Paul D. Allen

Inhibition of autophagy abrogates tumour necrosis factor α induced apoptosis in human T-lymphoblastic leukaemic cells

The pattern and the sequence of tumour necrosis factor‐α (TNFα) induced cell death in the acute T‐lymphoblastic leukaemic cell line CCRF‐CEM and its vinblastine‐resistant subline CEM/VLB100 have been studied. Previously, we found that the CEM/VLB100 cell line was more sensitive to TNFα‐induced killing than its parental CCRF‐CEM cell line. TNFα‐induced cell death showed an apoptotic pattern, as detected by agarose electrophoresis, flow cytometry and transmission electron microscopy (TEM). TEM images revealed that autophagy and condensed mitochondria occurred earlier than nuclear fragmentation. The specific inhibitor of autophagy, 3‐methyladenine (3MA), inhibited the formation of autophagosomes. TNFα‐induced DNA fragmentation and cytolysis were completely inhibited by 10 mM 3MA. Inhibition of the fusion of lysosomes with autophagosomes by asparagine did not block TNFα‐induced apoptosis. In addition, amino acid and protein deprivation enhanced TNFα‐induced autophagy but not apoptosis. We propose that the early stages of autophagy are required for, but do not necessarily result in, TNFα‐induced apoptosis.

The proteasome inhibitor bortezomib acts independently of p53 and induces cell death via apoptosis and mitotic catastrophe in B-cell lymphoma cell lines.

Bortezomib is a proteasome inhibitor with proven efficacy in multiple myeloma and non-Hodgkins lymphoma. This study reports the effects of bortezomib in B-cell lymphoma cell lines with differing sensitivity to bortezomib to investigate factors that influence sensitivity. Bortezomib induced a time- and concentration-dependent reduction in cell viability in five lymphoma cell lines, with EC(50) values ranging from 6 nmol/L (DHL-7 cells) to 25 nmol/L (DHL-4 cells) after 72 h. Bortezomib cytotoxicity was independent of p53 function, as all cell lines exhibited mutations by sequence analysis. The difference in sensitivity was not explained by proteasome or nuclear factor-kappaB (NF-kappaB) inhibition as these were similar in the most and least sensitive cells. NF-kappaB inhibition was less marked than that of a specific NF-kappaB inhibitor, Bay 11-7082. Cell cycle analysis showed a marked G(2)-arrested population in the least sensitive DHL-4 line only, an effect that was not present with Bay 11-7082 treatment. Conversely, in DHL-7 cells, bortezomib treatment resulted in cells moving into an aberrant mitosis, indicative of mitotic catastrophe that may contribute to increased sensitivity to bortezomib. These studies show that although bortezomib treatment had similar effects on apoptotic and NF-kappaB signaling pathways in these cell lines, different cell cycle effects were observed and induction of a further mechanism of cell death, mitotic catastrophe, was observed in the more sensitive cell line, which may provide some pointers to the difference in sensitivity between cell lines. An improved understanding of how DHL-7 cells abrogate the G(2)-M cell cycle checkpoint may help identify targets to increase the efficacy of bortezomib.

Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug‐induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine‐resistant leukaemic cell line CEM/VLB100 has a more active mitochondrial electron transport chain (ETC) than the parental CCRF‐CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine‐induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F1F0‐ATPase, oligomycin, rendered CEM/VLB100 cells more sensitive to vinblastine by inhibiting the energy‐dependent P‐glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine‐induced cell cycle changes in the CCRF‐CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB‐induced ETC depletion in CEM/VLB100 cells inhibited apoptosis in response to high concentration of vinblastine, but not G2M arrest. We suggest that: (1) over‐expression of Pgp by drug‐resistant cells may up‐regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down‐regulation of mitochondrial ETC activity may confer resistance to vinblastine‐induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine‐induced apoptosis downstream of microtubule disruption and cell cycle changes.

EFFECTS OF PARP INHIBITION ON DRUG AND FAS-INDUCED APOPTOSIS IN LEUKAEMIC CELLS

Poly (ADP-ribose) polymerase (PARP) is activated following binding to DNA strand breaks and is cleaved in cells undergoing apoptosis. Work predominantly in murine systems has suggested that inhibitors of PARP might potentiate the effects of chemotherapeutic agents and be used as adjuncts to cancer therapy. Therefore, we studied the role of PARP in drug-induced apoptosis in HL-60, myeloid leukaemia cells and found that pre-treatment with 3-aminobenzamide (3AB) or 6(5H)-phenanthridinone, inhibitors of PARP, resulted in resistance to, rather than potentiation of apoptotic death induced by DNA-damaging agents, idarubicin, etoposide and fludarabine, as determined by flow cytometry, following propidium iodide staining. 3AB treated CEM/VLB100, mdr-expressing human lymphoblastic leukaemia cells were also found to be more resistant to idarubicin compared to cells treated with idarubicin alone, however, apoptosis was not reduced in parental CCRF-CEM cells under the same conditions. Similar results were obtained using agents with primary modes of action which do not involve DNA damage, vinblastine and a fas-ligating antibody (CH11). The precise role of PARP has yet to be defined but might involve effects on cell cycle progression. We conclude that PARP activation appears to be involved in apoptosis in certain leukaemic cell lines and that these effects are independent of lineage or p-glycoprotein. Constitutive failure to activate PARP might be responsible for conferring resistance to apoptosis.

Etoposide-mediated deregulation of the G2M checkpoint in myeloid leukaemic cell lines results in loss of cell survival

Summary. The K562 leukaemic cell line expresses an inherent survival signal due to the antiapoptotic properties of Bcr‐abl, which is, in part, mediated by prolonging the G2M checkpoint and allowing DNA repair mechanisms to operate post genotoxic insult. Arrest of the cell cycle is mediated by retaining an inactivating state of phosphorylation of cyclin‐dependent kinase 1 (Cdk1) on tyrosine 15. Our data confirmed that cell survival in K562 was promoted by cell cycle arrest at G2M in response to the genotoxin etoposide. There was no predicted cell cycle arrest in Bcr‐abl‐positive derivative cell lines of K562 that did not survive the same genotoxic insult but, paradoxically, Cdk1 tyrosine phosphorylation was enhanced to a higher extent compared with the parental cell line where arrest of the cell cycle was observed. To ascertain that this was not an anomaly of the derivative lines, HL60 cells were treated with concentrations of etoposide that induced arrest of the cell cycle or apoptosis. Only HL60 cells that subsequently underwent apoptosis elicited the same effect of increased Cdk1 tyrosine phosphorylation. It is proposed that the augmented tyrosine phosphorylation status of Cdk1 is associated with the abolition of cell survival, in addition to the previously reported induction of cell cycle arrest in myeloid cell lines.

p53-mediated downregulation of Chk1 abrogates the DNA damage-induced G2M checkpoint in K562 cells, resulting in increased apoptosis

Summary. BCR–ABL confers apoptotic resistance to a range of genotoxic agents, and this protection is mediated in part by prolonging the G2 checkpoint. The p53 tumour suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. To investigate the effect of p53 on the BCR–ABL‐mediated G2M checkpoint response, we transiently transfected the BCR–ABL‐positive, p53‐negative cell line K562 with wild‐type human p53. The p53‐transfected cells showed a decreased ability to arrest in G2 and an increase in apoptosis in response to etoposide treatment, relative to the control mock‐transfected cells. p53‐transfected and control cells were treated with etoposide and trapped at mitosis with nocodazole. The mitotic index of p53‐transfected cells was higher than that of the control cells, which suggests that p53 abrogates the G2 checkpoint response to etoposide treatment in K562 cells. We found that the expression of the cell cycle checkpoint protein Chk1 was reduced in the etoposide‐treated p53‐transfected cells by 24 h, and this correlated with a reduction in the extent of etoposide‐induced phosphorylation of CDK1 at tyrosine 15 (Y15). We conclude, therefore, that p53 overrides the strong G2 checkpoint response to etoposide in K562 cells, by directly or indirectly downregulating Chk1 expression, which, in turn, contributes to the proapoptotic effect of p53.

Constitutive levels of cAMP-dependent protein kinase activity determine sensitivity of human multidrug-resistant leukaemic cell lines to growth inhibition and apoptosis by forskolin and tumour necrosis factor alpha.

The cyclic adenosine monophosphate (cAMP)‐dependent protein kinase (PKA) signal pathway regulates cell proliferation, differentiation and cell death. It may also regulate the multidrug resistance (MDR) phenotype in leukaemic cells. These data showed that MDR1+ K/Dau600 cells exhibited a higher basal level of PKA activity than MDR− parental cells. The significance of this on tumour necrosis factor α (TNFα)‐induced apoptosis and cytostasis was investigated further. In comparison with MDR1− parental cells, K/Dau600 cells had a higher expression of PKA regulatory subunit RIα and nuclear catalytic subunit PKAcα. They were also more susceptible to inhibition of proliferation and induction of apoptosis by TNFα and/or forskolin, but this could be attenuated by H89. An increase in cAMP was associated with the apoptosis in the K/Dau600 cell line. Forskolin inactivated NF‐κB in K/Dau600 cells but not in K562 cl.6 cells, whereas TNF activated NF‐κB in K562 cl.6 cells but not in K/Dau600 cells. 8‐Cl‐cAMP exhibited similar inhibitory effects on the proliferation of all of the cell lines used via its metabolite 8‐Cl‐adenosine, which indicates that these effects were independent of residual PKA or cAMP. Therefore, the differential sensitivity to apoptosis and/or growth inhibition could be mediated via cAMP, partly through PKA via NF‐κB and partly by PKA‐independent pathways.

Modulation of surface TNF expression by human leukaemic cells alters their sensitivity to exogenous TNF.

In this study, U937 leukaemic cells underwent apoptotic cell death following exposure to TNF. Pre-incubation of cells for 48 h with VitD(3) (10(-8)M) induced resistance to TNF, whereas incubation with tau-IFN or GM-CSF increased susceptibility to TNF. Resistance to exogenous TNF (exTNF) following culture with VitD(3) was associated with increased expression of endogenous TNF (enTNF). The TNF inhibitors pentoxifylline(PTF) and dichloroisocoumarin (DCI) inhibited TNF synthesis by U937 cells and abrogated the increase in resistance to TNF seen with VitD(3). The tau-IFN increased TNF expression, whereas GM-CSF had little effect. The data show that the sensitivity of leukaemic cells to exTNF can be modulated by cytokines. The protective effect of VitD(3) is mediated in part by directly upregulating enTNF synthesis.

8-Cl-adenosine mediated cytotoxicity and sensitization of T-lymphoblastic leukemia cells to TNFα-induced apoptosis is via inactivation of NF-κB

Abstract These data show that 8-Cl-cAMP is cytotoxic to the lymphoblastic leukemia cell line CEM and its vinblastine selected multidrug resistant derivative, CEM/VLB100 although PKA was not involved in these effects. The cytotoxic effects of 8-Cl-cAMP was abrogated by cotreatment with either ADA or IBMX which indicated a degradation form of 8-Cl-cAMP was needed for this cytotoxicity. CEM and CEM/VLB100 cells displayed a notable sensitivity to 8-Cl-adenosine-induced growth inhibition and apoptosis. 8-Cl-adenosine increased the cytosolic levels of IκBα which prevented NF-κB nuclear translocation. 8-Cl-adenosine also prevented TNFα-induced IkB decay and NF-κB activation in CEM and CEM/VLB100 cells.

Inhibition of FAS/FAS-Ligand Does Not Block Chemotherapy-Induced Apoptosis in Drug Sensitive and Resistant Cells

It has been suggested that one means by which chemotherapeutic agents exert their effect on leukaemic cells, is via autocrine induction of fas-ligand which then binds to fas (CD95), activates the caspase pathway and results ultimately in apoptotic death. In order to test this hypothesis, we have treated leukaemic cell lines with various chemotherapeutic agents (idarubicin, etoposide, fludarabine and 2-CdA) with and without pre-treatment with fas (ZB4) and fas-ligand (NOK-1) blocking monoclonal antibodies. Cell cycle analysis and quantitation of apoptosis were performed by flow cytometry following propidium iodide staining. HL-60 cells were found to be sensitive to the induction of apoptosis with all drugs tested but were highly resistant to treatment with a fas-ligating antibody (CH11). Apoptosis was neither inhibited in parental CEM cells nor their mdr-expressing drug resistant counterpart, CEM/VLB100 by pre-treatment with either ZB4 or NOK1. In addition, CEM/VLB100 were slightly more sensitive to treatment with CH11 (100 ng/ml) than parental CEM cells (% age apoptosis = 30.35 and 23.675, p = 0.024) and at least as sensitive to recombinant fas-ligand (50 ng/ml) (% age apoptosis = 26.6 and 20.2, p = NS). We conclude that it is unlikely that fas/fas-ligand interactions play a significant role in the induction of apoptosis by these chemotherapeutic agents in the leukaemic cell lines tested.