% between 4 and 6 hours after addition of DT388–GM-CSF. By 6 hours this was accompanied by activation of caspase-9, followed by activation of caspase-3, cleavage of caspase substrates, and chromatin fragmentation. Hygromycin B and emetine failed to elevate ceramide levels or induce apoptosis at concentrations that inhibited protein synthesis by 50%. Exposure to C6-ceramide inhibited protein synthesis (EC50∼5 μM) and decreased viability (EC50 ∼6 μM). Sphingomyelinase treatment depleted sphingomyelin by about 10%, while increasing ceramide levels and inhibiting protein synthesis. Diphtheria toxin increased ceramide and decreased sphingomyelin in U-937 cells, a cell line extremely sensitive to diphtheria toxin; exposure to DT388–GM-CSF showed sensitivity at less than 1.0 pM. Diphtheria toxin and conjugate trigger ceramide formation that contributes to apoptosis in human leukemia cells through caspase activation and inhibition of protein synthesis.

Abstract The adenosine triphosphate binding-site–directed agent STI571 and the tyrphostin adaphostin are undergoing evaluation as bcr/abl kinase inhibitors. The current study compared the effects of these agents on the survival of K562 cells, bcr/abl-transduced FDC-P1 cells, and myeloid progenitors from patients with chronic myelogenous leukemia (CML) compared with healthy donors. Treatment of K562 cells with 10 μM adaphostin resulted in decreased p210bcr/ablpolypeptide levels in the first 6 hours, followed by caspase activation and accumulation of apoptotic cells in less than 12 hours. By 24 hours, 90% of the cells were apoptotic and unable to form colonies. In contrast, 20 μM STI571 caused rapid inhibition of bcr/abl autophosphorylation without p210bcr/abl degradation. Although this was followed by the inhibition of Stat5 phosphorylation and the down-regulation of Bcl-xL and Mcl-1, only 7% ± 3% and 25% ± 9% of cells were apoptotic at 16 and 24 hours, respectively. Instead, the cytotoxic effects of STI571 became more pronounced with prolonged exposure, with IC90values greater than 20 μM and 1.0 ± 0.6 μM after 24 and 48 hours, respectively. Consistent with these results, 24-hour adaphostin exposure inhibited CML granulocyte colony-forming units (CFU-G) (median IC50, 12 μM) but not normal CFU-G (median IC50, greater than 20 μM), whereas 24-hour STI571 treatment had no effect on CML or normal CFU-G. Additional experiments revealed that STI571-resistant K562 cells remained sensitive to adaphostin. Moreover, the combination of STI571 + adaphostin induced more cytotoxicity in K562 cells and in CML CFU-G than either agent alone did. Collectively, these results identify adaphostin as a mechanistically distinct CML-selective agent that retains activity in STI571-resistant cell lines.

R115777 is a nonpeptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) that was developed as a potential inhibitor of Ras protein signaling, with antitumor activity in preclinical models. This study was a phase 1 trial of orally administered R115777 in 35 adults with poor-risk acute leukemias. Cohorts of patients received R115777 at doses ranging from 100 mg twice daily (bid) to 1200 mg bid for up to 21 days. Dose-limiting toxicity occurred at 1200 mg bid, with central neurotoxicity evidenced by ataxia, confusion, and dysarthria. Non–dose-limiting toxicities included reversible nausea, renal insufficiency, polydipsia, paresthesias, and myelosuppression. R115777 inhibited FT activity at 300 mg bid and farnesylation of FT substrates lamin A and HDJ-2 at 600 mg bid. Extracellular signal-regulated kinase (ERK), an effector enzyme of Ras-mediated signaling, was detected in its phosphorylated (activated) form in 8 (36.4%) of 22 pretreatment marrows and became undetectable in 4 of those 8 after one cycle of treatment. Pharmacokinetics revealed a linear relationship between dose and maximum plasma concentration or area under the curve over 12 hours at all dose levels. Weekly marrow samples demonstrated that R115777 accumulated in bone marrow in a dose-dependent fashion, with large increases in marrow drug levels beginning at 600 mg bid and with sustained levels throughout drug administration. Clinical responses occurred in 10 (29%) of the 34 evaluable patients, including 2 complete remissions. Genomic analyses failed to detect N-ras gene mutations in any of the 35 leukemias. The results of this first clinical trial of a signal transduction inhibitor in patients with acute leukemias suggest that inhibitors of FT may have important clinical antileukemic activity.

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal “committed stage” cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the “execution phase” extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.