R. Gitendra Wickremasinghe

Selective Apoptotic Killing of Malignant Hemopoietic Cells by Antibody-Targeted Delivery of an Amphipathic Peptide

The alpha-helical amphipathic peptide D-(KLAKLAK)2 is toxic to eukaryotic cells if internalized by a suitable targeting mechanism. We have targeted this peptide to malignant hemopoietic cells via conjugation to monoclonal antibodies, which recognize lineage-specific cell surface molecules. An anti-CD19/peptide conjugate efficiently killed 3/3 B lymphoid lines. However, an anti-CD33/peptide conjugate was cytotoxic to only one of three CD33-positive myeloid leukemia lines. The IC50 towards susceptible lines were in the low nanomolar range. Conjugates were highly selective and did not kill cells that did not express the appropriate cell surface cognate of the antibody moiety. Anti-CD19/peptide conjugates efficiently killed cells from patients with chronic lymphocytic leukemia but anti-CD33/peptide reagents were less effective against fresh acute myeloid leukemia cells. We therefore suggest that amphipathic peptides may be of value as targeted therapeutic agents for the treatment of a subset of hematologic malignancies.

Ligation of CD8α on human natural killer cells prevents activation‐induced apoptosis and enhances cytolytic activity

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric α/α form are more cytotoxic than their CD8– counterparts but the mechanisms behind this differential cytolytic activity remained unknown. Target cell lysis by CD8– NK cells is associated with high levels of effector cell apoptosis, which is in contrast to the significantly lower levels found in the CD8α+ cells after lysis of the same targets. We report that cross‐linking of the CD8α chains on NK cells induces rapid rises in intracellular Ca2+ and increased expression of CD69 at the cell surface by initiating the influx of extracellular Ca2+ ions. We demonstrate that secretion of cytolytic enzymes initiates NK‐cell apoptosis from which CD8α+ NK cells are protected by an influx of exogenous calcium following ligation of CD8 on the NK‐cell surface. This ligation is through interaction with fellow NK cells in the cell conjugate and can occur when the target cells lack major histocompatibility complex (MHC) Class I expression. Protection from apoptosis is blocked by preincubation of the NK cells with anti‐MHC Class I antibody. Thus, in contrast to the CD8– subset, CD8α+ NK cells are capable of sequential lysis of multiple target cells.

Okadaic acid-induced apoptosis of HL60 leukemia cells is preceded by destabilization of bcl-2 mRNA and downregulation of bcl-2 protein

We have studied the actions of the protein phosphatase inhibitor okadaic acid (OA) on the expression of bcl‐2 in HL60 human leukemia cells. OA induced downregulation of bcl‐2 mRNA and protein prior to the induction of apoptosis. Downregulation of bcl‐2 mRNA levels did not result from actions of OA on the bcl‐2 upstream negative response element. Nuclear run‐off analyses confirmed that OA did not affect bcl‐2 gene transcription. However, OA caused a rapid increase in the rate of degradation of bcl‐2 mRNA. Therefore, OA induces downregulation of bcl‐2 expression via destabilization of its transcript. The constitutive action of an OA‐sensitive protein phosphatase may therefore maintain HL60 cell survival by blocking bcl‐2 mRNA degradation.

The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment

Extensive evidence suggests that the malignant cells of chronic lymphocytic leukemia (CLL) patients are in close contact with activated T lymphocytes, which secrete a range of cytoprotective cytokines including interleukin-4 (IL-4). IL-4 induced the rapid phosphorylation and activation of the signal transducer and activator of transcription 6 transcription factor in CLL cells in vitro. Longer incubation with IL-4 resulted in up-regulation of the antiapoptotic proteins, Mcl-1 and Bcl-X(L). All of these events were blocked by the JAK3-selective inhibitor, PF-956980. A dye reduction cytotoxicity assay showed that IL-4 induced resistance to the cytotoxic drugs fludarabine and chlorambucil and to the novel p53-elevating agent nutlin 3. IL-4-induced drug resistance was reversed by PF-956980. These conclusions were confirmed by independent assays for apoptosis induction (annexin V binding, cleavage of poly[ADP-ribose] polymerase, and morphologic analysis). Coculture with bone marrow stromal cells in the presence of supernatants derived from activated T-lymphocyte cultures also protected CLL cells from apoptosis induction by chlorambucil. Protection by these combined signals was reversed by PF-956980. The data here provide a preclinical rationale for the possible therapeutic use of PF-956980 in conjunction with conventional cytotoxic drugs to achieve more extensive killing of CLL cells by overcoming antiapoptotic signaling by the microenvironment.

Autologous plasma activates Akt/protein kinase B and enhances basal survival and resistance to DNA damage-induced apoptosis in B-chronic lymphocytic leukaemia cells

We have studied the actions of autologous plasma on both basal and DNA damage‐induced apoptosis in B‐chronic lymphocytic leukaemia (B‐CLL) cells. Apoptosis was quantified using morphological criteria and Western blot analysis for the apoptosis‐specific p85 fragment of poly(ADP ribose) polymerase. Cell viability was estimated using the methyl thiazol tetrazolium bromide dye reduction assay. Plasma cultures showed lower rates of basal apoptosis as well as a decreased cytotoxic response to chlorambucil and γ‐radiation compared with cultures in fetal calf serum. Experiments using neutralizing antibodies suggested that the protective actions of plasma could not be accounted for by interleukin 4, the interferons α or γ or stromal cell‐derived factor 1, each of which have been shown to protect B‐CLL cells from apoptosis in vitro. Plasma addition to B‐CLL cells resulted in rapid activation of the Akt protein kinase, a key signalling enzyme that has been implicated in anti‐apoptotic signalling. LY294002, an inhibitor of phosphatidylinositol 3′‐kinase, blocked Akt activation by plasma. To the best of our knowledge, this is the first report to show that factors present in plasma promote basal survival of B‐CLL cells and resistance to cytotoxic drugs via stimulation of the Akt cytoprotective‐signalling pathway. Pharmacological blockade of this pathway may have potential in the development of novel therapeutic strategies for B‐CLL treatment.

Rapid down-regulation of protein kinase C and membrane association in phorbol ester-treated leukemia cells

Peripheral blood lymphocytes from patients with chronic lymphocytic leukemia (CLL) acquire after several days of exposure to 12‐O‐tetradecanoylphobol‐13‐acetate (TPA) several morphological, immunological and histochemical features of hairy cell leukemia. We have investigated the short term effects of TPA treatment on protein kinase C and its subcellular distribution. Within minutes of addition of TPA to CLL cells 20% of the cytosolic protein kinase C had associated with the particulate fraction. The remaining 80% of protein kinase C activity was down‐regulated. The association with the membrane dramatically increased the resistance of the enzyme to inhibition by the non‐ionic detergent, Triton X‐100. These results suggest that activation of protein kinase C causes multiple biological changes in CLL cells.

Cytotoxic drugs enhance the ex vivo sensitivity of malignant cells from a subset of acute myeloid leukaemia patients to apoptosis induction by tumour necrosis factor receptor-related apoptosis-inducing ligand

Summary. We have studied the actions of tumour‐necrosis‐factor‐related apoptosis‐inducing ligand (TRAIL) on cells isolated from patients with acute myeloid leukaemia (AML). Apoptosis induction was initially assessed by quantitative morphological analysis. Only 2/19 isolates showed a > 10% increase in apoptotic cells following TRAIL treatment. However, incubation with TRAIL combined with fludarabine, cytosine arabinoside or daunorubicin resulted in additive or super‐additive apoptosis induction in approximately half of the isolates. Molecular evidence of super‐additive apoptosis induction by TRAIL and cytotoxic agents was obtained by quantification of caspase 3 activation, detected by Western blot analysis of poly (ADP ribose) polymerase cleavage. The ability of TRAIL and daunorubicin to induce super‐additive apoptosis correlated with the ability of these agents to activate caspase 8 and to augment cellular levels of the truncated pro‐apoptotic form of the BCL‐2 family member BID. Our data suggest that co‐administration of TRAIL with conventional cytotoxic drugs may be of therapeutic value in some patients with AML.

Phytohemagglutinin treatment of T lymphocytes stimulates rapid increases in activity of both particulate and cytosolic protein kinase C

We have measured the activity of protein kinase C in particulate and cytosolic fractions prepared from lymphocytes following stimulation with phytohemagglutinin. Activity in the particulate fraction increased approximately three-fold within 5 min, and declined to nearly zero between 20 and 60 min. Cytosolic activity increased in a biphasic manner, with an initial increase at 5 min, a decline at 10 min, and a further increase by 20 min, which was sustained for at least 60 min. By contrast, 12-O-tetradecanoylphorbol-13-acetate caused a rapid translocation of protein kinase C from cytosol to the particulate fraction which was sustained for at least 1 h. The results suggest that agents, such as phytohemagglutinin, which both generate diacylglycerol and mobilize intracellular Ca2+ stores, result in changes in subcellular distribution and activity of protein kinase C which are different from those elicited by 12-O-tetradecanoylphorbol-13-acetate.

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.

Co-ordinated downregulation of bcl-2 and bax expression during granulocytic and macrophage-like differentiation of the HL60 promyelocytic leukaemia cell line

The bcl‐2 protein suppresses apoptosis and the bax protein opposes the cytoprotective effect of bcl‐2. A decrease in bcl‐2 levels has been implicated in the induction of apoptosis during the terminal differentiation of HL60 myeloid leukaemia cells. We show here that bax protein also declined with a time course similar to the downregulation of bcl‐2 following treatment of HL60 with phorbol myristate acetate (PMA), dimethyl sulphoxide (DMSO) or retinoic acid (RA). Decreased bcl‐2 protein expression in induced cells was associated with down‐regulation of its mRNA. By contrast, the decrease in bax occurred by a post‐transcriptional mechanism. Co‐ordinate downregulation of bcl‐2 and bax proteins may fine‐tune the induction of apoptosis during cellular differentiation.