The leukocyte integrins are cell adhesion molecules which play pivotal roles in the development of a variety of immune responses including T-cell-mediated cytotoxicity, lymphocyte proliferation, macrophage presentation of antigen, and adhesion of leukocytes to vascular endothelium. The relevance of lymphocyte function-associated antigen-1 (LFA-1) to leukocyte malignancies is currently under examination in a number of laboratories. Here, we present evidence demonstrating that LFA-1 plays a role during the in vitro invasion of human endothelium by JY lymphoma cells and during in vivo metastasis of two distinct models of murine leukemia: P815 mastocytoma and EL4 lymphoma. When assayed in vitro, a murine anti-human LFA-1 (alpha subunit) monoclonal antibody (mAb) inhibits up to 80% of JY lymphoma cell invasion. When assayed in vivo, a rat anti-LFA-1 (alpha subunit) mAb significantly inhibited the development of experimental metastases, when administered concomitantly with either P815 or EL4 tumor cells. The leukocyte integrins, particularly LFA-1, may represent useful targets for the therapeutic modulation of metastasis.

Animals↗

PubMed1992

Growth inhibition with reversible cell cycle arrest of carcinoma cells by flavone L86-8275.

BACKGROUND: Previous studies have shown that polyhydroxylated flavonoids such as quercetin and genistein can inhibit tumor cell growth in vitro, and preliminary in vivo studies of the flavone L86-8275 have shown growth inhibition of LX529 and A549 lung carcinomas. L86-8275 [(-)cis-5,7-dihydroxy-2-(2-chlorophenyl)-8[4-(3-hydroxy-1-methyl)- piperidinyl]-4H-1-benzopyran-4-one] is a flavone of novel structure. PURPOSE: The purpose of this study was to determine in vitro whether L86-8275 is a more potent inhibitor of growth in breast carcinoma and lung carcinoma cells than quercetin or genistein. METHODS: We studied the effects of L86-8275 on cell growth in seven breast carcinoma cell lines and five lung carcinoma cell lines. MDA468 breast carcinoma was then selected for further study. Cell proliferation was measured by a colorimetric dye reduction assay; synthesis of DNA, RNA, and protein by incorporation of the radioactive metabolic precursors thymidine, uridine, or leucine, respectively; adenosine triphosphate (ATP) content by a luciferase-mediated bioluminescence reaction; and cell cycle progression by the use of cell-synchronizing drugs (aphidicolin and nocodazole) and flow cytometry. RESULTS: L86-8275 was not cytotoxic to stationary-phase cells but reversibly inhibited the growth of cells in exponential growth phase. At concentrations of 25-160 nM, L86-8275 inhibited growth of human breast and lung carcinoma cell lines by 50%. MDA468 breast carcinoma cells were 60-fold and 400-fold more sensitive to L86-8275 than to quercetin and genistein, respectively. By 24 hours after addition of L86-8275, DNA synthesis in MDA468 cells was inhibited by greater than 95%, protein synthesis by 80%, and RNA synthesis by 40%-60%, under conditions that preserved cellular ATP levels at approximately 80%-90% of control values. When MDA468 cells released from aphidicolin-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed the S phase but arrested in G2. When cells released from nocodazole-induced cell cycle arrest were exposed to 200 nM L86-8275, they completed mitosis but arrested in G1. CONCLUSIONS: L86-8275 is a potent, yet reversible, growth-inhibitory flavone that can selectively block cell cycle progression in vitro at more than one point in the cell cycle. IMPLICATIONS: These findings suggest that L86-8275 is a candidate for further preclinical development, as well as a model for the synthesis of other flavonoids that might potently delay cell cycle progression to achieve inhibition of tumor growth. Future studies need to address optimal schedules for antiproliferative activity in vivo and inhibition of clonogenic activity.

Adenosine Triphosphate↗

PubMed1992

Application of an organic solvent extraction to the determination of catechol-O-methyltransferase activity by high-performance liquid chromatography in human mononuclear cells.

We report here a method for measuring mononuclear cell catechol-O-methyltransferase (COMT) activity which is ideally adapted to clinical studies. The method measures the O-methylation of dopamine to 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine. Whole mononuclear cell sonicate is incubated with saturating concentrations of dopamine, S-adenosyl-L-methionine and magnesium chloride in sodium-potassium phosphate buffer at pH 7.3. An organic solvent extraction using ethyl acetate is then used for product separation, followed by high-performance liquid chromatography with electrochemical detection for product separation and quantification. This method allows both O-methylated products, 3-methoxytyramine and 4-methoxy-3-hydroxyphenethylamine, to be isolated and quantified separately. The apparent Michaelis constants for dopamine and S-adenosyl-L-methionine using this method are similar to values reported previously (0.51 and 14 microM, respectively). The optimal concentration of magnesium chloride is eight to ten times higher than previously reported. No endogenous inhibitors were apparent using this assay. The within-day coefficient of variation using this method is 7% when measuring 3-methoxytyramine and 5% when measuring 4-methoxy-3-hydroxyphenethylamine. The between-day coefficient of variation is 11%. Mononuclear cell COMT activity can be detected using protein concentrations as low as 0.75 mg/ml, corresponding to 2-3 ml of whole blood. The small amount of blood required per sample allows multiple sample analysis from a single patient, including infants.

Adult↗

PubMed1992

Determination of platelet monoamine oxidase activity by high-performance liquid chromatography with electrochemical detection.

A procedure for determining human platelet monoamine oxidase (MAO) with dopamine (DA) as substrate is described. High-performance liquid chromatography (HPLC) with electrochemical detection (ED) was used to separate and detect components of the reaction mixture. The method for platelet preparation was also improved and only 2 ml of blood were required. Following a 10-min incubation of the platelet preparation with DA in 0.1 M Tris buffer (pH 9.0), excess DA substrate was removed by adsorption on a cation-exchange resin. The reaction product, 3,4-dihydroxyphenylacetaldehyde, was adsorbed on acid-washed alumina, eluted with 0.1 M perchloric acid and analyzed by HPLC. Simple, clean chromatograms were obtained with good reproducibility using 3,4-dihydroxybenzylamine as an internal standard. The within-sample, between-samples and between-day relative standard deviations were 0.9, 3.7 and 6.1%, respectively. The apparent Michaelis constant and maximum velocity were 0.10 mM and 0.37 nmol/min.mg protein, respectively. This HPLC-ED method offers a good alternative to methods using radioactivity.

Blood Platelets↗

PubMed1992

Phorbol ester reduces constitutive nuclear NF kappa B and inhibits HIV-1 production in mature human monocytic cells.

NF kappa B is a potent mediator of specific gene expression in human monocytes and has been shown to play a role in transcription of the HIV-1 genome in promonocytic leukemias. There is little information available on the response of NF kappa B to cytokines in normal human monocytes. We have used a 32P-labeled oligonucleotide derived from human immunodeficiency virus (HIV-1) long terminal repeat, which contains a tandem repeat of the NF kappa B binding sequence, as a probe in a gel retardation assay to study this transcription factor. Using this assay, we have detected NF kappa B in extracts of nuclei from normal human monocytes. Treatment of normal monocytes with 12-0-tetradecanoyl phorbol-13-acetate (TPA) for 4-24 h caused the complete disappearance of NF kappa B from nuclear extracts of monocytes. A similar result was obtained with the mature monocytic leukemia cell line THP-1. The constitutive transcription factor SP1 was unaffected by addition of TPA. The disappearance of NF kappa B from the nucleus was concentration dependent between 10 and 50 ng/ml of phorbol ester. In THP-1 cells, TPA also induced a new, faster-migrating NF kappa B species not induced in monocytes. Protein kinase C inhibitor staurosporine, but not cyclic nucleotide-dependent protein kinase inhibitor HA-1004, also dramatically reduced constitutive levels of nuclear NF kappa B. Finally, TPA addition to monocytes infected with HIV-1 inhibited HIV-1 replication, as determined by reverse transcriptase assays, in a concentration-dependent manner. These results are in striking contrast to the increase in nuclear NF kappa B and HIV-1 replication induced by phorbol esters in promonocytic leukemia cells U937 and HL-60, and emphasize the importance of studying cytokine regulation of HIV-1 in normal monocytes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Suppression of cytoplasmic senescence in Neurospora.

We have shown that senescence in Kalilo strains of Neurospora, caused by a linear mitochondrial plasmid called kalDNA, is suppressible by existing variants of the nuclear genome. The suppressors are manifested by 4:4 segregation of senescence and immortality in asci from crosses between senescent female strains and males chosen from non-senescent candidate stocks. In one case of suppression, the asci also show segregation at the plasmid level. There is a reduction of kalDNA to barely detectable levels in the four ascospores showing immortality, so this suppressor evidently influences the maintenance of the plasmid itself. In the other case of suppression, the phenotypic segregation is not correlated with segregation at the plasmid level, and all eight ascospores in the asci show both free and inserted forms of kalDNA. This suggests that the suppression genotype provides a way of tolerating the presence of the plasmid rather than diminishing it. However, the allele f, which provides an analogous kind of suppression for the cytoplasmic mutation poky, does not suppress Kalilo or Maranhar senescence. Suppression is hence shown to be a possible option for host strains to combat the plasmid in nature, but no examples of suppressors were found in a limited survey of natural isolates. In addition, we have shown that long-lived, presumably non-senescent, strains do not arise by suppressor mutation, but lose senescence plasmid DNA by another mechanism.

Cytoplasm↗

PubMed1992

Teaching trauma management.

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Emergency Service, Hospital↗

PubMed1992

Suramin: a novel growth factor antagonist with activity in hormone-refractory metastatic prostate cancer.

PURPOSE: Suramin is known to inhibit the growth of malignant prostate carcinoma cells in vitro. This led us to evaluate the effectiveness of suramin in the treatment of 38 patients with prostate carcinoma refractory to hormone therapy. PATIENTS AND METHODS: Suramin was administered by continuous infusion at a rate designed to reach a peak of 300 micrograms/mL at the end of 14 days. Patients were given 8 weeks to recover from any toxicity before beginning the second cycle. Subsequent cycles were administered in the same manner except the starting dose rate was 280 mg/m2. RESULTS: In 17 patients with measurable soft tissue disease, three had complete disappearance of soft tissue disease for 4, 5, and 11 months, whereas three patients had a greater than or equal to 50% decrease in the sum of the products of the diameters of all measurable disease for greater than or equal to 1 month. Of these 17 patients, pretreatment prostate-specific antigen (PSA) decreased by 75% or more in five (29%) and normalized in one (6%). The remaining 21 patients had disease limited to bone, and only one of these experienced resolution of more than 50% of all lesions on bone scan. Of these 21 patients, pretreatment PSA decreased by 75% or more in eight (38%) and normalized in five (25%). Median time to progression for all patients was 26.3 weeks, and median survival was 42.3 weeks. Patients with bone involvement alone exhibited a better survival than patients with soft tissue involvement (P2 = .02). Survival was strongly correlated (P2 = .0001) with a decline in the pretreatment PSA of greater than or equal to 75% by the eighth week on therapy, with nearly an 85% survival at 1 year compared with a 20% survival for those whose pretreatment PSA did not decline by that amount. CONCLUSION: We conclude that suramin is an active agent in hormone-refractory prostate carcinoma.

Aged↗

PubMed1992

The 'Jaykay' stoma flange cutter.

One of the many difficulties associated with stoma management is that of cutting the hole in the appliance to fit neatly and precisely around the mucocutaneous border. A new stoma flange cutter has been designed which facilitates the cutting or the enlargement of holes in stoma flanges to the exact size required. This equipment may be of use to partially sighted or manually disabled patients as well as to those able patients who would prefer an easier method of cutting the hole in their stoma flange. It may also be of use in operating theatres to enable theatre nurses to cut flanges more accurately and quickly for patients with colostomies, ileostomies or urinary conduits.

Colostomy↗

PubMed1991

Effect of Evans blue and trypan blue on syncytia formation and infectivity of human immunodeficiency virus type I and type II in vitro.

Polyanionic compounds were used to inhibit infectivity of human immunodeficiency virus in vitro. Suramin, Evans blue, and Trypan blue were shown to inhibit syncytia formation normally observed when HIV-1-infected cells are cocultured with CD4+ cells. The inhibition was more pronounced with Evans blue than with any of the other polyanions studied. The inhibitory effect was significantly weaker in HIV-2 systems. However, the reverse transcriptase activities of both types of viruses were inhibited by Evans blue. Another polyanionic compound, phosphorothioate 28-mer cytidine homopolymer (SdC28) was shown to inhibit syncytium formation induced by HIV-1-and HIV-2-infected cells in an identical manner. Evans blue showed partial blocking of gp120 binding to CD4 in a solid-phase enzyme-linked immunosorbent assay (ELISA). These results suggest that the polyanionic dyes may exert their antiviral effects, at least in part, by interfering with the binding and fusion of HIV with susceptible T cells.

Antiviral Agents↗

PubMed1991

Peptide growth factors: the parallel between fetal development and malignant transformation.

Normal embryonic development and tissue homeostasis depend upon cell-to-cell and tissue-to-tissue communication. Much of this depends upon the production and release of peptide growth regulators such as fibroblast growth factor, epidermal growth factor, etc. As the molecular basis of malignant transformation has been documented, it is apparent that a common means by which a tumor becomes autonomous of external control is by becoming independent of the need for external growth factor stimulation. This can occur through the cell producing and releasing the growth factors needed for its own growth. A similar end result can occur with growth factor receptor mutants in which the receptor gives a positive signal even when no growth factor is bound to the receptor. One potential therapeutic strategy is to interfere with this autocrine loop. Suramin is presented as an example of a drug capable of blocking such autocrine loops. This drug also reverses malignant transformation due to Platelet Derived Growth Factor and K-fgf/hst oncogenes on this basis. Another potential mechanism to accomplish the same end is through the use of monoclonal antibodies to either the growth factor or its receptor. Examples of this are available for TGF-alpha. We have discussed a range of practical issues involved in developing monoclonal antibodies as therapeutic agents.

Antibodies, Monoclonal↗

PubMed1991

Anthracyclines.

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Animals↗