Anne McGahon

The end of the (cell) line: Methods for the study of apoptosis in vitro

Publisher Summary This chapter describes techniques commonly used to study cell death using in vitro cell culture systems. These techniques include a range of simple dye exclusion assays that give crude information on cell viability but do not necessarily discriminate between apoptotic or necrotic modes of cell death. The chapter describes cell death assays that are based on the observation that apoptosis is accompanied by DNA fragmentation, either into the classical “ladder” pattern of 200 bp integer multiples, 50kb fragments, or single-stranded DNA cleavage. Assays used to measure this fragmentation include gel electrophoresis of total DNA, quantification of the release of radioactively labeled DNA, examination of cell cycle profile, and in situ nick translation. Because apoptotic cells are morphologically highly distinctive and are easily distinguishable from both viable and necrotic cells, most cell death assays should be coupled with direct morphological evaluation of the cell population under study to define with certainty the mode of cell death that is occurring.

Bcl-2-independent Bcr-Abl-mediated resistance to apoptosis: protection is correlated with up regulation of Bcl-xL.

Bcr–Abl is the molecule responsible for both the transformation phenotype and the resistance to chemotherapeutic drugs found in chronic myelogenous leukemia (CML) cells. Wild-type HL-60, a transformed pro-myelocytic cell line, is very susceptible to apoptosis-inducing agents. We show here that expression of Bcr–Abl in HL-60 cells rendered them extremely resistant to apoptosis induced by a wide variety of agents. The anti-apoptotic effect of Bcr–Abl was found to be independent of the phase of the cell cycle. Treatment with antisense oligonucleotides directed to bcr decreased the expression of the ectopic bcr–abl and restored susceptibility to apoptosis. Double mutations affecting the autophosphorylation site and the phosphotyrosine-binding motif (FLVRES) have been previously shown to impair the transforming activity of Bcr–Abl in fibroblasts and hematopoietic cells, however HL-60 cells expressing this double mutant molecule exhibited the same level of resistance to apoptosis as those expressing the wild-type Bcr–Abl. Interestingly, wild type and mutant Bcr–Abl induced in HL-60 cells a dramatic down regulation of Bcl-2 and increased the levels of Bcl-xL. The level of Bax did not change in response to the presence of Bcr–Abl. Antisense oligonucleotides targeted to bcl-x down-regulated the expression of Bcl-xL and increased the susceptibility of HL-60.Bcr–Abl cells to staurosporine. Importantly, HL-60 cells overexpressing Bcl-xL showed higher expression of Bcl-xL but lower resistance to apoptosis when compared to HL-60.Bcr–Abl cells. The results described here show that Bcr–Abl is a powerful mammalian anti-apoptotic molecule and can act independently of Bcl-2. Bcl-xL, however, seems to participate in part in Bcr–Abl-mediated resistance to apoptosis in HL-60 cells.

Regulation of the Fas Apoptotic Cell Death Pathway by Abl

Relatively little is known about oncogene involvement in the regulation of Fas-mediated apoptosis. Inhibition of Fas-induced cell death by the bcl-2 oncogene has been demonstrated to be only partial. In light of a growing body of evidence for the Abl kinase as a negative regulator of cell death, we sought to determine whether Abl expression could protect against Fas-mediated cell death. To address this question, we utilized two separate strategies. In the first, we expressed human Fas in K562, a chronic myelogenous leukemia cell line, which constitutively expresses bcr-abl and examined the effects of Fas ligation in these cells. Fas-positive K562 transformants (K562.Fas) were found to be protected against Fas-mediated cell death. However, down-regulation of Bcr-Abl protein levels in K562.Fas cells using antisense oligonucleotides targeted to bcr-abl mRNA rendered these cells highly susceptible to Fas-induced death. In the second approach we utilized a Fas-positive HL-60 cell line, which we transfected with a temperature-sensitive mutant of v-Abl. HL-60.v-Abl transfectants were found to be protected from Fas-induced apoptosis at the permissive but not the restrictive temperature for the Abl kinase. Taken together, these observations identify the Abl kinase as a negative regulator of Fas-mediated cell death. Since Abl was also found to block apoptosis mediated by ceramide, a recently proposed downstream effector of the apoptotic pathway initiated by Fas, we propose that Abl exerts its protective effects downstream of the early Fas-initiated signaling events.

Chemotherapeutic drug-induced apoptosis in human leukaemic cells is independent of the Fas (APO-1/CD95) receptor/ligand system

The potential role of the Fas (CD95/APO‐1) receptor/ligand system in chemotherapeutic drug‐induced apoptosis was examined in a number of human leukaemic cell lines. Flow cytometric profiles of doxorubicin‐treated HL‐60, K562, U937 and Jurkat cells failed to show any significant increase in Fas or Fas ligand expression over 24 h, despite the induction of significant levels of apoptosis in these cells. Although preincubation of human leukaemic cells with a neutralizing anti‐Fas IgG antibody blocked anti‐Fas IgM‐induced apoptosis, this strategy failed to inhibit chemotherapeutic drug‐induced apoptosis. To determine whether recruitment of the Fas/Fas ligand complex during drug‐induced apoptosis was a cell‐specific event we utilized the CEM cell line. Doxorubicin treatment of CEM cells over 24 h failed to show any up‐regulation in Fas or Fas ligand protein levels as detected by flow cytometry. Furthermore, neutralizing anti‐Fas IgG Ab failed to inhibit chemotherapeutic drug‐induced apoptosis in CEM cells. The present studies do, however, demonstrate a role for anti‐Fas IgM Ab in producing a cytotoxic synergistic effect when used in combination with chemotherapeutic drugs. Low‐dose anti‐Fas IgM treatment in combination with doxorubicin, methotrexate, camptothecin and etoposide produced an augmented cytoxicity in CEM cells. Taken together these observations demonstrate that although recruitment of the Fas/APO‐1/CD95 receptor/ligand system is not a necessary requirement for chemotherapeutic drug‐induced apoptosis, combination of anti‐Fas IgM and drug treatment produces a synergistic cytotoxic effect which may prove useful in the treatment of human leukaemias.

Sensitisation of HL60 human leukaemic cells to cytotoxic drug-induced apoptosis by inhibition of PI3-kinase survival signals.

Drug resistance remains a serious limiting factor in the treatment of acute myeloid leukaemia (AML) either at initial presentation or following primary or subsequent relapses. Using specific kinase inhibitors, this study has investigated the contribution of the Ras/PI3-kinase regulated survival pathways to drug resistance and suppression of apoptosis in a cell line derived from AML (HL60). Inhibition of the Raf/MAP-kinase (ERK) pathway with a specific MAP-kinase inhibitor, apigenin did not sensitise HL60 cells to drug-induced apoptosis, indicating a lack of involvement in chemoresistance. In contrast, the PI3-kinase inhibitors, LY294002 and wortmannin, did induce a significant increase in apoptosis in combination with cytotoxic drugs. The contribution of downstream mediators of PI3-kinase, p70S6-kinase and PKB/Akt were then investigated. While inhibition of p70S6-kinase with rapamycin did not increase drug-induced apoptosis, PI3-kinase inhibition resulted in notable dephosphorylation of PKB, suggesting that the PI3-kinase/PKB survival pathway may play a major role in chemoresistance in AML. This pathway has been reported to mediate heterodimer interactions with the proapoptotic regulator, Bad. In contrast to previous studies, we found no evidence of Bad binding to anti-apoptotic Bcl-2, Bcl-XLor Mcl-1, or of alterations in Bax heterodimers. This suggests that alternative targets of PI3-kinase/PKB, distinct from the Bcl-2 family may be responsible for contributing to survival factor-mediated drug resistance in AML.

Downregulation of Bcr-Abl in K562 cells restores susceptibility to apoptosis: characterization of the apoptotic death.

We examined the susceptibility of a variety of human leukemic cell lines to the induction of apoptosis. K562, a chronic myelogenous leukemic cell line which expresses the bcr-abl fusion gene, was found to be extremely resistant to apoptosis, irrespective of the inducing agent. This resistance can be attributed to the deregulated Abl kinase activity of bcr-abl, as downregulation of its expression using antisense oligodeoxynucleotides targeted to the beginning of the abl sequence in this chimeric gene rendered these cells susceptible to cytotoxic drug-induced apoptosis. Examination of the morphological and biochemical features of apoptosis in K562 cells revealed the typical membrane blebbing and chromatin condensation associated with this form of cell death. In situ TdT-mediated end labeling of the DNA revealed the presence of strand breaks in the treated cells and field inversion gel electrophoresis revealed the presence of large 10 – 50 kb fragments. However there was an absence of oligonucleosomal DNA fragmentation, whether or not Bcr-Abl was expressed. Thus, while inhibition of expression of Bcr-Abl renders K562 cells susceptible to apoptosis, the absence of oligonucleosomal DNA fragmentation in these cells is independent of the function of this molecule.

Inhibition of PI3-kinase sensitises HL60 human leukaemia cells to both chemotherapeutic drug- and Fas-induced apoptosis by a JNK independent pathway

Increasing resistance to chemotherapeutic regimes remains a serious problem in the treatment of acute myeloid leukaemia. We have shown that phosphatidylinositol (PI) 3-kinase inhibition significantly sensitises the AML derived cell line, HL60 to chemotherapeutic drug- and Fas-induced apoptosis. PI3-kinase inhibition significantly potentiates cytotoxic drug-induced c-jun N-terminal kinase (JNK) activation, reported to be a requirement for apoptosis. However, JNK inhibition does not enhance cell viability following treatment with drug and inhibitor. Furthermore, PI3-kinase inhibition significantly increases sensitivity to apoptosis mediated by an exogenous receptor agonist, again by a JNK independent mechanism. These results suggest that PI3-kinase inhibitors could be of significant therapeutic importance, lowering the threshold for apoptosis induced by both chemotherapy and cell-mediated immune response.