Intrinsic and chemo-sensitizing activity of SMAC-mimetics on high-risk childhood acute lymphoblastic leukemia.

SMAC-mimetics represent a targeted therapy approach to overcome apoptosis resistance in many tumors. Here, we investigated the efficacy of the SMAC-mimetic BV6 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In ALL cell lines, intrinsic apoptosis sensitivity was associated with rapid cIAP degradation, NF-κB activation, TNF-α secretion and induction of an autocrine TNF-α-dependent cell death loop. This pattern of responsiveness was also observed upon ex vivo analysis of 40 primograft BCP-ALL samples. Treatment with BV6 induced cell death in the majority of ALL primografts including leukemias with high-risk and poor-prognosis features. Inhibition of cell death by the TNF receptor fusion protein etanercept demonstrated that BV6 activity is dependent on TNF-α. In a preclinical NOD/SCID/huALL model of high-risk ALL, marked anti-leukemia effectivity and significantly prolonged survival were observed upon BV6 treatment. Interestingly, also in vivo, intrinsic SMAC-mimetic activity was mediated by TNF-α. Importantly, BV6 increased the effectivity of conventional induction therapy including vincristine, dexamethasone and asparaginase leading to prolonged remission induction. These data suggest SMAC-mimetics as an important addendum to efficient therapy of pediatric BCP-ALL.

Rapid engraftment of human ALL in NOD/SCID mice involves deficient apoptosis signaling.

Previously, we found that rapid leukemia engraftment (short time to leukemia, TTL(short)) in the NOD/SCID/huALL (non-obese diabetic/severe combined immuno-deficiency/human acute lymphoblastic leukemia) xenograft model is indicative of early patient relapse. As earlier intact apoptosis sensitivity was predictive for good prognosis in patients, we investigated the importance of apoptosis signaling on NOD/SCID/huALL engraftment. Intact apoptosome function as reflected by cytochrome c-related activation of caspase-3 (CRAC-positivity) was strongly associated with prolonged NOD/SCID engraftment (long time to leukemia, TTL(long)) of primary leukemia cells, good treatment response and superior patient survival. Conversely, deficient apoptosome function (CRAC-negativity) was associated with rapid engraftment (TTL(short)) and early relapse. Moreover, an intact apoptosis signaling was associated with high transcript and protein levels of the pro-apoptotic death-associated protein kinase1 (DAPK1). Our data strongly emphasize the impact of intrinsic apoptosis sensitivity of ALL cells on the engraftment phenotype in the NOD/SCID/huALL model, and most importantly also on patient outcome.

Acute lymphoblastic leukemia cells treated with CpG oligodeoxynucleotides, IL-4 and CD40 ligand facilitate enhanced anti-leukemic CTL responses.

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Although the majority of patients initially respond to upfront chemotherapy, relapses with poor prognosis occur in approximately 20% of cases. Thus, novel therapeutic strategies are required to improve long-term survival. B-cell precursor (BCP)-ALL cells express low levels of immunogenic molecules and, therefore, are poorly recognized by the immune system. In the present study, we investigated the effect of various combinations of potent B-cell stimulators including CpG, Interleukin (IL)-2 family cytokines and CD40 ligand (CD40L) on the immunogenicity of primary BCP-ALL cells and a series of BCP-ALL cell lines. The combination of CpG, IL-4 and CD40L was identified as most effective to enhance expression of immunogenic molecules on BCP-ALL cells, resulting in an increased capacity to induce both allogeneic and autologous cytotoxic T lymphocytes (CTL). Importantly, such CTL exhibited significant anti-leukemic cytotoxicity not only towards treated, but also towards untreated BCP-ALL cells. Our results demonstrate that the combination of CpG with other B-cell stimulators is more efficient than CpG alone in generating immunogenic BCP-ALL cells and anti-leukemic CTL. Our results may stimulate the development of novel adoptive T cell transfer approaches for the management of BCP-ALL.

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.

Apoptosis induction in peripheral leukemia cells by remission induction treatment in vivo: selective depletion and apoptosis in a CD34+ subpopulation of leukemia cells.

In vitro studies demonstrating the induction of programmed cell death by cytotoxic drugs used in anticancer chemotherapy suggested that antileukemic treatment eliminates leukemia cells by apoptosis. We therefore analyzed apoptosis induction and activation of apoptosis signaling molecules in patients receiving remission induction treatment for AML and ALL during the initial phase of leukemia cell reduction. A coexistence of distinct populations of CD34(+) and CD34(-) leukemia cells could be identified. During chemotherapy, CD34(+) leukemia cells were more rapidly depleted than CD34(-) cells. Furthermore, a significant increase in leukemia cell apoptosis ex vivo was detected in CD34(+) cells, while no such increase was observed in the CD34(-) subpopulation, suggesting that CD34(+) leukemia cells are the main targets for apoptosis induction through antileukemic treatment. No alterations in Bax and Bcl-2 expression were found during in vivo chemotherapy, and CD95 expression and sensitivity remained low, indicating the induction of apoptosis independent of the CD95 system or regulation of protein levels of Bax and Bcl-2. The data suggest that analysis of leukemia cell subpopulations is required for further identification of apoptosis signaling molecules relevant for response to treatment and assessment of drug efficacy in vivo and in vitro.

Fermentation and costs of fuel ethanol from corn with Quick-Germ process.

The Quick-Germ process developed at the University of Illinois at Urbana-Champaign is a way to obtain corn oil, but with lower capital costs than the traditional wet-milling process. Quick-Germ has the potential to increase the coproduct credits and profitability of the existing dry-grind fuel ethanol process, but the fermentability of the corn remaining after oil recovery has not been tested. Therefore, a series of pilot scale (50 L) fermentations was carefully controlled and monitored with unique methods for standard inoculation and automatic sampling. It was found that the concentration of suspended solids was significantly reduced in the Quick-Germ fermentations. When compared at the same concentration of fermentable sugars, the fermentation rate and yield were not statistically different from controls. When Quick-Germ was integrated into a state-of-the-art dry-grind fuel ethanol process, computer simulation and cost models indicated savings of approx $0.01/L of ethanol ($0.04/gal) with the Quick-Germ process. Additional savings associated with the lower suspended solids could not be quantified and were not included. However, the savings are sensitive to the price of corn oil.

Determination of carbonyl compounds in exhaust gas by using a modified DNPH-method.

The determination of carbonyl compounds in gaseous samples is usually accomplished by enrichment methods, in which 2,4-dinitrophenyl-hydrazine (DNPH) as a derivatization reagent has become established to a large extent. However, the conventional methods of DNPH-impingers and of DNPH-cartridges are applicable to emission measurements in a limited way only, depending on the NO(2)-concentration in the exhaust gas. It could be proved that DNPH-derivatives, as well as DNPH, are also decomposed by NO(2) at a different speed, in which the hydrazones of unsaturated carbonyl compounds are probably more sensitive than those of the saturated carbonyl compounds. In view of this fact, the collecting methods had to be modified to avoid losses with the enrichment. The analysis of the compounds is carried out by HPLC with an effective gradient-system which is able to separate and detect the carbonyl compounds in exhaust gas within 16 min. Furthermore, a simple working-up procedure is presented which facilitates a parallel analysis by GC.

Distribution of fumonisins in food and feed products prepared from contaminated corn.

The fate and distribution of the fumonisins B1 (FB1) and B2 (FB2) were determined in products obtained from naturally contaminated corn used for ethanol fermentation and wet milling operations. Fumonisins are stable to the conditions used in ethanol fermentations and tend to concentrate in the distillers dried grain, a fraction generally used for animal feed. No toxin was found in the ethanol. Starch from wet milling of corn, naturally contaminated at 13.9 micrograms fumonisin B1/g, was free of detectable toxin. The other fractions contained fumonisins at the following levels: gluten (5.1-5.8 micrograms FB1/g, 4.7-4.9 micrograms FB2/g); fiber (2.7-5.7 micrograms FB1/g, 2.1-3.1 micrograms FB2/g); and germ (1.3-3.1 micrograms FB1/g, 0.7-1.6 micrograms FB2/g). The steep water and process water contained 22% of the recoverable fumonisins. A combination of analytical methodologies was required to determine fumonisins in the different products from the wet milling process.