PU.1, a novel caspase-3 substrate, partially contributes to chemotherapeutic agents-induced apoptosis in leukemic cells.

PU.1 is one of key regulators of hematopoietic cell development, a tightly-regulated lineage-specific process. Here we provide the first evidence that PU.1 protein is cleaved into two fragments of 24 kDa and 16 kDa during apoptosis progression in leukemic cell lines and primary leukemic cells. Further experiments with specific capase-3 inhibitor Z-DEVD-fmk and the in vitro proteolytic system confirmed that PU.1 is a direct target of caspase-3. Using site-directed mutagenesis analyses, the aspartic acid residues at positions 97 and 151 of PU.1 protein were identified as capsase-3 target sites. More intriguingly, the suppression of PU.1 expression by small interfering RNAs (siRNAs) significantly inhibits DNA-damaging agents NSC606985 and etoposide-induced apoptosis in leukemic cells, together with the up-regulated expression of anti-apoptotic bcl-2 gene. These results would provide new insights for understanding the mechanism of PU.1 protein in hematopoiesis and leukemogenesis.

Synergistic mitosis-arresting effects of arsenic trioxide and paclitaxel on human malignant lymphocytes.

The treatment outcome of acute lymphoblastic leukemia (ALL) has improved steadily over the last 50 years. However, the cure rates are unlikely to be raised further with current therapies. Since increasing the dosage of chemotherapeutic agents could also elevate toxicity, a solution to how one could achieve maximum therapeutic effect with the minimum dosage possible is imminent. One possibility is the employment of combination drug therapies. Arsenic trioxide (ATO) is a widely used drug for acute promyelocytic leukemia (APL). Its combination with other drugs presented therapeutic activities in malignant cancers other than APL. Considering the fact that ATO induces mitotic arrest prior to apoptosis induction, we attempted to investigate the potential anti-cancer effects of ATO in combination with the microtubule-stabilizing agent, paclitaxel (PTX), using malignant lymphocytes as in vitro models. Three malignant lymphocytic cell lines and primary cells were treated with ATO and/or PTX. Using the Chou-Talalay analysis for evaluation of combined effect of ATO and PTX, we found a synergistic effect of the two drugs in the inhibition of cell growth. We also found that the combination of ATO and PTX at low concentrations synergistically induced mitotic arrest followed by apoptosis in malignant lymphocytes, which increased phosphorylated cyclin-dependent kinase 1 (Cdk1) on Thr(161) and promoted the dysregulated activation of Cdk1. The ATO/PTX combination also significantly enhanced the activation of spindle checkpoint by inducing the formation of the inhibitory checkpoint complex BubR1/Cdc20. Our study provided the first in vitro demonstration that low concentrations of ATO and PTX synergistically induce mitotic arrest in malignant lymphocytes.

Protein kinase Cdelta stimulates proteasome-dependent degradation of C/EBPalpha during apoptosis induction of leukemic cells.

BACKGROUND: The precise regulation and maintenance of balance between cell proliferation, differentiation and death in metazoan are critical for tissue homeostasis. CCAAT/enhancer-binding protein alpha (C/EBPalpha) has been implicated as a key regulator of differentiation and proliferation in various cell types. Here we investigated the potential dynamic change and role of C/EBPalpha protein during apoptosis induction. METHODOLOGY/PRINCIPAL FINDINGS: Upon onset of apoptosis induced by various kinds of inducers such as NSC606985, etoposide and others, C/EBPalpha expression presented a profound down-regulation in leukemic cell lines and primary cells via induction of protein degradation and inhibition of transcription, as assessed respectively by cycloheximide inhibition test, real-time quantitative RT-PCR and luciferase reporter assay. Applying chemical inhibition, forced expression of dominant negative mutant and catalytic fragment (CF) of protein kinase Cdelta (PKCdelta), which was proteolytically activated during apoptosis induction tested, we showed that the active PKCdelta protein contributed to the increased degradation of C/EBPalpha protein. Three specific proteasome inhibitors antagonized C/EBPalpha degradation during apoptosis induction. More importantly, ectopic expression of PKCdelta-CF stimulated the ubiquitination of C/EBPalpha protein, while the chemical inhibition of PKCdelta action significantly inhibited the enhanced ubiquitination of C/EBPalpha protein under NSC606985 treatment. Additionally, silencing of C/EBPalpha expression by small interfering RNAs enhanced, while inducible expression of C/EBPalpha inhibited NSC606985/etoposide-induced apoptosis in leukemic cells. CONCLUSIONS/SIGNIFICANCE: These observations indicate that the activation of PKCdelta upon apoptosis results in the increased proteasome-dependent degradation of C/EBPalpha, which partially contributes to PKCdelta-mediated apoptosis.