Low-density lipoprotein induced actin cytoskeleton reorganization in endothelial cells: mechanisms of action.

The inhibitory effects of the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, nordihydroguaiaretic acid (NDGA) and SKF525A, on the disruption of dense peripheral bands and formation of stress fibers in cultured human umbilical vein endothelial cells exposed to atherogenic low-density lipoprotein (LDL) levels has been investigated. Endothelial cells (EC) in vitro and in vivo exposed to high LDL-cholesterol levels have cytoskeletal remodeling with stress fiber formation and loss of dense peripheral bands. Cultured EC incubated with exogenously applied hydrogen peroxide (H2O2: 1 mM) have cytoskeletal structural changes much similar to those observed with high LDL exposure. Previous studies have 1) demonstrated that exposure to atherogenic LDL levels causes heightened EC H2O2 production, 2) identified the reactive oxygen species source, NADPH oxidase, in EC, and 3) shown that the specific NADPH oxidase inhibitor, apocynin, and non-specific NADPH oxidase inhibitors, NDGA and SKF525A, suppress H2O2 production increases in high LDL-perturbed EC. In the present study, the cytoskeletal structure of EC exposed to 330 mg/dl LDL-cholesterol, and incubated with or without apocynin, NDGA and SKF525A, was examined. Each of these compounds promoted the retention of dense peripheral bands and minimized stress fiber formation. These findings are consistent with NADPH oxidase and it's reactive oxygen species byproducts modulating the cytoskeleton reorganization observed in high LDL-induced EC perturbation.

Endothelial cell oxidant production: effect of NADPH oxidase inhibitors.

The effects of known leukocyte NADPH oxidase inhibitors on general cellular oxidant production in cultured human endothelial cells (EC) has been investigated. EC were stimulated with 10 nM phorbol 12-myristate 13-acetate and cellular oxidant production measured in the presence and absence of inhibitors that act on various substituents of the oxidase complex and its activation pathways. The effects of the cytosolic oxidase subunit translocation inhibitors, catechols (3,4-dihydroxybenzaldehyde, caffeic acid, and protocatechuic acid), ortho-methoxy-substituted catechols (apocynin, vanillin, and 4-nitroguaiacol), and quinone, 1,4-naphthoquinone; flavoprotein inhibitors, diphenylene iodonium and quinacrine; haem ligands, imidazole and pyridine; directly acting thiol reagents, disulfiram and penicillamine; NADPH analogue, Cibacron Blue; redox active inhibitors, quercetin and esculetin; intracellular calcium antagonist, TMB-8; and calmodulin antagonists, W-7 and trifluoperazine, were determined. All compounds reduced oxidant production in stimulated EC. These findings add to previous observations suggesting the presence of a functionally active NADPH oxidase in EC. Identifying the major cellular reactive oxygen species source in perturbed EC will provide new insights into our understanding of endothelial dysfunction, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.

Thrombin stimulated reactive oxygen species production in cultured human endothelial cells.

In order to study the major cellular source of reactive oxygen species (ROS) in perturbed human endothelial cells (EC), the effect of thrombin, a phospholipase A2 activator, on cultured EC ROS generation has been investigated. EC were incubated with 0.1-1 unit/ml thrombin and cellular superoxide anion (O(-)2) release and hydrogen peroxide (H2O2) production measured. Thrombin exposure caused an elevation in EC O(-)2 release and H2O2 production. The effects of protein kinase C, arachidonic acid metabolism, NADPH oxidase, and phospholipase A2 inhibitors on thrombin-induced EC H2O2 production were examined. EC were exposed to 0.5 unit/ml thrombin and cellular H2O2 production measured in the presence and absence of the protein kinase C inhibitor, H-7; arachidonic acid metabolism inhibitors, indomethacin, nordihydroguaiaretic acid, and SKF525A; NADPH oxidase inhibitor, apocynin; and phospholipase A2 inhibitor, 4-bromophenacyl bromide. All inhibitors, with the exception of H-7 and indomethacin, suppressed thrombin-induced EC H2O2 production. The pattern of effects of these metabolic antagonists on thrombin-induced EC ROS production is similar to that previously reported on ROS production in EC exposed to high low-density lipoprotein levels, and in stimulated leukocytes. These findings further implicate NADPH oxidase as a major ROS source in EC.

Chemical modification studies of the active centre of Candida albicans chitinase and its inhibition by allosamidin.

Allosamidin, a glycoside antibiotic, is shown to be a strong, competitive inhibitor of semi-purified chitinase from yeast cells of Candida albicans. The inhibitory potency of allosamidin was pH-dependent, with IC50 values of 280 nM at pH 5.0 and 21 nM at pH 7.5. At higher, micromolar, concentrations, allosamidin inactivated this chitinase in a time- and concentration-dependent manner. Kinetic studies of this inactivation provided evidence for the formation of a reversible complex between allosamidin and chitinase, characterized by Kinact = 5 microM, followed by irreversible modification of the enzyme with velocity constant k2 = 4.6 x 10(-3) s-1. Chemical modification studies with the use of group-specific reagents suggested the presence of Glu/Asp carboxyl group(s) at or near the active site, that were important for enzyme activity. The carboxyl-specific reagent, 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide, inactivated the chitinase in a single step process, with apparent second-order rate constant of 0.014 M-1 s-1.

Kluyveromyces lactis toxin has an essential chitinase activity.

The Kluyveromyces lactis toxin is a protein containing three subunits (alpha, beta and gamma) which causes sensitive yeast cells to arrest proliferation in the G1 phase of the cell cycle. Despite the toxin's complex structure, the gamma subunit appears to be the only component required for it to arrest proliferation since intracellular expression of the gamma polypeptide alone in a sensitive yeast strain mimics the effect of the exogenous native toxin. The toxin alpha subunit shows sequence similarity to a variety of chitinases and here we report that the toxin is a potent exochitinase. The exochitinase activity is absolutely required for its biological activity against sensitive Saccharomyces cerevisiae cells and allosamidin, a specific inhibitor of chitinases, abolishes the biological activity of the toxin. However, since the alpha subunit is not required for the G1 arrest induced by the toxin, the chitinase activity of the toxin cannot be directly responsible for the ultimate effect of the toxin and most likely plays a role in the initial interaction of the toxin with sensitive cells.

Renal cell carcinoma treated by vaccines for active specific immunotherapy: correlation of survival with skin testing by autologous tumor cells.

Eighteen patients with metastatic renal cell carcinoma, who were treated by vaccines for active specific immunotherapy, also completed skin testing with autologous tumor cells, both prior to and following vaccine treatment. All patients have now been followed for more than 5 years. Ten patients who remained skin-test-negative following treatment had no clinical responses, and all had expired by 22 months. Eight patients became skin-test-positive; three of these had clinical regressions and three remain alive after more than 69 months. The survival times of the skin-test-positive group were significantly superior to those of the skin-test-negative group. The results suggest that skin testing with autologous tumor cells may accurately identify those patients who have acquired antigen-specific cell-mediated antitumor immunity.

Influence of intravesical administration of tumor necrosis factor alpha and systemic gamma-interferon on murine bladder cancer: lack of dose response.

The effect of intravesical administration of high dose recombinant tumor necrosis factor-alpha (rTNF) and in combination with systemic recombinant gamma-interferon (rIFN) on murine bladder cancer was studied. RTNF was given at 12.5 micrograms/mouse on days 7, 11 and 15 after tumor instillation or at 2.5 micrograms/mouse on days 7, 9, 11, 13 and 15. Some groups were also injected i.v., 24-h prior to each rTNF treatment with rIFN at a dose of 1.3 micrograms/mouse. RTNF treatment suppressed tumor growth up to 48% of control, although the difference was not statistically significant. Combined administration of rIFN did not provide additional benefit.

Interleukin 2 suppression of a murine bladder cancer implanted into kidney, bladder and skin; its organ specificity.

Little is known about organ associated tumor response to systemic interleukin 2 (IL2) therapy. The effect of IL2 on bladder cancer growth in the skin and in the genitourinary tract was investigated. C3H mice were implanted with the syngeneic transitional cell carcinoma, MBT-2, intradermally (i.d.), beneath the left renal subcapsular area, and in one experiment, simultaneously in the bladder. IL2 (human recombinant form; Biogen Research Co) was given i.p. at 5000 U thrice daily for 5 consecutive days commencing on Day 3, or for 10 to 11 days commencing on Day 10 with some doses omitted at signs of toxicity. For comparison, mice bearing 3-d and 10-d tumors in the skin and subcapsular kidney were treated with chemotherapy (cisplatin, 6 mg./kg. X 3; mitomycin C, 3 mg./kg. X 3; cyclophosphamide, 75 mg./kg. X 1). IL2 therapy mediated growth suppression of 10-d tumors in the genitourinary organs and skin at a similar rate. In contrast to IL2, systemic chemotherapy mediated tumor suppression in an organ specific manner; renal subcapsular tumors responded to the chemotherapy, whereas i.d. tumors were insensitive. Three-day tumors (both i.d. and renal subcapsular tumor) responded relatively well to each treatment compared to 10-d tumors. These data suggest that in systemic immunotherapy with IL2, anatomic location of the tumor is less important for inducing an antitumor response than in chemotherapy.

Ineffectiveness of adoptive chemoimmunotherapy with lymphokine-activated killer cells, interleukin-2, and cyclophosphamide on palpable intradermal murine bladder cancer.

The effects of adoptive immunotherapy with lymphokine-activated killer (LAK) cells and human recombinant interleukin 2 (IL2), on palpable intradermal (i.d.) bladder tumor were studied. The murine transitional cell carcinoma MBT-2 was used in C3H mice. IL2 was given intraperitoneally at 5,000 U/injection three times a day for 5 consecutive days beginning on day 10. LAK cells were generated in vitro from normal splenocytes: 10(7)-10(8) LAK cells were transferred intravenously on day 10 and, in some experiments, also on day 13. IL2 alone, LAK cells alone (total 8 x 10(7], and both in combination showed little or no influence on intradermally growing MBT-2 tumors. Cyclophosphamide was also combined with adoptive immunotherapy (IL2 and LAK). CY (100 mg/kg, i.p. on day 9 or 10) alone was able to suppress i.d. MBT-2 growth significantly. The combination treatment of IL2 and LAK cells with CY caused additional tumor growth suppression in a manner dependent on the total number of LAK cells transferred. The amount of the additional tumor growth suppression was, however, relatively small when compared with CY plus IL2-treated groups. In comparison, experimentally induced 3-day and 10-day pulmonary metastases of MBT-2 cells were treated by the same protocol of IL2 and LAK cells but without CY. IL2 alone reduced the number of gross metastatic nodules in the lung. The addition of LAK cells to the IL2 almost entirely eradicated the 3-day metastatic nodules but was less effective against the 10-day metastases. The data suggest that adoptive immunotherapy with IL2 and LAK cells mediates tumor regression of micrometastases at a selected organ (lung), but is ineffective against the same tumor growing in the skin or in gross metastatic nodules. Host immune suppression by CY was not beneficial in this model in creating a successful therapeutic effect of LAK cells and IL2.

Eradication of palpable intradermal murine bladder tumors by systemic interleukin-2 and cyclophosphamide in C3H mice.

The effect of cyclophosphamide on interleukin-2 (IL-2) therapy was investigated using the intradermal MBT-2 tumor in C3H mice. Human recombinant IL-2 (Biogen) was given intraperitoneally at doses ranging from 5,000 to 30,000 U, three times a day for 5 or 11-13 consecutive days beginning on day 10 after tumor implantation. When IL 2 was given alone, mice could not tolerate greater than 5 days of 5,000 U IL 2/injection: Administration of 10,000 and 15,000 U/injection of IL-2 for 5 days resulted in 64% (9 of 14) and 100% (14 of 14) mortality, respectively. Chemotherapeutic agents (cisplatin at 6 mg/kg or JM-8 75 mg/kg) and daily administration of cortisone acetate (75 mg/kg) partially protected mice from IL-2-induced toxic deaths. Coadministration of mannitol showed no protective effect. The combination of IL-2 and CY at 75 or 100 mg/kg, however, dramatically reduced the mortality induced by IL-2 and made it possible to escalate the dose and long-term administration of IL-2. Combination administration of 75 mg/kg CY with 15,000 U/injection of IL-2 for 11-13 days caused tumor regressions and resulted in a 66% (8 of 12) cure rate.

Effect of intravesical administration of tumor necrosis serum and human recombinant tumor necrosis factor on a murine bladder tumor.

The effect of intravesical administration of tumor necrosis factor (TNF) on the implantability and growth of bladder cancer was studied using the murine tumor model, MBT-2. Crude TNF was prepared from serum (TNS) of BCG infected rats after injection of endotoxin or the recombinant product of human TNF-alpha was used. Treatment was begun 24 hr. or seven days after tumor instillation and repeated three times. Tumor implantability and tumor weight were determined on day 21. Low dose TNF (200 U), given in the form of TNS, failed to suppress the growth of established tumors (seven-day tumor). It did, however, significantly reduce tumor implantability. The growth of seven-day tumors was significantly suppressed by administration of higher dose of TNF (3,700 U) given in recombinant form.

Reduction of bladder cancer growth in mice treated with intravesical Bacillus Calmette Guerin and systemic interleukin 2.

The effect of systemic administration of Interleukin 2 (IL2) on intravesical Bacillus Calmette-Guerin (BCG) therapy was studied in an established murine bladder tumor, MBT-2. BCG (100 micrograms.) was administered intravesically on days 7 and 14 after seeding bladders with MBT-2 cells. IL2 (5,000 U/injection) was given intraperitoneally every eight hours for 10 times (days seven through 10 and 14 through 17). BCG or IL2 therapy alone failed to reduce incidence of tumor implantation and tumor weight; whereas, combined treatment with BCG and IL2 reduced tumor weight significantly compared to saline or BCG treated mice. Cytotoxicity was assessed in a four-hour 75Semethionine-release assay. Augmentation of natural killer cell activity was only observed in mice treated with BCG plus IL2. MBT-2 target cells were not lysed by spleen cells from mice treated with BCG or saline. IL2 therapy produced lymphokine-activated killer cell activity, though combining BCG with IL2 suppressed this activity after each course of treatment. The results suggest that combined treatment with IL2 enhances the therapeutic effect of BCG therapy. However, this enhancement of antitumor activity is not clearly explained by augmentation of natural killer or in vivo-generated lymphokine-activated killer cells.

Inhibition of suppressor T lymphocytes (Ts) by cimetidine.

Cyclophosphamide (CY) is the most extensively studied inhibitor of suppressor T lymphocyte (Ts) function. However, repeated administration of CY can abrogate sensitization. Therefore, we were interested in identifying noncytotoxic inhibitors of Ts function as adjuncts in the immunotherapy of Ts-inducing murine tumors. The effect of cimetidine (a histamine type 2 receptor antagonist) and diphenhydramine (a histamine type 1 receptor antagonist) on the Ts mediating tolerance to 2,4-dinitrofluorobenzene was studied. We report our data regarding the specific inhibition of Ts by cimetidine.

Human hybrid tumor cells: observations on their production and clinical effects.

Human hybrid tumor cells have been produced by fusing cells from freshly harvested tumor specimens with cells from a cultured human tumor line, D98OR. Fusions were performed with cells from 67 tumors and continuously growing hybrid lines were obtained from 16 (24%). A successful fusion usually produced 1 or 2 hybrid lines, but four easily fusable tumors produced from 6 to 26 lines. The parent cells and hybrids were analyzed by flow cytometry. Hybrids appeared to retain a high percentage of parental deoxyribonucleic acid. Ten patients participated in a clinical study in which they received intradermal immunization with semiautologous hybrids and Corynebacterium parvum as adjuvant. The only side effect was slight local tenderness at the injection sites. No tumor regressions occurred. Skin testing with parental and hybrid cells was performed prior to and following immunization with hybrids. Delayed cutaneous hypersensitivity was often achieved for hybrids but not for autologous tumor cells.

Specific immunotherapy with suppressor function inhibition for metastatic renal cell carcinoma.

In this pilot clinical investigation we have investigated the concept of modulating suppressor T lymphocyte (Ts) function to augment delayed type hypersensitivity (DTH) and antitumor immunity in patients with metastatic renal cell carcinoma. Cyclophosphamide (CY) was used for modulating Ts function. We used three doses of CY per 100 mg/m2, 500 mg/m2, and 1000 mg/m2. Cyclophosphamide was administered i.v. 24 h prior to the first of 6 weekly immunizations with irradiated autologous tumor cells mixed with Corynebacterium parvum. Twenty of 26 patients were evaluable for response. Five of these 20 (25%) evaluable patients had responses, one complete response and four partial responses. Fifteen patients had post-treatment skin testing with autologous tumor cells. Four of these 15 (26%) patients developed DTH to autologous tumor cells. Of the four patients acquiring skin test positivity three also had clinical responses, whereas among the 11 skin-test negative patients, only one clinical response was observed. Six of six (100%) patients who had serial T lymphocyte subset studies done had increases in their mean T helper/inducer:T suppressor/cytotoxic ratios after CY administration and immunization. These observations in an exploratory study suggest that further investigations of Ts modulation, autologous tumor cell skin testing, and T lymphocyte subsets may be of value.

In vivo enhancement of antitumor immunity by interleukin 2-rich lymphokines.

The ability of interleukin 2 (IL-2) to enhance in vivo antitumor immunity has been evaluated in the line 1 alveolar cell carcinoma (L1) model of BALB/c mice. A crude supernatant from phorbol myristate acetate exposed EL-4 cells rich in IL-2 plus other lymphokines (EL-4 IL-2), a concanavalin A-induced supernatant from murine splenocytes (Con A IL-2), and recombinant IL-2 (rIL-2) provided by Biogen were tested. Mice were immunized with a cloned population of L1 cells (10(6) irradiated L1 cells given s.c. in the left inguinal region) followed by s.c. injections of EL-4 IL-2, Con A IL-2, or rIL-2 given to the same site. Two immunizations of L1 cells each followed by IL-2 administration were given prior to challenge with live L1 cells s.c. on the right chest wall. Mice receiving EL-4 IL-2 survived significantly longer than those receiving L1 cells only. Daily administration of EL-4 IL-2 for 7 days after the last L1 immunization was significantly better than 3 days (P less than 0.01) which in turn was significantly better than 1 day (P less than 0.05). Among the doses tested (normalized in vitro to the Biologic Response Modifiers Program IL-2 standard) 404 units of IL-2/injection was optimal. The EL-4 IL-2 had to be injected adjacent to the site of L1 cells; s.c. injection at a distant site or i.p. was not effective. When rIL-2 or Con A IL-2 was substituted for EL-4 IL-2, survival was not prolonged; however, if Con A IL-2 (low IL-2 levels) was supplemented with rIL-2 to 404 units of IL-2, it augmented immunity as well as 404 units of EL-4 IL-2. The data suggest that IL-2 is not the only lymphokine active in augmenting antitumor immunity induced by L1 cells. Some preliminary experiments indicate that a multilymphokine approach may have potential clinical relevance.

Somatic cell hybridization of human tumor samples.

Human intraspecific hybrids were formed between tumor cells isolated from both primary and metastatic tumors and a tissue culture adapted cell line, D98OR, a HeLa derivative which is thioguanine and ouabain resistant. Five different tumor types in all were attempted: renal cell carcinoma, colon adenocarcinoma, melanoma, chrondrosarcoma, and hepatocarcinoma. The tumor tissue was either (1) immediately dissociated and fused, or (2) frozen and later thawed, dissociated, and fused. Two different PEG concentrations were used. The results reported here demonstrate that: (1) hybrid tumor cell lines can be made from several types of cancer, (2) unfrozen tumor tissue fused with D98OR by exposure to 50% PEG appears optimal, (3) chromosome loss, as determined by flow cytometry studies of hybrid DNA content, is minimal, and (4) hybrids have characteristics consistent with derivation from tumor cells rather than derivation from the nonmalignant cells of a tumor.

Effect of BCG immunotherapy on N-nitroso-N-methylurea-induced carcinogenesis of guinea pig colon.

A total of 480 guinea pigs each received twice weekly intrarectal instillations of 1 mg N-nitroso-N-methylurea (NMU) during 14-35 weeks (total dose NMU, 28-70 mg) to induce colon neoplasms. At 24, 28, or 35 weeks after the start of NMU instillation, the distal colon was exposed by a ventral laparotomy and suspected neoplastic lesions were treated by one of four methods: A, intratumoral instillation of an emulsion of killed BCG cell walls attached to oil droplets; B, intratumoral instillation of a control emulsion of killed BCG cell walls in an aqueous phase rather than lipid phase; C, surgical excision of the colon lesion with formation of a ventral diverting colostomy; or D, sham operation (no treatment). All guinea pigs were allowed to recover from surgery and were then observed for a period of 1 year for the study of the effects of these treatments on NMU-induced colon neoplasms. Colon neoplasms were produced in 76% of all sham-operated control guinea pigs, and the frequency of such neoplasms was dependent on the total dose of NMU. Most neoplasms were adenocarcinomas with invasion of the bowel wall but only approximately 5% of them metastasized. Treatment with BCG failed to alter the course of NMU-induced colon carcinogenesis, as determined by the frequency of colon neoplasia, the number, the gross, or microscopic characteristics of colon neoplasms, or the rate of survival.