Biomedical subjects

Susan J Zunino

Publications and source records attributed to Susan J Zunino.

8 recordsLinked to original sources

PubMed2005

Resveratrol-induced apoptosis is enhanced in acute lymphoblastic leukemia cells by modulation of the mitochondrial permeability transition pore.

We have previously shown that resveratrol can induce apoptotic cell death in cell lines established from patients with acute lymphoblastic leukemia (ALL). Cyclosporin A (CsA) and PK11195 are modulators of the mitochondrial permeability transition pore (MPTP) which has been proposed to play a critical role in regulating survival and death. Using SEM and RS4;11 lines with the t(4;11) translocation, the B-ALL line REH, and the T-ALL line Jurkat, we show that pre-treatment with CsA or PK11195 significantly enhances resveratrol-mediated apoptosis and mitochondrial membrane depolarization in these cells, as measured by annexin V and JC-1 staining, respectively. No significant multi-drug resistance efflux of the fluorescent substrate calcein was observed in these ALL lines, indicating that CsA and PK11195 were acting at the level of the mitochondria to enhance loss of mitochondrial membrane potential and induction of apoptosis. These data suggest targeting the MPTP sensitizes B- and T-cell ALL to the anti-cancer activity of resveratrol, and may be particularly useful for the treatment of high-risk t(4;11) ALL.

Antineoplastic Agents, Phytogenic↗

PubMed2004

Nitric oxide is synthesized in acute leukemia cells after exposure to phenolic antioxidants and initially protects against mitochondrial membrane depolarization.

We investigated the early events involved in loss of mitochondrial membrane potential (DeltaPsi(mt)) leading to apoptosis in cells derived from patients with acute lymphocytic leukemia after exposure to phenolic antioxidants. Using the nitric oxide binding dye diaminofluorescein-FM diacetate, we found that intracellular nitric oxide (NO) levels increased significantly within 4h after exposure to the antioxidants curcumin, carnosol, and quercetin. Inhibition of nitric oxide synthetase (NOS) activity with mercaptoethylguanidine increased the percentage of leukemia cells with depolarized mitochondria membranes after antioxidant treatment. These data suggest that NO production in the leukemia-derived cells may be a protective response to maintain DeltaPsi(mt) after antioxidant exposure and inhibition of NOS increases the disruption of mitochondrial homeostasis induced by the antioxidants.

Abietanes↗

PubMed2004

Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications.

Two sets of expression vectors were constructed that permitted the efficient expression of single-chain Fv fragments (scFvs) fused N-terminally to an enhanced mutant of the green fluorescent protein GFP+ or the red fluorescent protein DsRed in insect and mammalian cells. The vectors allowed rapid cloning of scFv fragments and secretion of the fusion proteins in a native conformation. Fluorescent scFv fusion proteins directed against a series of cluster of differentiation (CD) antigens were efficiently secreted by transiently transfected mammalian cells and insect cells infected with baculoviral expression constructs. Yields of the secreted proteins varied from 100 microg/l to 3 mg/l. The purified proteins were functionally active in flow cytometry, immunofluorescent microscopy, and competition binding experiments performed to delineate the epitopes recognized by different monoclonal antibodies against the same polypeptide. The use of two different scFv fragments fused with red and green fluorescent proteins and reacting with T- and B-cell lineage markers (CD7 and CD19), respectively, allowed a simplified quantitation of both subsets in two-color flow cytometry experiments with mixed populations of T- and B-lymphoid cells. Due to the lack of Fc domains in the scFv proteins, the fluorescent fusion proteins showed more than 20-fold reduced background fluorescence compared with whole antibodies of the same specificity in experiments with effector cells expressing the high affinity FcgammaRI receptor CD64. Thus, for a number of analytical applications, fluorescent scFv fusion proteins offer advantages over the use of complete primary antibodies and chemically labeled fluorescent secondary antibodies.

Animals↗

PubMed2004

Chimeric CD19 antibody mediates cytotoxic activity against leukemic blasts with effector cells from pediatric patients who received T-cell-depleted allografts.

Relapse is a major problem after transplantation in children with acute B-lineage leukemias, and new therapies are needed to increase graft-versus-leukemia (GvL) effects without inducing graft-versus-host disease (GvHD). Here, we studied the ability of effector cells recovered from patients after transplantation with positive-selected stem cells from alternative donors to induce antibody-dependent cellular cytotoxicity (ADCC). For this purpose, a chimeric CD19 antibody, CD19-4G7chim, was generated. This antibody efficiently mediated ADCC against primary acute lymphoblastic leukemia (ALL) blasts by using purified natural killer (NK) cells from healthy donors or mononuclear cells from patients as effector cells. Increased lysis was obtained after stimulation of effector cells with interleukin-2 (IL-2). ADCC was not prevented by inhibitory effects mediated by HLA class I. We propose that treatment with chimeric CD19 antibodies leading to ADCC by donor-derived NK cells may become a therapeutic option for the post-transplantation treatment of minimal residual B-lineage ALLs.

Antibodies↗

PubMed2004

A recombinant bispecific single-chain Fv antibody against HLA class II and FcgammaRIII (CD16) triggers effective lysis of lymphoma cells.

Bispecific antibodies offer the possibility of improving effector-cell recruitment for antibody therapy. For this purpose, a recombinant bispecific single-chain Fv antibody (bsscFv), directed against FcgammaRIII (CD16) and human leucocyte antigen (HLA) class II, was constructed and tested in functional assays. RNA from the hybridomas 3G8 and F3.3, reacting with CD16 and HLA class II, respectively, was used to generate phage display libraries. From these libraries, reactive phages were isolated and the bsscFv was constructed by connecting both single-chain Fv components through a 20 amino acid flexible linker. After expression in SF21 insect cells and chromatographic purification, the bsscFv bound specifically and simultaneously to both antigens. The affinities of the anti-CD16 and the anti-HLA class II scFv components of the bsscFv were 8.6 x 10(-8) mol/l and 13.7 x 10(-8) mol/l, respectively, which was approximately sevenfold lower than the F(ab) fragments of the parental antibodies. In antibody-dependent cellular cytotoxicity experiments with human mononuclear cells as effectors, the bsscFv-mediated specific lysis of both HLA class II-positive, malignant human B-lymphoid cell lines and primary cells from patients with chronic B-cell lymphocytic leukaemia. Optimal lysis was obtained at bsscFv concentrations of approximately 400 ng/ml, similar to the concentration required for maximum lysis by the corresponding chemically linked bispecific antibody. Thus, this recombinant bsscFv-antibody is an efficient molecule for effector-cell mediated lysis of malignant human B-lymphoid cells.

Animals↗

PubMed2003

Conditional cell suicide using dox-dependent caspase-2 expression.

BACKGROUND: Adoptive immune transfer is used as an efficient treatment modality to achieve a graft-versus-leukemia effect in persisting or relapsing residual leukemic disease. Safety considerations dictate the need for equipping the transferred cells with a conditional suicide mechanism to eliminate donor T cells when graft-versus-host disease occurs. We have examined in a model system using HeLa cells whether doxycycline (dox)-dependent expression of pro-apoptotic proteins could be used as a potential new strategy for conditional cell elimination. METHODS: Four constructs encoding pro-apoptotic proteins were tested in transient transfections to identify suitable cell death inducers. Murine caspase-2 placed under Tet-control was chosen for stable transfection into cell lines carrying different dox-dependent transregulators. The efficiency of cell death induction and the expression patterns of caspase-2 were analyzed in the respective clones. RESULTS: Different levels of induced cell death were obtained depending on the properties of the transregulators used to control target gene expression. High expression levels of caspase-2 in the presence of dox were required to achieve efficient induction of cell death, while tight repression in the absence of inducer was not necessary for cell survival. Dox treatment for 48 h resulted in 94% cell death indicating a very efficient conditional suicide mechanism. CONCLUSIONS: We propose that the principle of using pro-apoptotic cellular proteins placed under appropriate dox-dependent regulation may represent an alternative conditional suicide mechanism to the frequently used herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir-system, which harbors immunological and toxicological risks.

Animals↗

PubMed2002

A recombinant CD7-specific single-chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells.

A recombinant immunotoxin was constructed from the hybridoma antibody TH-69 directed against human CD7, a surface antigen of leukemic T cells. The antibody was subcloned as a single chain Fv (scFv) fragment and genetically linked to a truncated Pseudomonas exotoxin A fragment containing the catalytic domains II and III but lacking the receptor binding domain I. Domain I was replaced by the scFv, thus conferring restricted specificity for CD7-positive cells. The bacterially expressed and purified toxin retained binding specificity for CD7-positive cells. It promoted apoptosis in two CD7-positive cell lines derived from T-lineage acute lymphoblastic leukemias, CEM and Jurkat, but not in the CD7-negative B-lymphoid lines REH, Nalm-6, and SEM. Maximum killing in excess of 95% was reached after 96 h in CEM and Jurkat cells with a single dose of 100 ng/ml. Cells treated with a similarly constructed scFv-exotoxin A immunotoxin against melanoma-associated chondroitin sulfate proteoglycan, an antigen absent from leukemic T cells, remained unaffected. Lysis of target cells occurred via apoptosis as evidenced by staining with Annexin V and specific cleavage of poly(ADP-ribose) polymerase. Approximately 20% of leukemic cells from a patient with CD7-positive acute T-cell leukemia kept in long-term primary culture for 30 cell generations were killed within 96 h after treatment with the toxin. These findings justify further evaluation of the agent in view of potential therapeutic applications.

ADP Ribose Transferases↗

PubMed2002

Interferon-gamma increases the expression of glycosylated CD95 in B-leukemic cells: an inducible model to study the role of glycosylation in CD95-signalling and trafficking.

B-lineage acute leukaemia cells are generally resistant to CD95-mediated apoptosis. In this report, the CD95-resistant B-leukaemia lines SEM, RS4;11, and REH were used to investigate the mechanisms of resistance to CD95-signalling. We found that interferon-gamma (IFN-gamma) treatment increased the presence of high molecular weight forms of CD95 in these cells as judged by Western analysis, and treatment of protein extracts with Peptide: N -glycosidase F indicated that the majority of high molecular weight forms were due to N-linked glycosylation. Treatment of whole cells with neuraminidase from Vibrio cholerae substantially reduced the relative molecular mass of CD95 observed after IFN-gamma treatment and partially sensitized the three leukaemia lines to CD95-mediated death. To further characterize the different steps of oligosaccharide processing that may regulate CD95 signalling, the leukaemic cells were treated with IFN-gamma and the glycosidase inhibitors castanospermine, 1-deoxymannojirimycin (DMM), and swainsonine. Treatment with DMM, a mannosidase inhibitor, efficiently reduced the appearance of high molecular weight forms of CD95 after IFN-gamma treatment, and sensitized SEM and REH cells to CD95-mediated death. However, the IFN-gamma-induced increases of CD95 on the cell surface were not altered by treatment with any of the glycosidase inhibitors, suggesting that the generation of complex oligosaccharide structures is not required for trafficking of CD95, but may instead be used as a mechanism of partially blocking CD95 signalling in these cells. In conclusion, IFN-gamma treatment of the B-lineage leukaemia lines provides a novel, inducible system for the further characterization of post-translational modifications involved in modulating sensitivity to CD95-signalling.

1-Deoxynojirimycin↗