Cytosine arabinoside induces costimulatory molecule expression in acute myeloid leukemia cells.

Chemotherapeutic drugs kill cancer cells mainly by direct cytotoxicity, but they might also induce a stronger host immune response by causing the tumor to produce costimulatory cell surface molecules like CD80. We previously reported that in myeloid leukemic cells, gamma-irradiation induced CD80 expression. In this study, we show that cytosine arabinoside (Ara-C), even at low doses, induced CD80 expression in vitro in mouse DA1-3b leukemic cells, by a mechanism that involved reactive oxygen species. In vivo experiments in the mouse DA1-3b/C3H whole-animal acute myeloid leukemia (AML) model showed that injection of Ara-C induced expression of CD80 and CD86, and decreased expression of B7-H1, indicating that chemotherapy can modify costimulatory molecule expression in vivo, in a way not necessarily observed in vitro. Mouse leukemic cells exposed in vivo to Ara-C were more susceptible to specific cytotoxic lymphocyte (CTL)-mediated killing. Ara-C also induced CD80 or CD86 expression in 14 of 21 primary cultured human AML samples. In humans being treated for AML, induction chemotherapy increased CD86 expression in the leukemic cells. These findings indicate possible synergistic strategies between CTL-based immunotherapy and chemotherapy for treatment. They also suggest an additional mechanism by which chemotherapy can eradicate AML blasts.

Gamma-irradiation enhances transgene expression in leukemic cells.

The majority of immunotherapy-based gene therapy protocols consist of ex vivo gene transfer in tumor cells. To prevent further in vivo growth, modified cells must be irradiated before reinjection into patients. The present study examines the effects of gamma-irradiation on transgene expression in transduced leukemic cells. Human and murine leukemic cells were transfected with retroviral vectors or plasmids carrying beta-galactosidase, GM-CSF or CD80 genes. Fresh leukemic cells from patients with acute myeloid leukemia (AML) were transfected with AdZ.F(pK7) adenoviral vector. gamma-irradiation at various lethal doses enhanced transgene expression in leukemic cell lines and fresh AML cells when the gene of interest was under CMV promoter but not when SV40 promoter was used. Oxidative stress also enhanced transgene expression and both irradiation and oxidative stress effects were inhibited by addition of N-acetyl-L-cysteine, a thiol anti-oxidant, indicating the involvement of reactive oxygen species. Transgene expression was also enhanced in vivo 48 and 120 h after subcutaneous injection of irradiated leukemic cells in syngeneic mice. These results show that a cell vaccine protocol using ex vivo gene transfer of transduced cells might be feasible in acute leukemia even if leukemic cells must be irradiated at lethal doses prior to reinjection to patients.

[Gene therapy and ovarian cancer: update of clinical trials]. / Thérapie génique et cancer de l'ovaire: exposé des essais cliniques en cours.

Ovarian cancer is the first leading cause of death from gynecologic cancer. Advances in therapy are needed to obtain complete response after surgery and/or chemotherapy. Gene therapy is a new alternative therapeutic approach. 380 gene therapy clinical trials (3173 patients) are going to be assessed. 63% of these trials concern therapy of cancer. 16 gene therapy clinical trails are applied to ovarian cancer. These 16 clinical trials assess different treatment strategies: Mutation compensation by replacement of an altered tumor suppressor gene (p53, BRCA1); Molecular chemotherapy by transfer of a suicide gene (HSV-tk gene); Antitumoral immunotherapy by cytokine gene transfer (IL2, IL12); Oncogene inhibition (erb-B2 gene); Multi Drug Resistance gene transfer. A knowledge of basis concepts of gene transfer strategies, is needed to understand these different treatment strategies. Thus, the goals of this review are, first, to provide the basis concepts of gene transfer strategies to the obstetrician-gynecologist and second, to submit recent gene therapy clinical trials about ovarian cancer.

Gene transfer of GM-CSF, CD80 and CD154 cDNA enhances survival in a murine model of acute leukemia with persistence of a minimal residual disease.

Gene transfer of various cytokines and co-stimulatory molecules has been reported to induce a potent antileukemic immunity in murine models, however, the relative efficiency and possible synergistic effects between candidate genes have not been extensively investigated. We analyzed in a murine model of BCR/ABL acute leukemia whether gene transfer of CD154, CD80 or GM-CSF as a single agent or combination of CD154 + GM-CSF, CD80 + CD154 and GM-CSF + CD80 in leukemic cells could enhance survival. We observed that CD154 gene transfer induced a marked inhibition of leukemogenicity, and also that CD154 and combination of GM-CSF and CD80 gene transfer protected mice against subsequent challenge with leukemic cells and had a therapeutic effect for a pre-established leukemia disease. We also found minimal residual leukemic disease by RT-PCR for 6 to 12 months in 0 to 25% of animals injected with transduced leukemic cells and surviving the challenge without evidence of disease, except in the control empty plasmid group where very few mice survived the challenge but all of those were positive by RT-PCR. These findings suggest that leukemic cell vaccination by gene transfer can induce a tumor dormancy phenomenon compatible with long-term survival.

gamma-ray irradiation induces B7.1 expression in myeloid leukaemic cells.

Expression of B7 molecules provides co-stimulatory signals to T lymphocytes, which prevent the induction of anergy. It has been previously reported that B7.1 gene transfer in a murine leukaemia model induced a potent antileukaemic immunity and that relative expression of B7.1 and B7.2 in human acute myeloid leukaemia (AML) had prognostic significance. As ex vivo engineering of leukaemic cells for immunotherapy protocols would require prior irradiation of these cells before reinjection to the patient, we investigated in murine and leukaemic cell lines and in 20 ex vivo primary cultured acute myeloid leukaemic cells the effect of gamma-irradiation on the expression of B7 molecules. We observed that gamma-irradiation enhanced B7.1 molecule expression in murine leukaemic cell lines and in B7.2 molecules in human HL60 and K562 cell lines. gamma-Irradiation induced B7.1 molecule expression in 90% AML samples but only 21% showed B7.2 molecule expression enhancement. B7.1 expression was increased both at the protein and RNA level in human AML cells but only at the protein level in the DA1-3b murine cell line. Oxidative stress increased B7.1 expression in the murine DA1-3b cell line but human cell lines and AML samples remained unaffected both by heat shock and oxidative stress, suggesting different pathways of B7.1 induction between mouse and human cells. Our data show that B7.1 expression can be induced by ex vivo irradiation of AML cells, indicating that these cells can express co-stimulatory molecules without gene transfer.

Transduction of bone marrow cells by the AdZ.F(pK7) modified adenovirus demonstrates preferential gene transfer in myeloma cells.

Adenoviral vectors can efficiently infect myeloma cell lines, but transduction of fresh myeloma cells performed at low multiplicity of infections (MOIs) showed only partial efficacy. The modified adenoviral vector AdZ.F(pK7), through binding of polylysines to heparan sulfate-containing receptors, could increase virus adsorption and gene transfer efficiency in myeloma cells, which express heparan sulfate-containing receptors. Thus, we investigated the ability of AdZ.F(pK7) vector to achieve efficient gene transfer in primary cultured fresh myeloma cells. Transduction of 16 primary cultured myeloma samples showed that gene transfer was much more efficient with AdZ.F(pK7) than with control AdZ.F. Both addition of soluble heparin and cell treatment with heparinase I dramatically inhibited gene transfer in myeloma cells by AdZ.F(pK7) but had no effect with AdZ.F, while addition of recombinant fiber protein inhibited AdZ.F but not AdZ.F(pK7), confirming that AdZ.F(pK7) gene transfer in myeloma cells is mediated by the targeting of heparan sulfates. AdZ.F(pK7) transduction of bone marrow cells showed that myeloma cells and hematopoietic progenitor AC133-, CD34-, and CD33-positive cells were efficiently transduced at an MOI of 100, but that only myeloma cells were significantly transduced at an MOI of 12. Thus, AdZ.F(pK7) vector seems to be well suited for immunological approaches of gene therapy or bone marrow-purging applications in multiple myeloma.

Increased gene transfer in acute myeloid leukemic cells by an adenovirus vector containing a modified fiber protein.

Applications of gene transfer in acute myeloid leukemia (AML) blast cells have still not been developed, mostly due to the lack of an efficient vector. Adenoviruses have many advantages as vectors, but remain poorly efficient in cells lacking fiber receptors. A promising strategy is the retargeting of adenoviruses to other cellular receptors. We report the dramatic enhancement of gene transfer efficiency in AML blasts using AdZ.F(pK7), a modified adenovirus containing a heparin/heparan sulfate binding domain incorporated into the fiber protein of the adenovirus. We transduced 25 AML blast samples with efficiency reaching 100% of the cells in most samples. Optimal results were obtained at 8400 physical particles per cell, corresponding to a multiplicity of infection of 100 plaque forming units per cell. Control AdZ.F adenovirus efficiently transduced leukemic cell lines but gave poor results in AML samples. Both addition of soluble heparin and cell treatment with heparinase inhibited AdZ.F(pK7) gene transfer, showing that heparan sulfates are the major receptors mediating AdZ.F(pK7) transduction of AML blasts. Although adenoviruses can infect nondividing cells, we observed that a combination of growth factors (GM-CSF, IL-3, stem cell factor) was required for efficient transduction in order to maintain AML blast cell viability. This study demonstrates that retargeting the adenovirus fiber protein to heparan sulfates can overcome the low efficiency of adenovirus in AML blast cells and may provide a useful tool for gene therapy approaches in AML.

Methylation of the p15(INK4b) gene in myelodysplastic syndromes is frequent and acquired during disease progression.

p15(INK4b) gene is an inhibitor of cyclin-dependent kinase (CDK) 4 and CDK6 whose expression is induced by transforming growth factor (TGF)beta. Recent reports suggest frequent methylation of the p15(INK4b) gene promoter in leukemias, and it has been proposed that this methylation could be necessary for leukemic cells to escape TGF beta regulation. We investigated the methylation status of p15(INK4b) gene in 53 myelodysplastic syndromes (MDS) cases, including nine that had progressed to acute myeloid leukemia (AML), using a recently described sensitive method where polymerase chain reaction (PCR) is preceded by bisulfite modification of DNA (methylation specific PCR). p15(INK4b) methylation was observed in 20 of 53 (38%) of the cases. Twenty of the 24 patients with greater than 10% bone marrow blasts had p15(INK4b) methylation (including all nine patients who had progressed to AML) as compared with none of MDS patients with <10% bone marrow blasts. No correlation between karyotypic abnormalities and methylation status was found. Patients with p15(INK4b) methylation had a worse prognosis, but the prognostic significance of p15(INK4b) methylation was no more found by multivariate analysis, due to its strong correlation to the percentage of marrow blasts. In 10 MDS cases, sequential DNA samples were available. In five of them, methylation of the p15(INK4b) gene was detected at leukemic transformation, but not at diagnosis. Our results showed that methylation of the p15(INK4b) gene in MDS is correlated with blastic bone marrow involvement and increases with disease evolution toward AML. It suggests that proliferation of leukemic cells might require an escape of regulation of the G1 phase of the cell cycle, and possibly of TGF beta inhibitory effect.