Kanagasabai Ganeshaguru

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.

The effect of external deoxyribonucleosides on deoxyribonucleoside triphosphate concentrations in human lymphocytes

Abstract The effects of deoxyribonucleosides on the intracellular levels of deoxyribonucleoside triphosphates (dNTP) and on the rate of labelled thymidine incorporated into DNA of human phytohaemag-glutinin-stimulated lymphocytes have been studied. Thymidine (10−2–10−6M) expanded the dTTP and reduced dATP and dCTP levels. Deoxycytidine (10−3M) expanded the dCTP level and caused inhibition of [3H] thymidine incorporation into DNA but had no detectable effect on the other dNTP concentrations. Deoxyadenosine (10−3 M) expanded the dATP level, and reduced the other dNTP levels and deoxyguanosine (10−4M) expanded the dGTP level and reduced the dCTP level; both inhibited [3H] thymidine incorporation into DNA. The sensitivity of these cells to the addition of deoxynucleosides to their culture medium indicates that the plasma and tissue levels of nucleosides may profoundly influence DNA synthesis by human cells in vivo.

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.

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 >u200a10% 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.

Non‐benign sickle cell anaemia in western Saudi Arabia

Seventy‐one Saudi and Yemeni Arabs with sickle cell anaemia from western Saudi Arabia aged between 11/2 and 42 years were studied. The mean steady state haemoglobin concentration of 8.1 g/dl was lower than that of 10.7 g/dl reported previously for sickle cell anaemia in eastern Saudi Arabia. The patients were divided into an SSLF group with fetal haemoglobin (HbF) of 10.0% or below (44 patients) and an SSHF group having HbF above 10.0% (27 patients). No significant differences were found in the haemoglobin concentrations, haematological indices and incidences of bone changes of the two groups. SSLF patients were significantly more prone to infections (P<0.01), however. Also, there was an overall high incidence of hepatomegaly (69.0%) and splenomegaly (54.9%) and hepatomegaly was significantly more common in the SSLF group (P<0.02).

Glucosylceramide synthase inhibitors sensitise CLL cells to cytotoxic agents without reversing P-gp functional activity

Malignant B-cells from most chronic lymphocytic leukaemia (CLL) patients over-express MDR1 encoded P-glycoprotein (P-gp) multidrug efflux pump. Inhibition of glucosylceramide (GC) synthesis has been shown in cell lines to correlate with the expression and function of P-gp and sensitise cancer cells to cytotoxic agents. We investigated the hypothesis that reducing intracellular GC levels will reduce P-gp expression in malignant cells from CLL patients. We studied the ability of glucosylceramide synthase (GCS) inhibitors N-butyl-deoxygalactonojirimycin (OGB-1) and N-nonyl-deoxygalactonojirimycin (OGB-2) to sensitise CLL cells to conventional cytotoxic drug 2-chlorodeoxyadenosine (CdA) and the cytostatic drugs chlorambucil and fludarabine. The effect on P-gp activity was analysed using the calcein-AM accumulation assay where a multidrug activity factor (MAF) of >10 in the presence of a P-gp inhibitor denotes P-gp functional activity. The P-gp over-expressing cell line CEM-VLB showed a MAF value of 96.4 with the P-gp inhibitor Z.3HCL, which fell to 15.7 after co-incubation with OGB-1 and 45.9 with OGB-2. The IC(50) for vincristine fell from >10 microg/ml to 55.5 ng/ml in the presence of OGB-2. In P-gp(+ve) peripheral blood mononuclear cells from three normal volunteers, the mean MAF values for Z.3HCL, OGB-1 and OGB-2 were 23.86, 1.83 and 16.2 respectively. In 9/13 CLL samples the mean P-gp functional activity was 22.15 and P-gp was over-expressed in 12/13 samples. However, the MAF value with OGB-1 and OGB-2 was <10. Nevertheless, sensitisation in CLL cells was observed by a reduction in the IC(50) in the presence of OGB-1 and OGB-2 with the conventional drugs. We conclude that although GCS inhibitors sensitize CLL cells to cytotoxic and cytostatic drugs, they do not appear to have any effect on P-gp functional activity.

The mechanisms of action of three fluorine substituted cytosine analogs: Implications for cancer chemotherapy

Abstract The mechanisms of action in DNA synthesis of three fluorine substituted cytosine analogs have been studied in human phytohaemagglutinin-stimulated lymphocytes. 5-Fluorocytidine (over a wide range of concentrations) and 5-fluorocytosine (at high concentration) inhibited 3H-deoxyuridine incorporation into DNA without inhibiting 3H-thymidine incorporation. 5-Fluorocytidine caused reduction in the free cell concentration of TTP. In contrast, ara-fluorocytosine markedly inhibited both 3H-deoxyuridine and3H-thymidine incorporation into DNA but did not cause a consistent change in thymidine triphosphate concentration. These results indicate that 5-fluorocytidine, and to a lesser extent, 5-fluorocytosine block de novo TMP synthesis in human cells. It seems likely that they are both metabolized to 5-fluorodeoxyuridinc monophosphate which inhibits the enzyme TMP synthetase. On the other hand, the results suggest that ara-fluorocytosine has an action identical to that of cytosine arabinoside, inhibition of DNA polymerase. It is postulated that cells resistant to cytosine arabinoside which have high cytidine deaminase levels might be selectively sensitive to 5-fluorocytidine and to 5-fluorocytosine.

39 SELECTIVE TOXICITY OF PURINE NUCLEOSIDES TO HUMAN T-LEUKAEMIC CELLS

In vitro cytotoxicity of various purine nucleosides and purine enzyme inhibitors, alone or in combination, and of the alkylating agent mafosfamide incubated for 4/ 24h has been studied in 17 leukaemic cell lines of various phenotypes and normal bone marrow cells. The purine nucleosides/inhibitors included: 2′chlorodeoxyadenosine (CdAdo), 2′deoxyadenosine (dAdo), 3′deoxyadenosine (3′dAdo), adenosine, adenine arabinoside (ara-A), deoxyguanosine (dGdo), guanine arabinoside (Ara-G), 2-deoxycoformycin (dCF) and 8-aminoguanosine (8-AG). T-lymphoblastic cell lines were found to be the most sensitive to the toxic effects of the purine analogues. Marked and selective inhibition of T-cell growth was shown by the combinations dCF with either dAdo or ara-A, of 8-AG with dGdo and by CdAdo or Ara-G alone. These compounds even at high concentrations produced only partial inhibition of the growth of normal bone marrow cells in in vitro assays (CFU-GM and CFU-GEHM) except for CdAdo which inhibited the formation of CFU-GEMM colonies. dCF plus 3′dAdo was toxic to all the cell lines at the concentrations employed, as well as to CFU-GM and CFU-GEMM and so was mafosfamide. The high therapeutic index of some of the purine nucleosides after a relatively short exposure period makes them candidates for selective in vitro removal of residual neoplastic cells in autologous BMT for acute lymphoblastic leukaemia of T-cell origin.