Kaufmann, Scott H.

AbstractTumor necrosis factor- (TNF-) exerts two separate effects on neutrophils, stimulating effector functions while simultaneously inducing apoptosis. We examined here the involvement of caspases in neutrophil apoptosis and the effect of TNF-–induced apoptosis on reactive oxygen production. Immunoblotting and affinity labeling showed activation of caspase-8, caspase-3, and a caspase with a large subunit of 18 kD (T18) in TNF-–treated neutrophils. Active caspase-6 and -7 were not detectable in this cell type. Caspase-8 activated caspase-3 and T18 in neutrophil cytoplasmic extracts. zVAD-fmk blocked neutrophil apoptosis, in parallel with the inhibition of caspase activation. TNF-–induced caspase activation was accompanied by a decrease in the ability of neutrophils to release superoxide anion. Conversely, TNF- treatment in the presence of zVAD-fmk caused a prolonged augmentation of superoxide release. Granulocyte-macrophage colony-stimulating factor inhibited TNF-–induced caspase activation and apoptosis, while reversing the diminution in superoxide release. These observations not only suggest that a caspase cascade mediates apoptotic events and downregulates oxygen radical production in TNF-–treated neutrophils, but also raise the possibility that suppression of caspase activation with enhanced proinflammatory actions of TNF- may underlie the pathogenesis of inflammatory diseases.

AbstractBcl-2, Bcl-xL, and Mcl-1 are three related intracellular polypeptides that have been implicated as negative regulators of apoptosis. In contrast, the partner protein Bax acts as a positive regulator of apoptosis. Based on the observation that all four of these polypeptides are expressed in a variety of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines, cellular levels of these polypeptides were examined by immunoblotting in bone marrow samples harvested from 123 adult AML patients and 36 adult ALL patients before initial antileukemic therapy. Levels of Bcl-2, Mcl-1, Bcl-xL, and Bax each varied over a more than 10-fold range in different pretreatment leukemia specimens. When the 54 AML and 23 ALL samples that contained greater than 80% malignant cells were examined in greater detail, it was observed that pretreatment levels of Bcl-2 and Mcl-1 correlated with each other (R = .44,P < .001 for AML and R = .79,P < .0001 for ALL). In addition, a weak negative correlation between Bax expression and age was observed in AML samples (R = −0.35, P < .02) but not ALL samples. There was no relationship between pretreatment levels of these polypeptides and response to initial therapy. However, examination of 19 paired samples (the first harvested before chemotherapy and the second harvested 23 to 290 days later at the time of leukemic recurrence) revealed a greater than or equal to twofold increase in Mcl-1 levels in 10 of 19 pairs (7 of 15 AML and 3 of 4 ALL) at recurrence. In contrast, 2 of 19 pairs contained twofold less Mcl-1 at the time of recurrence. Approximately equal numbers of samples showed twofold increases and decreases in Bcl-2 (5 increases, 3 decreases) and Bcl-xL (1 increase, 4 decreases) at recurrence. Bax levels did not show a twofold decrease in any patient. These results, coupled with recent observations that cells overexpressing Mcl-1 are resistant to a variety of chemotherapeutic agents, raise the possibility that some chemotherapeutic regimens might select for leukemia cells with elevated levels of this particular apoptosis inhibitor.

AbstractThe Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1 . Likewise, affinity labeling with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK (bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or γ-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.

Abstract Recent studies have suggested that variations in levels of caspases, a family of intracellular cysteine proteases, can profoundly affect the ability of cells to undergo apoptosis. In this study, immunoblotting was used to examine levels of apoptotic protease activating factor-1 (Apaf-1) and procaspases-2, -3, -7, -8, and -9 in bone marrow samples (at least 80% leukemia) harvested before chemotherapy from adults with newly diagnosed acute myelogenous leukemia (AML, 42 patients) and acute lymphocytic leukemia (ALL, 18 patients). Levels of each of these polypeptides varied over a more than 10-fold range between specimens. In AML samples, expression of procaspase-2 correlated with levels of Apaf-1 (Rs = 0.52, P &lt; .02), procaspase-3 (Rs = 0.56,P &lt; .006) and procaspase-8 (Rs = 0.64, P &lt; .002). In ALL samples, expression of procaspases-7 and -9 was highly correlated (Rs = 0.90,P &lt; .003). Levels of these polypeptides did not correlate with prognostic factors or response to induction chemotherapy. In further studies, 16 paired samples (13 AML, 3 ALL), the first harvested before induction therapy and the second harvested at the time of leukemia regrowth, were also examined. There were no systematic alterations in levels of Apaf-1 or procaspases at relapse compared with diagnosis. These results indicate that levels of initiator caspases vary widely among different leukemia specimens but cast doubt on the hypothesis that this variation is a major determinant of drug sensitivity for acute leukemia in the clinical setting.

AbstractPrevious studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 μmol/L etoposide. After treatment with 17 μmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 μmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

Abstract Background : B-Chronic Lymphocytic leukemia (CLL) is the most common leukemia in North America. The standard current treatments use purine nucleoside analogues as single agent therapy or in combination with rituximab, steroids, and alkylating agents. Overall response rates with these treatments in previously untreated patients may reach 90 % with CR rates of 50–60%, however, most patients will relapse. Thus, despite refinements in therapy, CLL remains an incurable malignancy and there remains significant and urgent need to identify and develop new agents with novel mechanisms of action for the treatment of CLL. In line with this we have explored the mechanism and in vitro activity for a tyrphostin, adaphostin. Methods: We evaluated the in vitro efficacy of adaphostin to induce apoptosis in CLL B-cells. Peripheral blood was collected from patients with CLL (n=57). Highly purified CLL B-cells ((minimum 80% CD19+, mean 92.2% CD19+ and 94.4% CD19+/CD5+ positive)) were cultured with freshly prepared adaphostin for 24 – 120 hours. Cell death was analyzed by flow cytometry using Anexin V/Propidium iodide (PI). PARP cleavage and anti-apoptotic protein levels were measured by immunoblot techniques. The effect of combination treatment on CLL B cells with adaphostin and fludarabine on CLL B-cells was also assessed. Results: Analysis by Annexin V/PI staining revealed that the mean IC50 for adaphostin at 24 h was 4.2 uM (range 1.10 uM-11.25 uM; median = 4.25; n = 29) for CLL isolates and >10 uM for B and T-cells isolated from normal donors. Median IC50 levels for Adaphostin were not significantly different based on IgVH mutation status, level of CD38 expression, or cytogenetic abnormalities by FISH testing (Table 1). Immunoblots demonstrated adaphostin-induced PARP cleavage and cleavage of caspase 3 substrates, suggesting that adaphostin induces cell death through apoptosis. Adaphostin increased the intra-cellular level of reactive oxygen species (ROS) in CLL B-cells; and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis (mean reduction cell death=60%; range 23–99% reduction) suggesting generation of ROS is critical to adaphostin’s induction of apoptosis. Adaphostin also caused a decrease in the level of the anti-apoptotic proteins Bcl-2 in a majority of patients on both flow cytometry and immunoblots. When adaphostin was combined with fludarabine (F-ARA-ATP), a synergistic effect on cell death was observed in all 10 CLL samples when analyzed by mathematical modeling software. Summary: These findings indicate that adaphostin induces selective apoptosis in CLL B-cells from all risk categories through a mechanism that involves ROS generation. Importantly, we also demonstrate its ability to augment the effects of fludarabine. We continue to explore the preclinical development of adaphostin as a novel agent for the treatment of CLL. IC50 Adaphostin Dose Levels by Prognostic Groups FACTOR N Median IC 50 Adaphostin Range p-Value Rai Stage Group Low/Int (0–II) 19 4.7 1.8 – 11.25 0.018 High (III – IV) 10 3.05 1.1 – 5.4 - IgVH Mutation Status Mutated 11 4.45 1.1 – 11.25 0.72 Nonmutated 10 3.93 1.8 – 5.8 - CD38 Status Negative 20 4.55 1.1 – 11.25 0.74 Positive 9 4.25 1.8 – 6.6 - FISH defects 13q- 8 4.70 1.75 – 11.25 Normal 6 3.53 1.1 – 5.8 0.83 12+ 4 3.93 2.25 – 6.6 17p/11q 5 4.20 2.0 – 5.4

The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1 . Likewise, affinity labeling with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK (bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or γ-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.

Recent studies have suggested that variations in levels of caspases, a family of intracellular cysteine proteases, can profoundly affect the ability of cells to undergo apoptosis. In this study, immunoblotting was used to examine levels of apoptotic protease activating factor-1 (Apaf-1) and procaspases-2, -3, -7, -8, and -9 in bone marrow samples (at least 80% leukemia) harvested before chemotherapy from adults with newly diagnosed acute myelogenous leukemia (AML, 42 patients) and acute lymphocytic leukemia (ALL, 18 patients). Levels of each of these polypeptides varied over a more than 10-fold range between specimens. In AML samples, expression of procaspase-2 correlated with levels of Apaf-1 (Rs = 0.52, P < .02), procaspase-3 (Rs = 0.56,P < .006) and procaspase-8 (Rs = 0.64, P < .002). In ALL samples, expression of procaspases-7 and -9 was highly correlated (Rs = 0.90,P < .003). Levels of these polypeptides did not correlate with prognostic factors or response to induction chemotherapy. In further studies, 16 paired samples (13 AML, 3 ALL), the first harvested before induction therapy and the second harvested at the time of leukemia regrowth, were also examined. There were no systematic alterations in levels of Apaf-1 or procaspases at relapse compared with diagnosis. These results indicate that levels of initiator caspases vary widely among different leukemia specimens but cast doubt on the hypothesis that this variation is a major determinant of drug sensitivity for acute leukemia in the clinical setting.

Bcl-2, Bcl-xL, and Mcl-1 are three related intracellular polypeptides that have been implicated as negative regulators of apoptosis. In contrast, the partner protein Bax acts as a positive regulator of apoptosis. Based on the observation that all four of these polypeptides are expressed in a variety of acute myelogenous leukemia (AML) and acute lymphocytic leukemia (ALL) cell lines, cellular levels of these polypeptides were examined by immunoblotting in bone marrow samples harvested from 123 adult AML patients and 36 adult ALL patients before initial antileukemic therapy. Levels of Bcl-2, Mcl-1, Bcl-xL, and Bax each varied over a more than 10-fold range in different pretreatment leukemia specimens. When the 54 AML and 23 ALL samples that contained greater than 80% malignant cells were examined in greater detail, it was observed that pretreatment levels of Bcl-2 and Mcl-1 correlated with each other (R = .44,P < .001 for AML and R = .79,P < .0001 for ALL). In addition, a weak negative correlation between Bax expression and age was observed in AML samples (R = −0.35, P < .02) but not ALL samples. There was no relationship between pretreatment levels of these polypeptides and response to initial therapy. However, examination of 19 paired samples (the first harvested before chemotherapy and the second harvested 23 to 290 days later at the time of leukemic recurrence) revealed a greater than or equal to twofold increase in Mcl-1 levels in 10 of 19 pairs (7 of 15 AML and 3 of 4 ALL) at recurrence. In contrast, 2 of 19 pairs contained twofold less Mcl-1 at the time of recurrence. Approximately equal numbers of samples showed twofold increases and decreases in Bcl-2 (5 increases, 3 decreases) and Bcl-xL (1 increase, 4 decreases) at recurrence. Bax levels did not show a twofold decrease in any patient. These results, coupled with recent observations that cells overexpressing Mcl-1 are resistant to a variety of chemotherapeutic agents, raise the possibility that some chemotherapeutic regimens might select for leukemia cells with elevated levels of this particular apoptosis inhibitor.

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 μmol/L etoposide. After treatment with 17 μmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 μmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.