Reinforced cytotoxicity of lymphokine-activated killer cells toward glioma cells by transfection with the tumor necrosis factor-alpha gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human tumor necrosis factor (TNF)-alpha gene by means of novel liposomes with a positive change on their surface. The cells secreted significant amounts of TNF-alpha into the culture medium and exhibited reinforcement of cytotoxicity toward a human glioma cell line (U251-SP), being three times more cytotoxic than nontransfected LAK cells. The mechanism for the reinforcement of cytotoxicity is considered to involve not only an increase in TNF-alpha secretion from LAK cells but also its expression on their surface. Intratumoral or intrathecal injection of LAK cells transfected with the TNF-alpha gene may be useful for the treatment of patients with malignant gliomas.     

Specific cytotoxicity in vitro of lymphocytes sensitized in culture against tumor cells

The production of tumor-specific cell-mediated cytotoxicity following in vitro sensitization of C57BL spleen cells against a syngeneic 3LL Lewis lung carcinoma was studied. Lymphocytes were sensitized on monolayers of the tumor cells for 4-5 days. The cytotoxicity was assayed by measuring the reduction in 3H-leucine and 3H-thymidine incorporation by target cells after interaction with the sensitized lymphocytes. Spleen lymphocytes sensitized on monolayers of 3LL tumor cells caused a high extent of lysis; such cells tested on C57BL or C3H fibroblast targets evoked only a low level of cytotoxicity. C57BL spleen cells sensitized on C57BL fibroblasts caused a low level of cytotoxicity when tested on a 3LL target. Thus cytotoxicity appeared to be tumor specific. The reduced incorporation into protein and DNA of target tumor cells caused by the sensitized lymphocytes was a measure of cell injury, which was more sensitive than direct cell count or uptake of 51CR. Lymphocytes from syngeneic tumor-bearing mice, tested 13-25 days after tumor inoculation, did not manifest in vitro cytotoxicity. On the contrary, such lymphocytes sometimes appeared to have a promoting effect on the tumor cells.     

Enhancing effect of O6-alkylguanine derivatives on chloroethylnitrosourea cytotoxicity toward tumor cells

O6-Alkylguanine derivatives are well known as chemical modulators of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Depletion of the enzyme by these derivatives leads to increase sensitivity of tumor cells to chloroethylnitrosoureas. We tested the effect of O6-methylguanine, O6-benzylguanine, O6-(p-methylbenzyl)guanine, O6-(p-chlorobenzyl)guanine, O6-(p-methoxybenzyl)guanine, O6-methylhypoxanthine and O6-benzylhypoxanthine on the sensitivity of tumor cell lines to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) using a colorimetric cytotoxicity assay. The sensitivity of MGMT-proficient tumor cells including HeLA S3, C6-1, C6-2/ACNU, U-138 MG and U-373 MG cells was greatly enhanced by 2 hr pretreatment of 10-100 microM O6-benzylguanine, O6-(p-methylbenzyl)guanine and O6-(p-chlorobenzyl)guanine, but not by O6-methylguanine or O6-methylhypoxanthine. O6-(p-methylbenzyl)guanine moderately sensitized the 2 cell lines, HeLa S3 and C6-1, tested in our study to ACNU cytotoxicity. O6-Benzylhypoxanthine at the high concentration (100 microM) sensitized, to some extent, 3 MGMT-proficient cell lines. Lesser degrees of enhancement by the O6-benzylguanine derivatives were noted in MGMT-deficient tumor cells. Biological effects of O6-alkylguanine derivatives on enhancing ACNU cytotoxicity of tumor cells suggest that the exocyclic 2-amino and O6-benzyl groups in O6-benzylguanine skeleton are both essential for the inhibition of MGMT activity.     

Potentiation of photodynamic therapy by mitomycin C in cultured human colon adenocarcinoma cells

Severe ketotic diabetes induced in rats by streptozotocin resulted in a reduction in activity of the hepatic branched-chain alpha-ketoacid dehydrogenase complex, regardless of whether activity was expressed on the basis of liver wet weight, total liver, liver protein, or liver DNA. A decrease in enzyme specific activity (units of enzyme activity per mg of enzyme protein) was found responsible for the reduction in measurable enzyme activity of the complex. Insulin treatment reversed the decrease in enzyme specific activity. Treatment of tissue extracts with phosphoprotein phosphatase had no effect, indicating that activity of the complex was decreased by some mechanism other than reversible phosphorylation. Specific protein components of the complex were also not found reduced by the diabetic state. Induction of severe ketotic diabetes in rats previously fed a low-protein diet resulted in activation of the enzyme as a consequence of dephosphorylation. Nevertheless, the specific activity of the dephosphorylated enzyme of diabetic, low-protein-fed rats was decreased relative to that of control, low-protein-fed animals. Reconstitution studies with tissue extracts fortified with the purified E1 component indicate that severe diabetes induces a defect in this component of the hepatic branched-chain alpha-ketoacid dehydrogenase complex.     

Synergistic effect and possible mechanisms of tumor necrosis factor and cisplatin cytotoxicity under moderate hyperthermia against gastric cancer cells

Photosensitization induces intracellular free calcium changes ([Ca2+]i) in some eukaryotic cell systems which either contribute to or protect against cell inactivation. We have investigated whether or not similar changes can be induced in prokaryotes. The skin bacterium Propionibacterium acnes was sensitized using protoporphyrin IX (PP IX) or 5-aminolevulinic acid (ALA). Exogenous ALA resulted in either a preferential accumulation of protoporphyrin (ALA-PP) or of coproporphyrin and/or uroporphyrin (ALA-CP/UP) in P. acnes. For PP IX or ALA-PP sensitization, exposure to broad-band red light resulted in an increase in [Ca2+]i. For ALA-PP sensitization, this increase was transient and [Ca2+]i returned to basal levels within 5-10 min after irradiation. However, the elevated [Ca2+]i levels obtained after PP IX sensitization were maintained for at least 1 h after irradiation. In both cases, the reduction in the external calcium concentration led to an enhancement in the cell survival, indicating that induced [Ca2+]i changes may participate in photoinactivation. Sensitization by hydrophilic coproporphyrin and/or uroporphyrin (ALA-CP/UP) did not affect the [Ca2+]i levels, but higher levels of cell inactivation were obtained. It therefore appears that damage to membrane-associated components is at least partly responsible for [Ca2+]i alterations after photosensitization.     

Protein replacement by receptor-mediated endocytosis corrects the sensitivity of Fanconi anemia group C cells to mitomycin C

Current methods for direct gene transfer into hematopoietic cells are inefficient. Here we show that functional complementation of Fanconi anemia (FA) group C cells by protein replacement can be as efficacious as by transfection with wild-type FAC cDNA. We expressed a chimeric protein (called His-ILFAC) consisting of the mature coding portion of gibbon interleukin-3 (IL-3) and full-length FAC in Escherichia coli. The purified bacterial protein is internalized by hematopoietic cells via IL-3 receptors. The intracellular half-life of His-ILFAC is approximately 60 minutes, which is comparable to that of the transgene-encoded FAC protein. In this cell-culture model His-ILFAC completely corrects the sensitivity of FA group C cells to mitomycin C, but it has no effect on FA cells that belong to complementation groups A and B. We suggest that receptor-mediated endocytosis of cytokine-fusion proteins may be of general use to deliver macromolecules into hematopoietic progenitor cells.     

Enhancement of cyclophosphamide cytotoxicity in vivo by the benzamide analogue pyrazinamide

Bovine conglutinin is a serum lectin that agglutinates erythrocytes preincubated with antibodies and complement. This agglutination occurs through the binding of conglutinin to iC3b, a fragment of the complement component C3. It was reported that conglutinin binds fluid-phase C3b and C3c as well as iC3b. We re-investigated the reactivity of conglutinin towards fluid-phase C3 degradation products. ELISA wells were coated with conglutinin and reacted with C3 split products generated in normal human serum, in factor I-deficient serum, or in factor I-depleted serum. Conglutinin-bound C3 fragments were detected with anti-C3c and anti-C3d antibodies. An increased signal was observed during the activation of complement in normal human serum with the peak response after 1-2 hr, following which the signal decreased, reaching background level after 72 hr. The oligosaccharides on C3c, generated in serum, are thus not recognized by conglutinin. No signal was observed when factor I-deficient serum or factor I-depleted serum was used instead of normal serum. Reconstitution with purified factor I re-established the normal pattern. Examination of the conglutinin-bound C3 molecules by SDS-PAGE and Western blotting with anti-C3c and anti-C3d antibodies revealed bands characteristic for iC3b, and no bands corresponding to C3b or C3c. Reduction of the disulphide bonds prior to the incubation of the activated serum with the conglutinin-coated wells revealed a band of 63,000 MW, characteristic of the N-terminal fragment of the alpha-chain of iC3b. We also investigated the binding to the solid-phase conglutinin of purified C3 and degradation products generated with enzymes. In this case, C3 as well as C3b and C3c were bound, suggesting conformational changes in C3 during purification. In conclusion, when C3 conversion takes place at near physiological conditions, conglutinin interacts specifically with the oligosaccharide on the alpha-chain of iC3b.     

Identification of nonproliferating but viable hypoxic tumor cells in vivo

We have used the combination of pimonidazole labeling of hypoxic cells, bromodeoxyuridine labeling of proliferating cells, and cell sorting based on Hoechst 33342 perfusion to directly study hypoxia and proliferation in human tumor xenografts and transplantable murine tumors in vivo. Hypoxia was largely confined to cells in regions with the least perfusion, although in tumors exhibiting transient blood flow, hypoxic cells were not as highly localized. Similarly, proliferation and hypoxia were mutually exclusive except in areas of a tumor subjected to transient changes in perfusion. By determining the clonogenic potential, pimonidazole labeling intensity, and radiosensitivity of sorted tumor cell subpopulations, we have provided direct evidence that pimonidazole identifies hypoxic tumor cells of therapeutic relevance in vivo. Given that pimonidazole exhibits few diffusion or delivery problems and no apparent cytotoxicity, it appears to be a versatile and useful label for hypoxic cells in solid tumors.     

Evaluation of 64Cu-ATSM in vitro and in vivo in a hypoxic tumor model

We have evaluated Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), an effective marker for the delineation of hypoxic but viable tissue, in vitro in the EMT6 carcinoma cell line under varying degrees of hypoxia and compared it with the flow tracer 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) and the hypoxic tracer 18F-fluoromisonidazole (MISO). We have also compared the uptake of Cu-ATSM and Cu-PTSM in vivo and ex vivo in a murine animal model bearing the EMT6 tumor. METHODS: Uptake of 64Cu-ATSM, 64Cu-PTSM and 18F-MISO in vitro into EMT6 cells was investigated at the dissolved oxygen concentrations of 0, 1 x 10(3), 5 x 10(3), 5 x 10(4) and 2 x 10(5) ppm. Biodistribution performed at 1, 5, 10, 20 and 40 min compared 64Cu-ATSM with 64Cu-PTSM in BALB/c mice bearing EMT6 tumors. To determine long-term retention of 64Cu-ATSM, biodistribution was also performed at 1, 2 and 4 h. Ex vivo autoradiography of tumor slices after co-injection of 60Cu-PTSM (60Cu, T1/2 = 23.7 min) and 64Cu-ATSM (64Cu, t1/2 = 12.7 h) into the same animal was performed. RESULTS: After 1 h, 64Cu-ATSM was taken up by EMT6 cells: 90% at 0 ppm, 77% at 1 x 10(3) ppm, 38% at 5 x 10(3) ppm, 35% at 5 x 10(4) ppm and 31% at 2 x 10(5) ppm. 18F-MISO also showed oxygen concentration dependent uptake, but with lower percentages than 64Cu-ATSM. 64Cu-PTSM showed 83%-85% uptake into the cells after 1 h, independent of oxygen concentration. Biodistribution data of 64Cu-ATSM and 64Cu-PTSM showed optimal tumor uptake after 5 and 10 min, respectively (0.76% injected dose (ID)/organ for 64Cu-ATSM and 1.11%ID/organ for 64Cu-PTSM). Ex vivo imaging experiments showed 60Cu-PTSM uniform throughout the EMT6 tumor, but heterogeneous uptake of 64Cu-ATSM, indicative of selective trapping of 64Cu-ATSM into the hypoxic tumor cells. CONCLUSION: Cu-ATSM exhibits selectivity for hypoxic tumor tissue both in vivo and in vitro and may provide a successful diagnostic modality for the detection of tumor ischemia.     

Spermatogenic cell apoptosis induced by mitomycin C in the mouse testis

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity.     

Natural cell-mediated cytotoxicity (NCMC) against NK-sensitive tumours in vitro by murine spleen Ly-6C+ natural T cells

Ly-6C+ cells constitute 13 +/- 3% of freshly isolated (CBA x C57BL/6)F1 mouse spleen leukocytes. Three distinct populations were identified: CD3 epsilon +NK-1.1- conventional T cells (6%), CD3 epsilon -NK-1.1- granulocytes (5%) and CD3 epsilon +NK-1.1+ T cells (approximately 2%). The CD3 epsilon +NK-1.1+ cells displayed a predominantly large granular leukocyte morphology and were the only Ly-6C+ cell subset identified by MAb 2B6-F2 to spontaneously lyse the NK-sensitive YAC-1 tumour in vitro. On further phenotypic analysis, these cells co-expressed high levels of TCRV beta 8.1/8.2 and CD11b, moderate levels of CD90 and low levels of CD4 or CD8. The removal of CD4+ and CD8+ cells prior to Ly-6C+ cell sorting showed that it was the CD4-CD8- double-negative (DN) CD3 epsilon +NK-1.1+ T-cell subset which was responsible for killing YAC-1. These results indicate that we have identified a DN Ly-6C+ subset of the recently designated NK-1.1+TCR alpha beta low natural T (NT) cells, which are capable of natural cell-mediated cytotoxicity (NCMC) against the NK-sensitive YAC-I tumour in vitro. Additionally, these cells mediated the in vitro killing of 2 further NK-sensitive tumours, murine B16 melanoma and human Jurkat T lymphoma. YAC-1 and Jurkat expressed Fas and were susceptible to anti-Fas MAb or rhuman Fas ligand (rhFasL)-induced lysis. Furthermore, anti-human Fas MAb M3 was shown to block sorted Ly-6C+ splenocyte in vitro killing of Jurkat. In contrast, B16 did not express cell-surface Fas and was resistant to anti-Fas MAb-induced lysis. Taken together, these results show that not only do Ly-6C+ NT cells kill NK-sensitive tumours in vitro but they mediate this activity via multiple cytotoxic mechanisms including Fas.     

Generation of in vitro natural cytotoxicity of horse lymphocytes against sarcoid-derived tumor cells not expressing major histocompatibility complex antigens

OBJECTIVE: To analyze in vitro lymphocyte-mediated immune responses of horses with sarcoids against allogeneic sarcoid cells containing endogenous retrovirus but not expressing major histocompatibility complex antigens. DESIGN: Lymphocyte-mediated immune reactions were assessed by means of proliferative responses in mixed lymphocyte tumor cell culture (MLTC) assay and lymphocyte-mediated cytotoxicity against various equine target cells. ANIMALS: 12 horses with sarcoid tumors and 15 control horses. PROCEDURE: Blood lymphocytes were cocultured in MLTC with allogeneic sarcoid cells (Mc-1, BayMc-1), equine testis cells, or normal equine dermal fibroblasts. Lymphocytes were assayed for proliferative responses by [3H]thymidine uptake and for cytotoxicity against the same targets by 51Cr release assay. The lymphocyte populations were analyzed for some common surface markers. RESULTS: Lymphocytes from horses with sarcoids exerted an anamnestic proliferative response in MLTC against Mc-1 cells, but this procedure never generated cytotoxic lymphocytes. However, lymphocytes from all horses cultured in medium with 10% allogeneic serum only had selective. natural cytotoxicity against Mc-1 that was generated without DNA synthesis. Approximately 80% of the lymphocytes disappeared during culture; however the remaining population of small, viable lymphocytes indicated a decrease of CD4+ T lymphocytes, but numbers of T cells with receptors for Helix pomatia A hemagglutinin were unaffected. Few lymphocytes had Fc-receptors for IgG, were complement-reactive positive cells or were B cells expressing surface immunoglobulin. CONCLUSIONS: Results may indicate a natural defense system, which preferentially recognizes and lyses tumor cells that are deficient in surface expression of major histocompatibility complex antigens, without intervention of conventional T-cell receptors or antibodies.     

The role of effector cells and antiserum in the inhibition of cell-mediated cytotoxicity of allogeneic tumor cells

Previous experiments in this laboratory demonstrated a progressive decrease in cell-mediated cytotoxicity (CMC) against allogeneic tumor cells by immune spleen cells from mice repeatedly immunized with those tumor cells. In the present study, immune spleen cells, obtained at specified intervals during the course of multiple immunizations of BALB/c mice with EL-4 lymphoma cells, were tested for CMC against EL-4 target cells pretreated with anti-EL-4 serum which had been obtained from singly or repeatedly immunized animals. Cytolysis of EL-4 cells was measured by a 51Cr-release assay. The results indicate that blocking of CMC in an allogeneic tumor model may occur by two pathways. First, antigen or antigen-antibody complexes present in the immunized animal may bind in vivo to the antigen receptor sites of of sensitized effector cells that are used in the in vitro CMC assay, thereby blocking their interaction with tumor cells. Second, immune serum that is added to the in vitro CMC assay may contain highly avid antibodies, as well as antigen-antibody complexes, that bind to tumor cells and thereby block interaction with sensitized effector cells. The identification of these elements may be of prognostic significance in certain clinical situations.     

Enhancement of terminal B lymphocyte differentiation in vitro by fibroblast-like stromal cells from human spleen

Stromal elements are major components of lymphoid tissues contributing to both tissue architecture and function. In this study we report on the phenotype and function of fibroblast-like stromal cells obtained from human spleen. These cells express high levels of CD44 and ICAM-1 and moderate levels of VLA-4, VCAM, CD40 and CD21. They fail to express endothelial, epithelial, lymphocyte and monocyte/macrophage markers. We show that these cells interact with B cell blasts induced in vitro by anti-CD40 and anti-mu stimulation. As a result of these interactions both IL-6 and IgG secretion into culture medium is increased. The enhanced secretion of IgG is partly inhibited by abolishing B cell blaststromal cell contact or by anti-IL-6, anti-VCAM or anti-CD49d antibodies. Our studies also suggest that the ability of stromal cells to promote B cell survival is most likely the underlying mechanism of the enhanced immunoglobulin secretion. Comparison of stromal cells from different lymphoid and non-lymphoid organs revealed that bone marrow- and spleen-derived stromal cells are the most effective in promoting B cell blast differentiation.     

In vitro cytotoxicity of paclitaxel-transferrin conjugate on H69 cells

Transferrin is a serum glycoprotein involved in iron transport. Transferrin acts also in cell growth regulation through membrane receptors. The number of transferrin receptors is increased in tumor and other rapidly dividing cells. This renders transferrin suitable for use in cytotoxic drugs targetting tumor cells. Paclitaxel was derivatized on 2' carbon and coupled with trasferrin using glutaraldehyde. The cytotoxicity of the conjugate was evaluated on small cell carcinoma of the lung cell line (H69). As compared to paclitaxel, the conjugate exhibited a slight decrease in cytotoxicity.     

Enhancement of thermal effect in virally infected mammalian cells in vitro and its modification by chlorpromazine

Somatostatin receptor-expressing tumours are potential targets for therapy with radiolabelled somatostatin analogues. We have synthesized a number of such analogues in the past and identified [DOTA-dPhe1, Tyr3]octreotide (SMT 487) as the most promising candidate molecule because of its advantageous properties in cellular and in vivo tumour models. In the current paper we describe the radiotherapeutic effect of yttrium-90 labelled SMT 487 in Lewis rats bearing the somatostatin receptor-positive rat pancreatic tumour CA 20948. SMT 487 binds with nanomolar affinity to both the human and the rat somatostatin receptor subtype 2 (sst2) (human sst2 IC50=0.9 nM, rat sst2 IC50=0.5 nM). In vivo, 90Y-SMT 487 distributed rapidly to the sst2 expressing CA 20948 rat pancreatic tumour, with a tumour-to-blood ratio of 49.15 at 24 h post injection. A single intravenous administration of 10 mCi/kg 90Y-SMT 487 resulted in a complete remission of the tumours in five out of seven CA 20948 tumour-bearing Lewis rats. No regrowth of the tumours occurred 8 months post injection. Control animals that were treated with 30 microg/kg of unlabelled SMT 487 had to be sacrificed 10 days post injection due to excessive growth or necrotic areas on the tumour surface. Upon re-inoculation of tumour cells into those rats that had shown complete remission, the tumours disappeared after 3-4 weeks of moderate growth without any further treatment. The present study shows for the first time the curative potential of 90Y-SMT 487-based radiotherapy for somatostatin receptor-expressing tumours. Clinical phase I studies with yttrium-labelled SMT 487 have started in September 1997.     

Enhancement of antitumor cytotoxicity of hepatic lymphocytes by oral administration of PSK

Neuroimaging in epilepsy now includes magnetic resonance imaging, positron emission tomography (PET), single-photon emission tomography (SPECT), magnetoencephalography, and magnetic resonance spectroscopy. Recent advances in neuroimaging include improved rates of detection of lesions on structural imaging using quantitative methods, identification of metabolic patterns on functional imaging with PET, and recognition of the value of ictal SPECT in seizure localization.