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Biomedical subjects

A Mazumder

Publications and source records attributed to A Mazumder.

At least 55 records · Page 3Linked to original sources
PubMed1996

Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures.

An oligonucleotide (T30177) composed entirely of deoxyguanosine and thymidine has previously been shown to fold upon itself in the presence of potassium into a highly stable four-stranded DNA structure containing two stacked deoxyguanosine quartets (G4s). T30177 also protects host cells from the cytopathic effects of human immunodeficiency virus type 1 (HIV-1). We report that this G4 oligonucleotide is the most potent inhibitor of HIV-1 integrase identified to date, with IC50 values in the nanomolar range. Both the number of quartets formed and the sequence of the loops between the quartets are important for optimal activity. T30177 binds to HIV-1 integrase without being processed and blocks the binding of the normal viral DNA substrate to the enzyme. The normal DNA substrate was not able to compete off T30177 binding to HIV-1 integrase, indicating a tight binding of G4s to the enzyme. Experiments with truncated HIV-1 integrases indicate that the N-terminal region containing a putative zinc finger is required for inhibition by T30177 and that T30177 binds better to full-length or deletion mutant integrases containing the zinc finger region than to a deletion mutant consisting of only the central catalytic domain. The N-terminal region of integrase alone is able to bind efficiently to T30177, but not the linear viral DNA substrate, in the presence of zinc. Hence, G4s represent the first class of compounds that inhibit HIV-1 integrase by interacting with the enzyme N-terminal domain. The greater inhibitory potency of T30177 in buffer containing magnesium versus manganese suggests that divalent metal ion coordination along the phosphodiester backbone may play a role in the inhibitory activity. T30177 inhibited HIV-2 integrase with similar potency as HIV-1 but inhibited feline and simian immunodeficiency virus integrases at higher concentrations, suggesting selectivity can be achieved. We propose that novel AIDS therapies could be based upon guanosine quarters as inhibitors of HIV-1 integrase.

Binding, Competitive↗
PubMed1996

DNA sequence- and structure-selective alkylation of guanine N2 in the DNA minor groove by ecteinascidin 743, a potent antitumor compound from the Caribbean tunicate Ecteinascidia turbinata.

Ecteinascidin 743 is one of several related marine alkaloids isolated from the Caribbean tunicate Ecteinascidia turbinata. It is remarkably active and potent in a variety of in vitro and in vivo systems and has been selected for development as an anticancer agent. The present study investigates the interactions of ecteinascidin 743 with DNA. Ecteinascidin 743 retarded the electrophoretic migration of both supercoiled and relaxed simian virus 40 DNA even in the presence of sodium dodecyl sulfate and after ethanol precipitation, consistent with covalent DNA modifications. Similar results were obtained in a DNA oligonucleotide derived from ribosomal DNA. However, DNA denaturation reversed the DNA modifications. The homopolymeric oligonucleotide dG/dC was modified while neither the dI/dC nor the dA/dT oligonucleotides were, consistent with covalent attachment of ecteinascidin 743 to the exocyclic amino group at position 2 of guanine. Ecteinascidin 743 was then compared to another known DNA minor groove alkylating agent, anthramycin, which has also been shown to alkylate guanine N2. Footprinting analyses with DNase I and 1,10-phenanthroline-copper and exonuclease III digestions showed that ecteinascidin 743 covers three to five bases of DNA and exhibits a different sequence specificity than anthramycin in the Escherichia coli tyrosine tRNA promoter (tyrT DNA). The binding of ecteinascidin to DNA was abolished when guanines were substituted with inosines in this promoter. A band shift assay was designed to evaluate the influence of the bases flanking a centrally located guanine in an oligonucleotide containing inosines in place of guanines elsewhere. Ecteinascidin 743 and anthramycin showed similarities as well as differences in sequence selectivity. Ecteinascidin 743-guanine adducts appeared to require at least one flanking guanine and were strongest when the flanking guanine was 3' to the targeted guanine. These data indicate that ecteinascidin 743 is a novel DNA minor groove, guanine-specific alkylating agent.

Alkylation↗
PubMed1996

Antiretroviral agents as inhibitors of both human immunodeficiency virus type 1 integrase and protease.

The human immunodeficiency virus type one integrase (HIV-1 integrase) is required for integration of a double-stranded DNA copy of the viral RNA genome into a host chromosome and for HIV replication. We have previously reported that phenolic moieties in compounds such as flavones, caffeic acid phenethyl ester (CAPE), tyrphostins, and curcumin confer inhibitory activity against HIV-1 integrase. We have investigated the actions of several recently described protease inhibitors, possessing novel structural features, on HIV-1 integrase. NSC 158393, which contains four 4-hydroxycoumarin residues, was found to exhibit antiviral, antiprotease, and antiintegrase activity. Both the DNA binding and catalytic activities (3'-processing and strand transfer) of integrase were inhibited at micromolar concentrations. Disintegration catalyzed by an integrase mutant containing only the central catalytic domain was also inhibited, indicating that the binding site for these compounds resides in the central 50-212 amino acids of HIV-1 integrase. Binding at or near the integrase catalytic site was also suggested by a global inhibition of the choice of attacking nucleophile in the 3'-processing reaction. NSC 158393 inhibited HIV-2, feline, and simian immunodeficiency virus integrases while eukaryotic topoisomerase I was inhibited at higher concentrations, suggesting selective inhibition of retroviral integrases. Molecular modeling studies revealed that the two hydroxyls and two carbonyl moieties in NSC 158393 may represent essential elements of the pharmacophore. Antiviral efficacy was observed with NSC 158393 derivatives that inhibited both HIV protease and integrase, and the most potent integrase inhibitors also inhibited HIV protease. Hydroxycoumarins may provide lead compounds for development of novel antiviral agents based upon the concurrent inhibition of two viral targets, HIV-1 integrase and protease.

4-Hydroxycoumarins↗
PubMed1996

Inhibitors of Escherichia coli RNA polymerase specific for the single-stranded DNA of transcription intermediates. Tetrahedral cuprous chelates of 1,10-phenanthrolines.

Single-stranded DNA of the lacUV-5 promoter formed at the active site of Escherichia coli RNA polymerase during transcription is specifically cleaved by the redox active tetrahedral cuprous chelates of 1,10-phenanthroline and its derivatives. The cleavage sites are observed in the open, initiating, and elongating complexes. Redox-inert, tetrahedral cuprous chelates of neocuproine (2,9-dimethyl-1,10- phenanthroline) and its 5-phenyl and 4-phenyl derivatives protect the template strand of DNA from scission within these steady state intermediates and inhibit transcription. Although these cuprous chelates of neocuproine bind at multiple sites within three distinct enzyme intermediates, the highest affinity site is within the elongation complex. The I50 of 5 microM for the 2:1 5-phenylneocuproine cuprous complex ((5 phi NC)2Cu+) in runoff transcription therefore primarily reflects its intermediate. The neocuproine cuprous chelates are novel transcription inhibitors because they bind to single-stranded DNA sites generated during the course of catalysis by RNA polymerase.

Base Sequence↗
PubMed1996

Differential induction of programmed cell death in CD8+ and CD4+ T cells by the B subunit of cholera toxin.

We have recently demonstrated that a short-term treatment of parental splenocytes with the B subunit of cholera toxin (CT-B) abrogates the development of acute GVHD in F1 hybrid mice transplanted with these cells. In order to obtain better insight into the mechanism of the action of CT-B, we studied the effect of CT-B on survival of purified murine T cells and their subsets. We show that treatment with B subunit stimulates apoptosis in T cells, detectable following incubation in vitro. Although apoptosis was noticed in both CD8+ and CD4+ T cell subsets, the treatment preferentially stimulates programmed cell death (PCD) in CD8+ population. Thus, immunosuppressive action of CT-B in vivo may be in part due to its ability to eliminate CD8+ T cells.

Animals↗
PubMed1996

Probing the active site residues in aromatic donor oxidation in horseradish peroxidase: involvement of an arginine and a tyrosine residue in aromatic donor binding.

The plausible role of arginine and tyrosine residues at the active side of horseradish peroxidase (HRP) in aromatic donor (guaiacol) oxidation was probed by chemical modification followed by characterization of the modified enzyme. The arginine-specific reagents phenylglyoxal (PGO), 2,3-butanedione and 1,2-cyclohexanedione all inactivated the enzyme, following pseudo-first-order kinetics with second-order rate contents of 24M(-1.)min(-1), 0.8M(-1.)min(-1) and 0.54M(-1.)min(-1) respectively. Modification with tetranitromethane, a tyrosine-specific reagent, also resulted in 50% loss of activity following pseudo-first-order kinetics with a second-order rate constant of 2.0M(-1.)min(-1). The substrate, H2O2, and electron donors such as I- and SCN- offered no protection against inactivation by both types of modifier, whereas the enzyme was completely protected by guaiacol or o-dianisidine, an aromatic electron donor (second substrate) oxidized by the enzyme. These studies indicate the involvement or arginine and tyrosine residues at the aromatic donor site of HRP. The guaiacol-protected phenylglyoxal-modified enzyme showed almost the same binding parameter (Kd) as the native enzyme, and a similar free energy change (deltaG')for the binding of the donor. Stoicheiometric studies with [7-14C]phenylglyoxal showed incorporation of 2 mol of phenylglyoxal per mol of enzyme, indicating modification of one arginine residue for complete activation. The difference absorption spectrum of the tetranitromethane-modified against the native enzyme showed a peak at 428 nm, characteristic of the nitrotyrosyl residue, that was abolished by treatment with sodium dithionite, indicating specific modification of a tyrosine residue. Inactivation stoicheiometry showed that modification of one tyrosine residue per enzyme caused 50% inactivation. Binding studies by optical difference spectroscopy indicated that the arginine-modified enzyme could not bind guaiacol at all, whereas the tyrosine-modified enzyme bound it with reduced affinity (Kd 35mM compared with 10mM for the native enzyme). Both the modified enzymes, however, retained the property of the formation of compound II (one-electron oxidation state higher than native ferriperoxidase) with H2O2, but reduction of compound II to native enzyme by guaiacol did not occur in the PGO-modified enzyme, owing to lack of binding. No non-specific change in protein structure due to modification was evident from circular dichromism studies. We therefore suggest that the active site of HRP for aromatic donor oxidation is composed of an arginine and an adjacent tyrosine residue, of which the former plays an obligatory role in aromatic donor binding whereas the latter residue plays a facilitatory role, presumably by hydrophobic interaction or hydrogen bonding.

Arginine↗
PubMed1996

(-)-Arctigenin as a lead structure for inhibitors of human immunodeficiency virus type-1 integrase.

The natural dibenzylbutyrolactone type lignanolide (-)-arctigenin (2), an inhibitor of human immunodeficiency virus type-1 (HIV-1) replication in infected human cell systems, was found to suppress the integration of proviral DNA into the cellular DNA genome. In the present study 2 was tested with purified HIV-1 integrase and found to be inactive in the cleavage (3'-processing) and integration (strand transfer) assays. However, the semisynthetic 3-O-demethylated congener 9 characterized by a catechol substructure exhibited remarkable activities in both assays. Structure-activity relationship studies with 30 natural (1-6), semisynthetic (7-21), and synthetic (37-43, 45, 46) lignans revealed that (1) the lactone moiety is crucial since compounds with a butane-1,4-diol or tetrahydrofuran substructure and also lignanamide analogues lacked activity and (2) the number and arrangement of phenolic hydroxyl groups is important for the activity of lignanolides. The congener with two catechol substructures (7) was found to be the most active compound in this study. 7 was also a potent inhibitor of the "disintegration" reaction which models the reversal of the strand transfer reaction. The inhibitory activity of 7 with the core enzyme fragment consisting of amino acids 50-212 suggests that the binding site of 7 resides in the catalytic domain.

4-Butyrolactone↗
PubMed1996

Allogeneic bone marrow transplantation in a patient with hypereosinophilic syndrome.

We describe a 32-year-old man with idiopathic hypereosinophilic syndrome (HES) who presented with pulmonary dysfunction, thrombocytopenia, lymphadenopathy, and hepatosplenomegaly. The patient developed progressive disease on prednisone and hydroxyurea therapy, and he underwent a successful allogeneic bone marrow transplantation (BMT). The patient is asymptomatic with no evidence of eosinophilia 30 months after transplantation. There is currently no cure for patients with HES, and BMT should be considered in selected patients.

Adult↗
PubMed1996

Immunomodulation following chemotherapy.

In the last decade, immunomodulation has emerged as a mode of therapy capable of mediating the regression of cancer in some patients. This article reviews our experience with immunomodulation following transplant and non-transplant chemotherapy. We used interferon and cyclosporine A following conventional chemotherapy in a non-transplant setting for a B16 melanoma in a murine model. This combination generated cells with MHC-unrestricted cytotoxicity. We have also used immunotherapy in the transplant setting with IL-2 activated PBSC in patients with breast cancer. Of the 28 patients treated, 20 developed GVHD and the average time to reconstitution was 12 days (comparable to a control group). This article also raises the possibility of extending immunomodulation to breast cancer patients in the nontransplant setting to induce an antitumor immune response following cytoreductive chemotherapy.

Animals↗
PubMed1996

Heregulin induces increase in sensitivity of an erbB-2-overexpressing breast cancer cell type to lysis by lymphokine-activated killer cells.

The erbB-2 oncoprotein is overexpressed in 30% of tumors from breast and ovarian cancer patients and it is related to poor overall and disease-free survival. In vitro studies on erbB-2-overexpressing cells have found a strong correlation between this oncogene overexpression and relative resistance to lymphokine-activated killer (LAK) cell lysis. gp30/heregulin/NDF (neu differentiation factor), indirect activators of erbB-2, are able to induce a more differentiated phenotype on erbB-2-overexpressing, erbB-3- and/or erbB-4-positive breast cancer cells. We tested the ability of these highly homologous growth factors to stimulate LAK cell lysis of breast cancer cells. Our experiments demonstrated a marked increase in LAK cell cytotoxicity towards an erbB-2-overexpressing, erbB-3-positive cell line by treatment of these cells with heregulin for 72 h. In contrast we did not observe any enhancement of lysis of MCF-7, a cell line that does not overexpress erbB-2 and is positive for the erbB-3 and erbB-4 receptors, after treatment with heregulin. The increased lysis was associated with upregulation of intercellular adhesion molecule 1 (ICAM-1), down-regulation of erbB-2 and increased binding between breast cancer cells and LAK cells. Pre incubation of target (SKBR3) cells with blocking anti-ICAM-1 antibody completely abrogated the enhanced cytotoxicity. A similar effect was observed by pretreatment of the effector (LAK) cells with antibodies directed against LFA-1, the receptor for ICAM-1. These results suggest the possible utilization of gp30/heregulin in the treatment of breast cancer patients by its ability to stimulate patient immune responses.

Breast Neoplasms↗
PubMed1996

Modulation of B16 melanoma growth and metastasis by anti-transforming growth factor beta antibody and interleukin-2.

Earlier evidence suggests that transforming growth factor beta (TGF beta) plays a significant role in tumor progression and metastasis. The most likely mechanism of the action of TGF beta is induction of immunosuppression in the host, allowing for unchecked tumor growth and metastasis. We attempted to test that hypothesis and to compare antitumor effects of anti-TGF beta antibody alone and in combination with interleukin-2 (IL-2). Six- to 8-week-old female C57B1-6 mice were induced with murine B16 melanoma by tail vein injection. Therapy was started 48 h after tumor injections. Monoclonal anti-TGF beta antibody (2G7) was administered intraperitoneally (i.p.) at 500 micrograms every other day, and IL-2 at 10,000 U i.p. twice daily, for 21 days. A threefold decrease in the number of lesions in the anti-TGF beta/IL-2 treatment group compared with the control group was observed, a highly significant statistical difference (p = 0.002). No statistically significant differences were seen between the control group and other studied groups (IL-2 alone, anti-TGF beta alone). Analysis of TGF beta levels in plasma by the TGF beta-1 Quantikine assay indicated normal levels in the control and IL-2 groups, and significantly diminished levels in the two groups that received TGF beta antibody. However, acid-ethanol extraction of plasma (to reverse antibody binding before assay) showed normal plasma TGF beta levels in all groups, suggesting that the antibody may alter the availability of TGF beta in vivo. Microscopic analysis of metastases revealed a decrease in the average size of lesions in the groups treated with IL-2. Thus, combination therapy using anti-TGF beta antibody and IL-2 may be a novel, less toxic approach to tumor immunotherapy.

Animals↗
PubMed1996

Effects of nucleotide analogues on human immunodeficiency virus type 1 integrase.

We extended our previous study with 3'-azido-3'-deoxythymidine nucleotides [Proc. Natl. Acad. Sci. USA 91:5771-5775 (1994)] and examined the effects on human immunodeficiency virus type 1 (HIV-1) integrase of the nucleotides of three nucleoside analogues currently under evaluation in clinical trials: beta-D-2',3'-didehydro-3'-deoxythymidine, beta-D-2'-ara-fluoro-2', 3'-dideoxyadenosine, and beta-L-2',3'-dideoxy-3'-thiacytidine. Beta-D-2',3'-Didehydro-3'-deoxythymidine and beta-D-2'-ara-fluoro-2',3'-dideoxyadenosine nucleotides had IC50 values for strand transfer of 100 and 200 microM, respectively, whereas the corresponding 2',3'-dideoxynucleoside triphosphates, ddT triphosphate and ddA triphosphate, did not inhibit the integrase at 800 and 200 microM, respectively. Beta-L-2',3'-Dideoxy-3'-thiacytidine triphosphate had no effect up to 500 microM. The L-enantiomers of 5-fluoro-2',3'-dideoxycytidine monophosphate and triphosphate had IC50 values of approximately 40 microM, whereas their D-enantiomer isomers showed no inhibition at 200 microM. NAD, pyridoxal phosphate, and coumermycin A1, which exhibit no antiviral activity but are typically used to probe nucleotide binding sites, were also tested. NAD was inactive, and its etheno derivative exhibited activity at 1 mM. In contrast, pyridoxal phosphate (IC50 = 18 microM and coumermycin A1 (IC50 = 5 microM were potent inhibitors. None of the coumermycin monomeric derivatives were active integrase inhibitors. The physiological ribonucleotides ATP and GTP inhibited HIV-1 integrase at or near cellular concentrations, suggesting that they may regulate HIV-1 integrase activity in cells. In general, the active nucleotides tested inhibited binding of HIV-1 integrase to its substrate DNA an inhibited an integrase deletion mutant containing only amino acids 50-212, indicating that nucleotides bind to the enzyme catalytic core. Consisently, the choice of nucleophile in the 3'-processing reaction was blocked to the same extent regardless of the nucleotide used (water, glycerol, or the viral DNA hydroxyl) by the enzyme. These observations suggest new strategies for antiviral drug development that could be based on nucleotide analogues as inhibitors of HIV-1 integrase.

Antiviral Agents↗
PubMed1996

Stem cell transplantation with chemoradiotherapy myeloablation and interleukin-2.

Interleukin 2 (IL-2) stimulates the proliferation of T-cells both in vitro and in vivo. When murine or human peripheral blood (PB) or bone marrow (BM) mononuclear cells are incubated with IL-2 in vitro for 24 hours, cytotoxic T-cells are generated. If these activated cells are infused into mice, the enhanced cytotoxicity continues if low dose IL-2 is administered. This combination of administering activated cells with the subsequent low dose IL-2 infusion results in enhanced tumor cell destruction and improved survival rates in mice with acute myeloid leukemia. The encouraging results of these laboratory experiments prompted the initiation of phase I clinical trials in patients with refractory/relapsed hematologic malignancies and patients with breast cancer (Stages II-IV). Results from these trials demonstrate that stem cell transplantation with IL-2 activated stem cells (either PB or BM) with or without parenteral administration of IL-2 results in hematopoietic reconstitution with mild-to-moderate toxicities. This regimen also generates cutaneous and visceral autologous graft versus host disease (AuGVHD). The majority of our patients with relapsed/refractory hematologic malignancies or breast cancer developed either clinical and/or histological evidence of AuGVHD. Further studies are being conducted to determine if patients who develop AuGVHD experience improved disease-free survival from a possible autologous graft versus tumor (GVT) effect. Current laboratory evaluations include the elucidation of the pathogenesis of AuGVHD and molecular evaluation of the purging efficacy of IL-2.

Animals↗
PubMed1996

The effect of hydroxychloroquine on alloreactivity and its potential use for graft-versus-host disease.

Hydroxychloroquine (HCQ) interferes with antigen processing and with receptor loading and recycling by raising the pH of lysosomes and endosomes. HCQ thus inhibits MHC class II-restricted antigen presentation by blocking the binding of peptides to MHC molecules. Additionally, HCQ has been shown to diminish the release of several cytokines. In light of these mechanisms of action, we felt that HCQ might be useful in the bone marrow transplant setting and therefore investigated its effect on alloreactivity in vitro. We have demonstrated that HCQ causes a dose-dependent reduction of the cytotoxicity, proliferation, and TNF alpha production resulting from allorecognition in mixed lymphocyte culture (MLC). HCQ does not mediate its effect solely through antigen processing and presentation and other early events since addition of HCQ as late as 120 h after the initiation of the MLC still has a suppressive effect on cytotoxicity. HCQ also inhibits the cytotoxicity of previously primed effectors. These results support an effect of HCQ on terminal mechanisms of cytotoxicity that have not been previously reported. HCQ's ability to reduce the cytotoxicity, proliferation, and TNF alpha production resulting from allorecognition suggests that it may be useful in the prevention and treatment of graft-versus-host disease (GVHD).

Cyclosporine↗
PubMed1996

Marrow engraftment and clinical manifestations of capillary leak syndrome.

A capillary leak syndrome occurs frequently in bone marrow transplant patients in addition to graft-versus-host-disease and infection. The underlying pathology is poorly understood but the clinical manifestations of excessive weight gain, ascites, and edema associated with kidney and liver abnormalities suggest a common injury to multiple organs. The clinical courses of 55 allogeneic and autologous marrow transplant recipients were retrospectively reviewed with regard to the presentation of a capillary leak syndrome (CLS). Twenty-nine patients (53%) developed non-cardiogenic pulmonary edema with or without concurrent pleural effusions; the incidence was comparable in allogeneic and autologous recipients. Pulmonary features were accompanied by hepatic dysfunction in 28, renal dysfunction in 22, and central nervous system abnormalities in 17. There was a strong correlation between time of engraftment and the first manifestations of CLS, both of which occurred earlier in allogeneic than in autologous transplant recipients. These data confirm that CLS occurs in both allogeneic and autologous transplant recipients and suggest a pivotal contribution by circulating leukocytes.

Bone Marrow Transplantation↗
PubMed1996

Hematopoietic potential of IL-2-cultured peripheral blood stem cells from breast cancer patients.

Patients receiving autologous transplants for various malignancies generally experience an increased incidence of relapse compared with patients receiving unmanipulated allogeneic transplants. We initiated a protocol for IL-2 activation of peripheral blood stem cells (PBSC) for induction of in vitro and in vivo autologous graft-versus-tumor (GVT) activity in patients with breast cancer. In this study we analyzed the effects of 24 h of IL-2 incubation on the hematopoietic potential of PBSC from these patients. Cells collected by leukapheresis were first cryopreserved and stored in liquid nitrogen, then thawed rapidly and incubated with IL-2 in a serum-free system for 24 h, with samples analyzed before and after incubation. Although there was a significant drop in mononuclear cells (MNC) (from 4.5 to 3.7 x 10(8)/kg) and CD34+ cells (from 12.3 to 7.5 x 10(6)/kg) after 24 h in culture, there was no significant change in colony-forming units (CFU) (from 12.5 to 11.5 x 10(5)/kg). Time to engraftment (neutrophils: < 0.5 x 10(9)/l; platelets: > 20 x 10(9)/l) was comparable to a cohort of similar patients receiving non-cultured PBSC transplants. These results indicate that mobilized frozen/thawed PBSC which have been cultured in IL-2 for 24 h retain adequate potential for hematopoietic reconsistution in this group of patients.

Breast Neoplasms↗
PubMed1996

Towards endothelial-cell-directed cancer immunotherapy: in vitro expression of human recombinant cytokine genes by human and mouse primary endothelial cells.

Recent studies have demonstrated the feasibility of cytokine gene transfer to enhance the antitumor activities of host immune cells. Endothelial cells forming the vascular supply of tumors may be useful vehicles for the delivery of cytokine molecules in order to effect tumor immunotherapy. In order to determine whether primary endothelial cells can express cytokine transgenes efficiently, we constructed two retroviral vectors containing a cDNA encoding either recombinant human interleukin-1 alpha (rhIL-1 alpha) or recombinant human interleukin-2 (rhIL-2), called LNCIL-1 alpha and LNCIL-2 respectively, and studied the expression of the two cytokines in vitro in non-immortalized endothelial cells. Human umbilical vein endothelial cells (HUVEC) transduced with LNCIL-1 alpha or LNCIL-2 secreted 1.8-33 ng/10(6) cells/24 h and 40-246.7 ng/10(6) cells/24 h of biological active rhIL-1 alpha and rhIL-2 respectively. Mouse microvascular endothelial cells (MMEC) transduced with LNCIL-1 alpha and LNCIL-2 secreted 1.5 ng/10(6) cells/24 h and 5.8-24.7 ng/10(6) of biologically active rhIL-1 alpha and rhIL-2 proteins respectively. Cocultivation of HUVEC/IL-2 and MMEC/IL-2 with normal human bone marrow cells generated potent cytotoxic activity against K562, Daudi and other cell targets in a 51Cr-release assay. While IL-2 transgene-expressing HUVEC and MMEC retained their normal morphology, rhIL-1 alpha transgene expression inhibited the growth and altered the morphology of both HUVEC and MMEC in culture. The cytokine-gene-transduced endothelial cells retained other endothelial cell features, including uptake of acetylated low-density lipoprotein (Ac-LDL) and expression of von Willebrand factor, and were euploid as shown by flow cytometry. These results demonstrate that endothelial cells, by sustaining the production of biologically active rhIL-2 at levels that are sufficient for the activation of potent cytotoxic lymphocyte activity, may be useful agents for cancer gene therapy.

Animals↗