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Clinical trials using vaccine measles virus (MV) as anticancer therapy are already underway. We compared the oncolytic potential of MV in two B-cell malignancies; adult acute lymphoblastic leukemia (ALL, an aggressive leukemia) and chronic lymphocytic leukemia (CLL, an indolent leukemia overexpressing Bcl-2) using patient-derived material. In vitro, distinct cytopathological effects were observed between MV-infected primary ALL and CLL cells, with large multinucleated syncytia forming in ALL cultures compared to minimal cell-to-cell fusion in infected CLL cells. Cell viability and immunoblotting studies confirmed rapid cell death in MV-infected ALL cultures and slower MV oncolysis of CLL cells. In cell lines, overexpression of Bcl-2 diminished MV-induced cell death providing a possible mechanism for the slower kinetic of MV oncolysis in CLL. In vivo, intratumoral MV treatment of established subcutaneous ALL xenografts had striking antitumor activity leading to complete resolution of all tumors. The antitumor activity of MV was also evident in disseminated ALL xenograft models. In summary, both ALL and CLL are targets for MV-mediated lysis albeit with different kinetics. The marked sensitivity of both primary ALL cells and ALL xenografts to MV oncolysis highlights the tremendous potential of MV as a novel replicating-virus therapy for adult ALL.

Chronic lymphocytic leukaemia (CLL) is the commonest haematological malignancy in the western world and is incurable by cytotoxic therapy. Considerable research effort has identified the signal transduction pathways in CLL cells that contribute to anti-apoptotic signalling. Some pathways are constitutively activated in CLL cells but upregulated in normal cells only when protein tyrosine kinases (PTKs) are activated by ligands. This review describes which PTKs are aberrantly activated in CLL cells and are potential targets for inhibition. Additional potential targets within pathways downstream of these PTKs include Mek/Erk, mTorc1, protein kinase C, PI-3 kinase/Akt, nuclear factor-κB and cyclin-dependent protein kinase. Numerous studies have identified chemical agents and antibodies that selectively kill CLL cells, irrespective of their genetic resistance to conventional chemotherapeutic agents, and which can overcome cytoprotective microenvironmental signalling. These studies have resulted in identification of novel therapies, some of which are currently undergoing clinical trials. In vitro and animal model studies and clinical trials could determine which inhibitors of which targets are the likely to be most effective and least toxic either singly or in combination.

The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin α, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21CIP1. Surprisingly, pifithrin α dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.

It has been previously shown that the subset of human natural killer (NK) cells which express CD8 in a homodimeric alpha/alpha 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 CD8 alpha(+) cells after lysis of the same targets. We report that cross-linking of the CD8 alpha 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 CD8 alpha(+) 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, CD8 alpha(+) NK cells are capable of sequential lysis of multiple target cells.

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.

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 gamma-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 alpha or gamma 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.

G ENETIC CHANGES involving oncogenes and tumor suppressor genes contribute to the deregulated expansion of malignant cells. While some of these changes result in increased proliferation, others contribute to an increase in cell numbers by inhibiting apoptosis (programmed cell death).[1][1] Because

We have investigated the molecular mechanisms underlying the ability of peripheral blood monocytes to block apoptosis induction in endothelial cells. Monocytes stimulated the expression of the bcl-2 homologue A1 in serum-starved endothelial cells after 6 h of coincubation, with elevated A1 levels persisting for up to 21 h. IL-1 and TNF also stimulated A1 expression at 6 h, but A1 transcript levels fell by 21 h. Direct cellular contact with monocytes was required for stimulation of A1 mRNA in endothelial cells. Stimulation of endothelial cell A1 mRNA by monocytes was not inhibited by anti-β2 integrin Abs, but anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) mAb reduced A1 transcript levels at 21 h. Studies employing either TNF on its own, or anti-TNF in endothelium/monocyte cocultures showed that TNF plays a role in the early (6-h) stimulation of A1, but is less important for the sustained elevation of A1 levels at 21 h. Serum-starved endothelial cells demonstrated increased survival and decreased apoptosis after coculture with monocytes. IL-10 reduced A1 mRNA expression in, as well as survival of, endothelial cells that were cocultured with monocytes. In comparison with A1, Bcl-2 was expressed at low levels and was up-regulated by monocytes only at 21 h, while neither Bax nor Bcl-xL levels were altered by monocytes. The interaction of monocytes with endothelium during the course of an inflammatory reaction may provide survival signals to endothelial cells.

Mitogenic stimulation of lymphocytes from persons infected with human immunodeficiency virus (HIV) resulted in massive cell death. In addition to early apoptosis, a second wave of cell death occurred 48-72 h after stimulation. At that time, the cells were enlarged, leaked content, and had plasma membrane damage-all indicative of necrosis. Furthermore, DNA fragmentation as determined by TUNEL assay was virtually absent. This activation-associated necrosis could not be prevented by interfering with CD95/CD95-ligand interactions or by blocking caspase activity and was unaffected by neutralizing antibodies to tumor necrosis factor-alpha or interferon-gamma. Necrosis was also induced by activation of normal lymphocytes in the presence of ribavirin, which inhibits the de novo pathway of nucleotide synthesis. Lymphocytes from HIV-infected persons are defective in their ability to synthesize nucleotides via this pathway, indicating one possible mechanism for the activation-associated necrosis seen in HIV infection.

Philadelphia chromosome (Ph)-positive leukaemia cells express the chimeric bcr/abl oncoprotein, whose deregulated protein tyrosine kinase (PTK) activity antagonizes the induction of apoptosis by DNA damaging agents, Treatment of Ph-positive K562, TOM 1 and KCL-22 cells with etoposide for 2d induced cytosolic vacuolation, but not nuclear condensation or DNA fragmentation, The bcr/abl kinase-selective inhibitor herbimycin A increased the induction of nuclear apoptosis by etoposide or gamma-radiation. The concentration of herbimycin required to synergize with etoposide was similar to that required to decrease the level of tyrosine phosphorylated proteins or of the protein tyrosine kinase activity of anti-abl immune complexes in K562 cells, The ability of herbimycin A to sensitize K562, TOM 1 or KCL-22 cells to apoptosis induction correlated with its ability to decrease the cellular content of phosphotyrosyl proteins in these Philadelphia-positive lines. Enhancement of nuclear apoptosis by herbimycin was not attributable to downregulation of the bcl-2 or bcl-X-L anti-apoptotic proteins, In contrast, herbimycin protected Philadelphia-negative HL60 cells from apoptosis induction by etoposide and did not affect killing of NC37 and CEM cells, The data suggest that the induction of apoptosis is blocked in cells expressing the bcr/abl oncoprotein and that herbimycin A increases induction of programmed cell death following DNA damage, Selective PTK inhibitors may therefore be of value in securing the genetic death of Ph-positive leukaemia cells.

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.

Cytokine deprivation from activated T cells leads to apoptosis associated with down-regulation of the bcl-2 gene product. It is not clear, however, how cytokines other than interleukin-2 (IL-2) may affect this process and regulate the involvement of other apoptosis-modulating genes. We show that a group of cytokines including IL-2 (IL-2R gamma), prevent the apoptosis of IL-2-deprived activated T cells. This rescue involves the induction of the anti-apoptosis genes bcl-2 and bcl-xL), but causes little change in expression of bax and bcl-xS, which promote apoptosis. Furthermore, the prevention of apoptosis and induction of proliferation by the common gamma chain cytokines can be dissociated. Thus, when proliferation is blocked, the common gamma chain cytokines still induce up-regulation of bcl-2 relative to bax and retard apoptosis. These cytokines can thus regulate the persistence or removal of effector T cells by coordinating the balance between genes which promote and those which inhibit apoptosis, events which are probably mediated at least in part by signals through the common gamma chain. These data also implicate inappropriate T cell apoptosis resulting from a dysfunctional common gamma-chain as part of the pathophysiological defect in patients with X-linked severe-combined immunodeficiency (SCID).

Malignant B lymphocytes from patients with B-chronic lymphocytic leukaemia (B-CLL) or hairy cell leukaemia (HCL) are refractory in vitro to mitogenic stimulation by several agents which trigger proliferation of normal B cells. Tumour necrosis factor (TNF) is a growth factor for these malignant cells, although the proliferative response is usually small. TNF regulates some of its cellular responses via induction of the transcription factors NF kappa B and AP-1 (jun/ fos). The induction of NF kappa B by TNF is mediated via a novel signalling pathway involving the generation of reactive oxidative intermediates. Induction of jun and fos proteins (polypeptide components of AP-1) are mediated via pathways involving protein kinase C and the protein kinase encoded by the raf proto-oncogene. Here we have used an electrophoretic mobility shift assay to show that TNF induced NF kappa B in malignant cells isolated from 3/3 HCL and 15/15 B-CLL patients. By contrast, phorbol myristate acetate (PMA), a direct activator of protein kinase C, failed to activate this transcription factor in 1/1 HCL and 5/5 B-CLL isolates. The induction of jun and fos proteins (as detected by Western blot analysis) showed greater heterogeneity. Nuclear jun was induced by TNF in 5/12 chronic B cell leukaemia isolates. PMA induced this protein in 4/5 samples. Nuclear fos was induced by TNF in only 2/12 isolates and by PMA in 2/5. The data suggest that the pathways for the activation of jun and fos by TNF are defective in some B-CLL and HCL cells and that these defects may be heterogeneous. The induction of AP-1 is crucial in securing the mitogenic response to TNF. It is therefore plausible that these lesions may contribute to the refractory nature of B-CLL and HCL cells to proliferative stimuli in vitro.

Diacylglycerol is a potent second messenger which is generated via the cleavage of inositolor choline-containing phospholipids and is involved in the transduction of proliferative signals. We have previously obtained evidence that the constitutive breakdown of inositol lipids may contribute to signalling the continuous proliferation of HL60 leukaemia cells (Porfiri E., Hoffbrand A. V. & Wickremasinghe R. G. (1991) Blood 78, 1069–1077). In order to assess the role of choline lipids as potential sources of growth-regulating second messengers, we have studied the pathways of constitutive breakdown of radiolabelled phosphatidylcholine in intact HL60 cells. Neither exponentially growing HL60 cells nor HL60 cells which had been induced to cease proliferation by treatment with dimethylsulphoxide degraded choline lipids via phospholipase C- or phospholipase D-catalysed pathways. Both pathways were, however, activated by phorbol myristate acetate irrespective of proliferation status. The data here suggest that, unlike inositol lipids, choline lipids are not a source of second messenger molecules with potential roles in the regulation of HL60 cell proliferation.

The proliferation of malignant hemopoietic cell lines is inhibited by antagonists of 5-lipoxygenase, suggesting that the endogenous generation of leukotrienes via the action of this enzyme may play some role in the proliferation of these cells (Snyder D. S., Castro R. & Desforges J. F. (1989), Expl Hemat.17, 6). Here we have confirmed that the lipoxygenase inhibitors piriprost, nordihydroguiaretic acid and BW755C decreased DNA synthesis and proliferation of leukemic cell lines. However, the concentrations of these drugs required for half-maximal inhibition of proliferation were significantly greater than their ic 50 values for 5-lipoxygenase inhibition. We therefore studied the actions of two novel, potent lipoxygenase inhibitors, BWA4C and MK886, on proliferation (as measured by estimating the number of viable, trypan blue-excluding cells) and DNA synthesis (measured by the incorporation of radiolabeled thymidine) in the leukemia cell lines HL60, K562 and Jurkat. Neither parameter was affected by concentrations of these drugs which were shown in parallel studies to substantially inhibit leukotriene generation in whole blood. The data show that endogenous leukotriene generation does not play a significant role in the regulation of proliferation of these leukemic cell lines and suggest that conclusions about leukotriene involvement in the control of cellular metabolic pathways based on the use of lipoxygenase inhibitors should be re-assessed.

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-XL. 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.

We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 μM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.

The α-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.

We studied the actions of geldanamycin (GA) and herbimycin A (HMA), inhibitors of the chaperone proteins Hsp90 and GRP94, on B chronic lymphocytic leukemia (CLL) cells in vitro. Both drugs induced apoptosis of the majority of CLL isolates studied. Whereas exposure to 4-hour pulses of 30 to 100 nM GA killed normal B lymphocytes and CLL cells with similar dose responses, T lymphocytes from healthy donors as well as those present in the CLL isolates were relatively resistant. GA, but not HMA, showed a modest cytoprotective effect toward CD34+ hematopoietic progenitors from normal bone marrow. The ability of bone marrow progenitors to form hematopoietic colonies was unaffected by pulse exposures to GA. Both GA and HMA synergized with chlorambucil and fludarabine in killing a subset of CLL isolates. GA- and HMA-induced apoptosis was preceded by the up-regulation of the stress-responsive chaperones Hsp70 and BiP. Both ansamycins also resulted in down-regulation of Akt protein kinase, a modulator of cell survival. The relative resistance of T lymphocytes and of CD34+ bone marrow progenitors to GA coupled with its ability to induce apoptosis following brief exposures and to synergize with cytotoxic drugs warrant further investigation of ansamycins as potential therapeutic agents in CLL.

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 a10% 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.

Ligation of the cell-surface Fas molecule by its ligand (Fas-L) or agonistic anti-Fas monoclonal antibodies results in the cleavage and activation of the cysteine protease procaspase 8 followed by the activation of procaspase 3 and by apoptosis. In some leukemia cell lines, cytotoxic drugs induce expression of Fas-L, which may contribute to cell killing through the ligation of Fas. The involvement of Fas, Fas-L, and caspase 8 was studied in the killing of B-cell chronic lymphocytic leukemia (B-CLL) cells by chlorambucil, fludarabine, or γ radiation. Spontaneous apoptosis was observed at 24-hour incubation, with additional apoptosis induced by each of the cytotoxic treatments. Although Fas mRNA expression was elevated after exposure to chlorambucil, fludarabine, or γ radiation, Fas protein levels only increased after irradiation. Therefore, Fas expression may be regulated by multiple mechanisms that allow the translation of Fas mRNA only in response to restricted cytotoxic stimuli. None of the cytotoxic stimuli studied here induced Fas-L expression. An agonistic anti-Fas monoclonal antibody (CH-11) did not significantly augment apoptosis induction by any of the death stimuli. A Fas-blocking antibody (ZB4) did not inhibit spontaneous, chlorambucil-, fludarabine-, or radiation-induced apoptosis. However, procaspase 8 processing was induced by all cytotoxic stimuli. These data suggest that the Fas/Fas-L signaling system does not play a major role in the induction of apoptosis in B-CLL cells treated with cytotoxic drugs or radiation. However, Fas-independent activation of caspase 8 may play a crucial role in the regulation of apoptosis in these cells.

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.

Abstract 2906 Chronic Lymphocytic leukemia (CLL) is currently incurable using conventional therapies. CLL cells can evade killing by various therapeutic strategies. However the precise mechanisms are currently unknown. Autophagy is regulated by a complex system of proteins, and is used by both normal and malignant cells as a protective mechanism against cellular stress induced by starvation, hypoxia, reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress. In malignant cells autophagy was shown to promote tumorigenesis and/or resistance to chemotherapy. Therefore we hypothesized that autophagy may play a role in CLL biology. Autophagy can also promote cell death when stress signals are elevated above a particular threshold for a prolonged period of time. In this study we investigated the basal expression levels of autophagy specific genes and the effect of autophagy specific inhibitors (Bafilomycin, 3-methyladenine and hydroxychloroquine) and inducers (Phenethyl isothiocyanate) on CLL survival. Phenethyl isothiocyanate (PEITC) is about to enter clinical trials for CLL (NCT00968461). We have investigated induction of components of the autophagic pathway following treatment of CLL cells in vitro with a range of chemical inhibitors. Immunoblotting was carried out to investigate components of the autophagy pathway using phosphorylation state-specific and pan-reactive antibodies. Bafilomycin (BAF), 3-methyladenine (3-MA) and hydroxychloroquine (HCQ) toxicity towards CLL samples were evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form. PEITC was used at concentrations between 2.5 and 25μM to investigate its effect on signaling. Autophagy was quantitated by immunoblotting of LC3-I and LC3-II. Lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy and is essential for autophagasome formation. Immunoblotting was also performed for ATG3, ATG5 and ATG7, key components of the autophagy pathway. Monodansylcadaverine (MDC) was used with immunofluorescence and FACS analysis to investigate increases in autophagasome formation. Transmission electron microscopy (TEM) was used to confirm double membrane bound autophagosomes. Co-immunoprecipitation was used to evaluate if Beclin-1 was sequestered by Bcl-2 preventing autophagy. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation. LC3-I was lipidated to LC3-II (p=0.019) and ATG3 (p=0.021) was upregulated to a greater extent in CLL samples compared with normal B-cell controls at basal levels. This suggested that autophagy was active to a greater extent in CLL samples compared with normal individuals. In addition Beclin was dissociated from Bcl-2 in CLL samples indicating that autophagy was active. Autophagy appears to be a pro-survival mechanism in untreated CLL cells as inhibiting basal levels of autophagy with autophagy inhibitors BAF (50–200nM), 3-MA (5–10mM) and hydroxychlorquine (5–10μM) resulted in CLL apoptosis as shown by MTT, Annexin V/PI analysis and PARP cleavage. Interestingly augmenting autophagy was also capable of inducing apoptosis in CLL samples. Treatment with PEITC caused an increase in punctate staining using MDC which is suggestive of autophagosome formation. We went on to determine that PEITC further induced LC3-II lipidation using immunoblotting and showed a substantial increase in overall LC3 protein expression. PEITC also induced the expression of ATG3, a key protein in the autophagy pathway. We then evaluated autophagosome formation using TEM (Figure 1). Our data showed greater numbers of autophagosomes in the PEITC treated samples compared to the untreated controls. Therefore autophagy in CLL sits on a knife-edge, such that perturbations that either increase pro- death or decrease pro-survival autophagy signals can result in CLL cell death, depending on the duration and intensity of the signal. Figure 1. Transmission electron microscopy of CLL cells CLL cells were treated with 10μM PEITC. Double membrane bound organelles were found in the CLL cells after treatment which were not present in the no addition control (depicted by the arrows). These organelles are autophagsomes. Magnification (left picture) ruler is 500nM, (right picture) ruler is 100nM Figure 1. Transmission electron microscopy of CLL cells . / CLL cells were treated with 10μM PEITC. Double membrane bound organelles were found in the CLL cells after treatment which were not present in the no addition control (depicted by the arrows). These organelles are autophagsomes. Magnification (left picture) ruler is 500nM, (right picture) ruler is 100nM Disclosures: No relevant conflicts of interest to declare.

2492 Chronic Lymphocytic leukaemia (CLL) is the most common leukemia in the western world but is currently incurable using conventional therapies. Therefore novel agents which can kill CLL cells either alone or in combination with conventional agents are being continually sought. Activation of the MAPK pathway has been implicated as one of the protective signaling pathway in CLL and is characterised by expression of phosphorylated ERK. In CLL cells ERK can be activated by phosphorylation after BCR activation with αIgM. However in a proportion of CLL isolates ERK is constitutively active. MEK inhibitors (MEKi) are currently being used in clinical trials to treat relapsed or refractory leukemia including CLL (www.clinicaltrials.gov, NCT00920140). Therefore we hypothesised that inhibiting the ribosomal S6 kinase p90RSK which is immediately downstream of the MAPK pathway, may be of therapeutic value for the treatment of CLL. Since p90RSK regulates the majority of the anti-apoptotic signaling of the ERK pathway, we investigated the effect of inhibiting p90RSK with its specific inhibitor BI-D1870 (BI-D) on apoptosis in CLL. Immunoblotting was carried out to investigate phosphorylation of components of the ERK/p90RSK pathway using phosphorylation state-specific and pan-reactive antibodies. BI-D toxicity was evaluated by Annexin V/PI staining, MTT assay and immunoblotting for cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP) from its 116KDa to its 85KDa form. BI-D treatment of CLL cells decreased S6 phosphorylation, and this down regulation correlated with apoptosis. BI-D killed CLL cells rapidly, with a mean IC50 of 12μM following 18h incubation. Treatment of CLL cells with BI-D induced cytochrome C release indicating inhibition of p90RSK induced apoptosis by the intrinsic death pathway. However its toxicity was not due to inhibition of protein translation since inhibition of mTOR, a parallel pathway which also regulates S6 phosphorylation, was minimally toxic to CLL cells at concentrations as high as 500nM. However apoptosis did coincide with a reduction of the antiapoptotic protein Mcl-1. We further demonstrated that BI-D augmented apoptosis induction by cytotoxic drugs, as shown by PARP cleavage and by annexin V/PI binding assays. Analysis of cytotoxicity data using CalcuSyn software provided evidence for synergistic killing of the majority of CLL isolates by combinations of BI-D with either chlorambucil, fludarabine or nutlin 3. Constitutive or antigen induced activation of p90RSK via the MAPK pathway in CLL cells may contribute to apoptosis resistance and its inhibition may be of value in devising novel therapeutic strategies, either alone or in conjunction with conventional cytotoxic drugs. p90RSK inhibition led to apoptosis independently of protein translation inhibition and may be attributable in part to downregulation of Mcl-1. Given the recent interest in using MEK inhibitors (MEKi) for the treatment of CLL, these data suggests MEKi may induce apoptosis in CLL in part by inhibiting pathways downstream of p90RSK. In addition BI-D is a highly specific inhibitor of p90RSK and therefore may negate the off target effects shown with some MEKi. This is the first report on the consequences of inhibition of p90RSK in CLL cells and suggests that inhibiting p90RSK may be of considerable interest for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

Background: Macroautophagy (autophagy) has been shown to be both a pro-survival and pro-death signalling pathway. The decision between life or death is dependent on the type and developmental stage of the cell and the duration and intensity of the signal. Autophagy is used by cancer cells as a mechanism to overcome cellular stresses such as starvation, reactive oxygen species (ROS) generation and misfolded protein accumulation and can result in tumorigenesis and resistance to chemotherapy. One of the pathways which regulate responses to ROS/ER stress is the unfolded protein response (UPR). Briefly, upon ER stress, the HSP70 family member BiP is sequestered away from IRE1 , PERK and ATF6, resulting in IRE1 and PERK phosphorylation and activation of downstream signalling pathways. These signals can result in cell survival by upregulating AKT or cell death via induction of CHOP and noxa. However this pathway has not been fully investigated in chronic lymphocytic leukaemia (CLL). Interestingly the UPR is also known to regulate autophagy. The lipidation of LC3 from LC3-I to LC3-II is a surrogate marker of autophagy, since LC3-II is essential for formation of the autophagasome and the initiation of autophagy. Beclin-1 is another essential protein involved in canonical autophagy and is sequestered by Bcl-2 preventing autophagy in a number of cell types. Its release from Bcl-2 enables Beclin-1 to interact with other autophagy specific proteins and initiates autophagasome formation. Methods: Twenty CLL samples were treated with the HSP70 inhibitor 2-phenylacetylenesulphonamide (PAS; 10-20 M), an activator of the UPR, in the presence of the pan-caspase inhibitor ZVAD. In addition 4 control samples (normal B cells) were treated with PAS for the indicated times. Immunoblotting was performed for BiP, HSP70, Phosphorylated eIF2 (eIF2 -P), JNK (JNK-P), Jun (Jun-P), p38MAPK, LC3-I/II, Beclin expression and its co-immunoprecipitation with Bcl-2. Ten CLL cell isolates were treated with 2 autophagy inhibitors bafilomycin (Baf) (50-200 nM) and 3-methyl adenine (3-MA) (2.5-10 mM) and their effects on cell killing evaluated using PARP cleavage, a surrogate marker of caspase 3 activation. Results: CLL cells expressed a greater basal level of LC3-II in a proportion of samples relative to normal B cells. In addition Beclin-1 did not coimmunoprecipitate with Bcl-2 above that shown with the isotype control in CLL cells. Treatment with PAS resulted in an increase in stress which resulted in the upregulation of BiP and HSP70 protein. This subsequently led to phosphorylation of eIF2 indicative of PERK activation, coincidental with an increase in the eIF2 targets LC3-I/II and noxa expression. Treatment with PAS also resulted in an increase in JNK, Jun and p38MAPK phosphorylation, 3 proteins involved in regulating autophagy in mammalian cells. Furthermore, CLL cells treated with the autophagy inhibitors 3-MA and Baf rapidly underwent apoptosis within 24 h. Conclusion: These data suggest that autophagy is active at a basal level in a proportion of CLL cells and is greater than that seen in control B cells. Activation of the UPR with PAS led to an upregulation of proteins known to regulate autophagy and resulted in an increase in LC3-II, a protein routinely used as an indicator of autophagy. Inhibitors of autophagy also rapidly induced apoptosis in CLL cells. These data suggested that autophagy may have a pro-survival role in the biology of CLL. However pro-survival autophagy is balanced on a knife-edge, such that perturbation by inducing or inhibiting the pathway results in CLL cell death.

Abstract 2460 Poster Board II-437 Replicating viruses that selectively lyse transformed cells are promising agents for cancer therapy. Attenuated measles virus (MV) has particular tropism for B cells and is oncolytic in murine models of myeloma and lymphoma. These results have led to phase 1 studies in myeloma. Here we investigated the anti-tumour potential of MV in adult B lineage acute lymphoblastic leukaemia (ALL) and Chronic lymphocytic leukaemia (CLL). GFP expressing attenuated MV derived from the Edmonston vaccine lineage (MV-Edm) was used to infect ALL (n = 6) or chronic lymphocytic leukaemia (CLL, n = 7) primary specimens. All CLL and ALL cells expressed the MV receptor CD46. The second MV receptor SLAM was consistently expressed in all the CLL cells but only in 5/6 of the ALL cells. Both ALL and CLL cells were efficiently infected by MV-Edm as indicated by quantitation of viral nucleocapsid mRNA by RQ-PCR and immunoblotting of viral proteins N,H and F. Large multinucleated syncytia, characteristic of MV- induced cytopathology, were found in all infected ALL cultures (Figure 1), by contrast syncitium formation was less prominent in the infected CLL specimens. Despite this, both CLL and ALL cells were efficiently killed by MV-Edm, as characterised by FACS and trypan blue based assays as well as immunoblotting for PARP cleavage. Specificity of lysis for tumor cells was confrmed by infection of normal mononuclear cell controls, which showed minimal syncitium formation and death even after prolonged infection. To investigate the contribution of cell-to-cell fusion in the pathogenesis of MV-induced oncolysis we investigated a relatively non-fusogenic MV:MV-Moraten in our models. Surprisingly, both CLL and ALL cultures infected with MV-Moraten demonstrated a reduction in cell viability compared to uninfected controls despite the absence of typical features of MV cytopathology. Our data suggest that both ALL and CLL are targets for MV-mediated lysis and that cell-cell- fusion might not be an important determinant of this effect in-vitro. Ongoing in vivo studies of MV oncolysis in B-cell malignancy should further inform the therapeutic potential of vaccine MV for these neoplasms. Figure 1 MV-Edm infection results in prominent syncytia formation in primary ALL cells (A and B). Figure 1. MV-Edm infection results in prominent syncytia formation in primary ALL cells (A and B). Disclosures: No relevant conflicts of interest to declare.

p56 lck is a well-characterized tyrosine protein kinase (TPK) which is thought to play a role in mitogenic signal transduction in T lymphocytes. Immunoblot analysis of human lymphocyte proteins using an antiserum cross-reactive with phosphotyrosine resulted in the detection of a 55-60 kDa protein band (presumably p56 lck) as well as several additional phosphotyrosyl proteins in lymphocyte extracts. All of these phosphotyrosyl proteins were down-regulated following mitotic stimulation. Autophosphorylation of lymphocyte microsomal fractions in the presence of [gamma-32P] ATP resulted in the labelling of p56 lck as well as other proteins of different molecular weights. Analysis of these labelled proteins by tryptic digestion resulted in strikingly similar peptide maps. The data suggest that lymphocytes may contain a family of TPKs structurally related to p56 lck. The down-regulation of the putative TPKs following mitogenic stimulation of lymphocytes with phytohaemagglutinin suggests that this family of TPKs may participate in mitotic signalling events, followed by their down-regulation.

The p53 protein plays a key role in triggering DNA damage-induced apoptosis. The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins Puma and Noxa. However, recent studies identified a non-transcriptional mechanism, involving direct association of p53 with mitochondrial anti-apoptotic proteins Bcl-2 or Bcl-XL. We studied the relative contributions of transcription-dependent and -independent mechanisms of p53-mediated apoptosis in CLL cells in vitro. The cytotoxic drugs chlorambucil (chl) and fludarabine (flu) were used to induce p53, as was nutlin 3a, which augments p53 levels by inhibiting interaction with its negative regulator MDM2. Using differential detergent fractionation to isolate cytosolic (cyt), mitochondrial (mt) and nuclear (nuc) fractions, we found that a significant proportion (>50%) of the induced p53 was stably associated with mitochondria in cells treated with nutlin 3a (Fig 1), chl or flu. Co-immunoprecipitation studies established that p53 bound to Bcl-2. PFTα, an inhibitor of p53-mediated transcription, blocked upregulation of the known p53 targets p21CIP1, MDM2 and Puma. Surprisingly, apoptosis induction by nutlin 3a (Figure 2), chl or flu, quantified by western blot analysis of cleavage of poly(ADP ribose) polymerase (PARP), was augmented by PFTα. This observation was confirmed by morphological analysis of apoptosis. Cytochrome c release from mitochondria following p53 induction was also enhanced by PFTα. PFTα did not augment nutlin-, chl- or flu-induced killing of CLL cells lacking functional p53. Furthermore, PFTα did not augment killing by the p53-independent drug parthenolide and failed to increase apoptosis of normal T lymphocytes treated with chl or nutlin 3a. Our observations suggest that p53 induces apoptosis of CLL cells principally via its transcription-independent function, involving direct association with mitochondrial Bcl-2. p53’s transcriptional function apparently blocks apoptosis at a point between p53 association with Bcl-2 and subsequent release of cytochrome c. Therefore, therapeutic strategies aimed at blocking p53-mediated transcription may be of value in enhancing the action of agents which induce apoptosis of CLL cells via p53 upregulation. Figure 1 Figure 1. Figure 2 Figure 2.

The classical pathway of p53-mediated apoptosis involves transcriptional upregulation of pro-apoptotic proteins including Puma and Noxa. However, recent studies have identified a novel non-transcriptional mechanism mediated by direct binding of p53 to Bcl-2 or Bcl-XL at the mitochondrial surface and consequent neutralization of their anti-apoptotic function (Chipuk and Green, Cell Cycle, 2004; 3: 429–431). Induction of apoptosis of chronic lymphocytic leukaemia (CLL) cells by cytotoxic agents is strongly dependent on a functional p53 system and genetic changes which compromise p53 function result in drug resistance. We are therefore attempting to identify novel therapeutic agents which induce selective apoptosis of CLL cells by mechanisms independent of p53. Here we present data suggesting that 2-phenylacetylenesulfonamide (PAS; trivial name pifithrin μ) is a potential therapeutic agent for CLL. PAS was toxic to CLL cells, with a median LC50 of 10.72 μM (SD 3.8 μM n=20) as measured by a dye reduction cytotoxicity assay. Killing of CLL cells was by apoptosis, documented by the ability of 10–20 μM PAS to induce cytochrome c release from mitochondria (Fn2) to the cytosol (Fn1; Figure 1, upper), conformational change of Bax to a pro-apoptotic conformation, translocation of Bax from the cytosol to mitochondria (Figure 1, lower) and cleavage of the caspase 3 substrate poly(ADP ribose) polymerase (PARP). PAS toxicity was p53-independent, since p53-deleted CLL isolates were susceptible to killing. Furthermore, PAS was toxic towards leukemic cell lines with either inducible (Daudi), non-functional (Raji) or deleted (K562) p53. PAS treatment of CLL cells resulted in upregulation of Noxa and the subsequent displacement by Noxa of the pro-apoptotic Bim long (L) and extra-long (EL) isoforms from Bcl-2, as determined by western blot analysis of Bcl-2 immunoprecipitates (Figure 2). These observations contrast with those of Strom et al (Nat Chem Biol, 2006; 2: 474–479), who reported that PAS caused the displacement of p53 from mitochondria to the cytosol and thereby protected the cells from p53-dependent apoptosis. These differences may result from ectopic viral expression of p53 in the latter study whereas this protein was under normal cellular regulation in CLL cells. Normal T lymphocytes were relatively resistant to PAS, with IC50 values four times greater than those observed for CLL cells. In conclusion, our data suggest that PAS represents a novel class of agent which is toxic towards CLL cells via a mechanism involving p53-independent upregulation of Noxa and the subsequent unleashing of pro-apoptotic Bim from anti-apoptotic Bcl-2. This p53-independent toxicity may be of particular value in the treatment of drug-resistant CLL patients with compromised p53 pathways. Figure 1. Figure 1. Figure 2. Figure 2.

B-chronic lymphocytic leukaemia (CLL) is the most frequent leukaemia in the western world. CLL is incurable using conventional therapy. While the disease is controlled by treatment with chlorambucil (CHL) or fludarabine, extended exposure to these agents results in drug resistance. It is therefore important to identify novel agents which rapidly and selectively induce apoptosis of cells isolated from patients who have become resistant to conventional drugs. The sesquiterpene lactone parthenolide (PTL) is the active component of several medicinal plants which have been used for hundreds of years for the treatment of migraines, pain and inflammatory conditions. In some cell lines, PTL has been shown to block components of the NFκB pathway including the IκB kinase complex and/or the p65 subunit of NFκB. Here we investigated the sensitivity of isolates from 25 CLL patients (representing stages A, B and C) to PTL (1.25–80 μM) and CHL (3.125–200μM). IC50 values were evaluated by the 4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) dye reduction assay. Apoptosis induction was quantified by FACScan analysis of annexin V binding and by western blot detection of cleavage of the caspase 3 substrate poly (ADP ribose) polymerase (PARP). CHL resistance varied across the cohort from highly sensitive to highly resistant, with IC50 values in the range 5.3 to >200 μM. In contrast, all isolates tested were sensitive to PTL, with IC50 values in the range 3 to 20 μM. Strikingly, isolates from extensively-treated patients which were highly resistant to elevated levels of CHL in vitro were sensitive to PTL (Figure 1). PTL induced very rapid PARP cleavage which became clearly evident between 3–6 h of exposure to 5 μM PTL. In contrast, PARP cleavage was not detected until >12h exposure to 75 μM CHL in isolates which were sensitive to this drug. Furthermore, pulse/wash-out experiments showed that a very brief (1-2h) exposure to PTL was sufficient to induce commitment to the apoptotic programme. The annexin V-binding assay revealed that CLL cells were strikingly more sensitive to apoptosis induction by 2–5 μM PTL than were normal T or B lymphocytes or CD34+ bone marrow progenitor cells (Figure 2). Western blot analysis revealed that PTL-induced PARP cleavage was preceded by downregulation of anti-apoptotic mcl-1, a key mediator of the survival of B lineage lymphoid cells. However, we failed to detect a PTL-induced decrease in IκBαphosphorylation, suggesting that inhibition of IκB kinase did not contribute to the mechanism of CLL cell killing. In conclusion, the rapid, selective action of PTL and its ability to kill CLL isolates refractory to cytotoxic drugs suggest that this agent may be of value in the treatment of CLL patients who have become resistant to conventional therapeutic regimes. Download : Download high-res image (138KB) Download : Download full-size image Figure

Piriprost and nordihydroguiaretic acid (NDGA), specific inhibitors of arachidonate lipoxygenase, inhibited phytohaemagglutinin (PHA)-stimulated breakdown of inositol lipids in human T lymphocytes. The dual inhibitors eicosatetraynoic acid (ETYA) and BW 755C, which inhibit both lipoxygenase and cyclooxygenase, also had similar actions, whereas indomethacin and acetylsalicyclic acid, which inhibit cyclooxygenase alone, did not. The effects of lipoxygenase inhibitors and dual inhibitors were reversible. These agents did not inhibit phosphatidylinositol-4,5-bisphosphate-specific phospholipase C (PIP2-PLC) in vitro. Bromophenacyl bromide, and irreversible inhibitor of phospholipase A2, also abolished PHA-stimulated inositol lipid breakdown without affecting PIP2-PLC in vitro. The results are consistent with a role for the PHA-stimulated generation of arachidonic acid and its conversion to lipoxygenase metabolites (e.g. leukotrienes and/or hydroxyeicosatetraenoic acids) as intermediate steps in the signal transduction pathway between cell-surface mitogen receptors and the stimulation of PIP2-PLC in lymphocytes.

1. Autoradiographic experiments revealed that the average size of the replicating unit (replicon) in human phytohaemagglutunin-stimulated lymphocytes is 45 (+/- 1.3) micron. 2. A 5 min pulse of [3H]thymidine labelled DNA chains of approximately 40 S (15 micron) in control lymphocytes as revealed by velocity sedimentation in alkaline sucrose density gradients. Upon chasing in the absence of [3H]-thymidine the labelled DNA increased in size. By 6 h the bulk of the label co-sedimented with full-sized chromosomal DNA. 3. In untreated lymphocytes from patients with megaloblastic anaemia due to vitamin B-12 or folate deficiency or lymphocytes treated with methotrexate (10(-5) M) or hydroxyurea (5 . 10(-4) M) the increase in size of pulse-labelled DNA was slower than in control cells. 4. The block in maturation of pulse-labelled DNA to bulk DNA was not permanent. At 24 h of chase 75-80% of the pulse-label in both control and megaloblastic lymphocytes co-sedimented with bulk DNA. 5. We conclude that the lesions seen in DNA synthesis in megaloblastic anaemia due to folate or vitamin B-12 deficiencies occur through impaired biosynthesis of nucleotide precursors of DNA. Possible explanations of why the defects in DNA synthesis cause altered morphology of proliferating cells in megaloblastic anaemia are suggested.

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 Ca 2+ 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.

Human lymphocyte tyrosine protein kinases (TPKs) have been analyzed by gel-filtration chromatography. The major TPK species with activity towards an exogenous tyrosine-containing peptide had molecular masses of 70–100 kDa (TPK I) and 35–40 kDa (TPK II). TPKs I and II were distinct from the well-characterized autophosphorylating lymphoid cell TPK, pp56lck [(1983) J. Biol. Chem. 258, 10738–107421]. Both TPK I and TPK II were down-regulated following mitogenic stimulation of lymphocytes with phytohae-magglutinin. By contrast, pp56lck remained clearly detectable in stimulated lymphocytes. We suggest that TPKs I and II may play a role in the regulation of the lymphocyte cell cycle.

1. Gently lysed human lymphoblastoid cells (HPB-ALL) catalysed incorporation of the distal DNA precursors [3H]thymidine, [3H]dTMP, and [3H]dUMP into DNA. Measurement of the [3H]dTTP, produced during the incorporation reactions, provided kinetic evidence for the channelling of the distal precursors into DNA by a multienzyme complex consisting of precursor-synthesizing enzymes and of DNA polymerase. 2. In the presence of the DNA polymerase inhibitor, 1-beta-D-arabinofuranosylcytosine triphosphate, incorporation of [3H]dTMP into DNA was abolished. However, there was no accumulation of [3H]dTTP, suggesting that the flow of substrates through the complex was regulated by the activity of DNA polymerase. 3. Multienzyme complexes were detected by chromatography of a lysate on a column of Sepharose 6B. These complexes retained the ability to channel [3H]thymidine and [3H]dTMP into DNA.

A method is described for the preparation of DNA polymerase purified about 800-fold from rat liver nuclei. The yield of enzyme is about 140–200 μg from 200 g liver. Sodium dodecylsulphate-polyacrylamide gel electrophoresis of the enzyme in the final step, shows a main band corresponding to a polypeptide of molecular weight of 29000 ± 3%. Sephadex G-100 column chromatography indicates the enzyme to have an apparent molecular weight of approximately 60000 ± 2% at an ionic strength of 0.15, suggesting that the enzyme is a dimer as isolated. In 2 M NaCl, the apparent molecular weight is 42000. The enzyme prefers double-stranded DNA templates but utilises most efficiently those activated by deoxyribonuclease I. It has the ability to carry out limited synthesis using only one deoxynucleoside-5′-triphosphate in the assay. The final preparation of DNA polymerase has nucleoside diphosphate kinase associated with it.

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.

The cell-surface receptor for interleukin-2 (IL-2) consists of two unlinked polypeptides of 55 and 75 kDa (p55, p75). The monoclonal antibody antiTac binds to p55 alone. We show here that the binding of either IL-2 or antiTac to the surface of T lymphocytes triggered the generation of cAMP. Reagents which activate adenyl cyclase by stimulation of its guanine nucleotide-binding protein (Gs) also stimulated increases in cAMP. All of the above reagents, and cAMP itself, stimulated the turnover of phosphate residues bound to serine and threonine residues of an 85 kDa protein. The data provide evidence that the binding of ligands to the p55 component of the IL-2 receptor generates a biochemical signal by the stimulation of adenyl cyclase via Gs, and that the consequent generation of cAMP and activation of cAMP-dependent protein kinase modulates the turnover of p85-bound phosphate groups.

Human T lymphocytes stimulated with phytohaemagglutinin undergo a single round of cell division. Further proliferation is dependent on the lymphokine interleukin-2 (IL2) [(1987) Immunology 60, 7–12]. We show here that binding of IL2 to its receptors on the lymphocyte surface triggers the generation of cyclic AMP. In contrast, generation of inositol phosphates from the breakdown of inositol lipids was not detected. We suggest that cyclic AMP may play a role in the transduction of the IL2 proliferative signal in T lymphocytes.

A multienzyme complex consisting of DNA polymerase and several DNA precursor-synthesizing enzymes was solubilized by gentle lysis of cultured human cells. This complex channelled the distal precursor [3H]dTMP into DNA. The patterns of inhibition of the complex by aphidicolin and dideoxythymidine triphopshate (ddTTP) suggested that the complex contained the replicative DNA polymerase, polymerase α. Inhibition by ddTTP was competitive with dTTP. This was exploited to estimate the effective concentration of [3H]dTTP at the site of DNA synthesis during channelling of [3H]dTMP into DNA. The estimated concentration (about 50 μM) was so high as to suggest that the solubilized complex was able to functionally compartmentalize DNA precursors.

We have quantitated tyrosine protein kinase (TPK) activity in particulate and cytosolic fractions from human leukemic cells. Slowly proliferating cells from patients with chronic lymphocytic leukemia (CLL) had levels of TPK similar to those of quiescent normal lymphocytes. Cells from patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and chronic granulocytic leukemia (CGL) contained markedly lower levels of TPK activity, similar to the levels in phytohaemagglutinin-stimulated (proliferating) normal lymphocytes and in bone marrow cells. This suggested that TPK is part of a mechanism for transducing growth signals and is down-regulated following signal transmission. We also identified endogenous substrates for TPK in leukemic cells. Particulate fractions from ALL, CLL and AML cells contained substrates identical to those previously detected in normal lymphocytes. In particular, a 38kD substrate thought to be involved in early stages of growth signal transduction in normal lymphocytes was found in all samples of these groups examined. Cytosolic fractions from these groups of leukemia cells contained higher molecular weight substrates not found in resting or proliferating normal lymphocytes or bone marrow cells. In contrast, TPK substrates in both particulate and cytosolic fractions from CGL cells resembled those of normal bone marrow cells in that only proteins with molecular weight below 40kD were labelled on tyrosine. We conclude that leukemic cells do not contain higher levels of TPK than do normal hemopoietic cells. Qualitative differences in TPK species or in their substrates may result in aberrant regulation of proliferation in leukemic cells. However, we cannot exclude the possibility that additional TPK substrates detected in leukemic cells were a feature of the normal equivalent hematopoietic cells from which the leukemia cells were derived.

We have studied tyrosine phosphorylation in particulate fractions from 11 leukaemic cell lines by using as substrate either a synthetic tyrosine containing peptide or the endogenous proteins. The results were compared with those obtained using similar fractions from normal lymphocytes and bone marrow cells. Particulate fractions from all the leukaemic cell lines and normal bone marrow cells exhibited lower levels of tyrosine protein kinase activity compared to normal lymphocytes. When the phosphorylation of endogenous substrates was assayed, proteins were phosphorylated on tyrosine residues (rather than serine or threonine residues) to a larger extent in normal lymphocytes than in leukaemic cell lines. Separation of labelled endogenous substrates on sodium dodecyl sulfate-polyacrylamide gels showed a number of phosphorylated alkali-resistant bands in the range 14–175kd in the lymphoid cell lines; normal lymphocytes exhibited a smaller number of strongly phosphorylated bands. Normal lymphocytes from different individuals showed reproducible patterns of phosphorylated substrates. Normal bone marrow cells and myeloid leukaemia lines showed weak, if any, phosphorylation. Among the leukaemic cell lines no particular pattern of phosphorylated substrates common to cells of similar phenotype could be detected. We suggest that the level of overall tyrosine protein kinase activity in these fractions reflects their position in the cell cycle rather than their normal or malignant status.

Three agents which are mitogenic for T lymphocytes (phytohaemagglutinin, monoclonal antibody UCHT 1 and 12- O -tetradecanoylphorbol-13-acetate) stimulated rapid phosphorylation of a 42 kDa protein in per-meabilized T lymphocytes. Phosphorylation occurred on tyrosine and serine residues. A non-mitogenic monoclonal antibody (RFT11) did not stimulate phosphorylation of this protein. Furthermore, the dose response of 42 kDa protein phosphorylation and of mitogenesis to increasing amounts of phytohaemagglutinin were closely similar. We therefore propose that mitogen-stimulated phosphorylation of the 42 kDa protein is part of the mechanism for transduction of mitogenic signals in lymphocytes. To our knowledge, this is the first report of rapid, ligand-stimulated tyrosine protein phosphorylation in T lymphocytes.

We have studied the role of guanine-nucleotide binding regulatory proteins (G proteins) in the stimulation of inositol lipid breakdown during mitogenic activation of normal human T lymphocytes. The effect of the mitogen phytohemagglutinin (PHA) was compared with the action of two G-protein activators, fluoroaluminate (AlF4-) and guanosine-5'-O-thiotriphosphate (GTP gamma S). PHA and AlF4- stimulated the breakdown of inositol lipids via both the phospholipase A and C pathways when added to intact lymphocytes. PHA, AlF4- and GTP gamma S also triggered both these pathways when added to permeable lymphocytes. The magnitude of the response obtained with AlF4- and GTP gamma S was about four-fold less than with PHA. This difference was attributable to increases in cAMP elicited by AlF4- and GTP gamma S which inhibited the phospholipase pathways. AlF4-, GTP gamma S, and PHA all stimulated the phosphorylation of a 42 kDa protein on tyrosine residues. We propose a model for the early steps following mitogen binding, including sequential activation of a G protein, phospholipase C, protein kinase C and a tyrosine protein kinase. A parallel pathway involving G protein mediated activation of phospholipase A is also implicated.

1. 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). 2. The isolated complex incorporated the distal DNA precursors [ 3 H]thymidine or [ 3 H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [ 3 H]dTTP. Measurement of the apparent overall concentrations of [ 3 H]dTTP produced during incorporation of [ 3 H]thymidine and of [ 3 H]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. 3. The DNA polymerase inhibitor 1-β- d -arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [ 3 H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. 4. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.