Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0025202 (melanoma)
 69,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously isolated a monoclonal antibody, designated as 1-3-1, specific for tissue-type plasminogen activator (t-PA). We have shown that t-PA dissociates from 1-3-1 in the presence of the lysine analogue 6-aminohexanoic acid (6-AHA). Here we describe a method for the one-step immunoaffinity purification of t-PA from conditioned melanoma cell medium, using 1-3-1 immobilised on Sepharose under mild elution conditions, favourable for t-PA. The yield of t-PA (antigen or total protein) from a 1-3-1-Sepharose column, when eluted using a buffer supplemented with 0.2 M 6-AHA at neutral pH, was as effective as other buffers that involve a strong pH-change, i.e., pH 2-3. However, the enzymatic activity of the t-PA purified with 6-AHA was 25 to 30% higher, as compared with t-PA eluted using a pH change. This resulted in a markedly higher specific activity of t-PA purified with 0.2 M 6-AHA, as compared with t-PA purified using a strong pH-change. The purity of t-PA, purified using the present method, was very high, as determined by gel electrophoresis. An additional advantage of the present procedure is that the mild elution conditions prolong the column life.
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PMID:One-step purification of tissue-type plasminogen activator using affinity chromatography with a special monoclonal antibody under mild conditions. 152 79

In an attempt to improve bifunctional chelate labelling of Mab, we investigated the use of a polyamino acid backbone for multiple DTPA substitutions. Poly-L-lysine (PL) (3.8 Kd, n = 25) was partially acetylated with MADTPA to yield 11 moles of DPTA per mole of PL. The average numbers of DTPA on PL were directly quantified with MADTPA-C-14. The remaining epsilon amino groups on PL-DTPA (I) were measured with TNBS reagent. A selective maleimide derivatization of (I) with S-SMPB yielded (II), which contains 2.3 moles of maleimide groups per mole of (I). The sulfhydryl activation of Mab-TP41.2F(ab')2 with 2-Iminothiolane hydrochloride produced (III), containing 1.3 moles of sulfhydryl groups per mole of Mab. Compounds (II) and (III) were combined to form a single thioether-spaced chain linkage of Mab-PL-DTPA (IV), which was subsequently chelated with 111In to yield (V), which was the compound of interest. Indium-111-PL-DTPA (VI) and 111In-DTPA-MabTP41.2F(ab')2 (VII) also were prepared for control studies. Direct cell binding assay revealed the mean immunoreactivity of (V) to be 79.4% and that of (VII) to be 39.5%. In a biodistribution study on melanoma tumor-bearing athymic mice at 4, 24, and 48 hr postinjection, the tumor/blood and tumor/liver ratios at 48 hr were 11.6 and 1.2 for (V), compared to 3.7 and 0.13, respectively, with (VII). Thus, the PL configuration for radiolabeled antibodies seems to result in decreased hepatic accumulation and retained tumor activity. The findings suggest that further studies of this new compound are warranted.
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PMID:Reduced hepatic accumulation of radiolabeled monoclonal antibodies with indium-111-thioether-poly-L-lysine-DTPA-monoclonal antibody-TP41.2F(ab')2. 155 42

The relationship between transglutaminase activity, apoptosis and the propensity of a tumour to metastasise, was investigated in a series of metastatic variants of an HSV-2 induced hamster fibrosarcoma and two metastatic variants of the B16 mouse melanoma. The data suggest an inverse relationship between metastatic potential and cytosolic transglutaminase activity. A direct relationship was found between measured cytosolic activity and the levels of the endogenous product of transglutaminase, the protein crosslink epsilon(gamma-glutamyl)lysine. Increasing metastatic potential and decreasing cytosolic transglutaminase activity was accompanied by a corresponding decrease in the number of detergent-insoluble apoptotic envelopes isolated from variant cell lines. These apoptotic envelopes were found to be highly crosslinked structures, containing more than 85% of the cells content of epsilon(gamma-glutamyl)lysine. These data are in keeping with the idea that a major role for the cytosolic transglutaminase is in the formation of the highly crosslinked apoptotic envelope during programmed cell death and that perturbation of this function may be an important determinant in the development of the metastatic phenotype.
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PMID:Apoptosis: a potential role for cytosolic transglutaminase and its importance in tumour progression. 167 3

Antibody-morpholinodoxorubicin conjugates were prepared for targeted immunotherapy of human melanoma. Spacer molecules that differ in hydrolytic stability were employed between the C-13 of the drug and amino residue of lysine on the monoclonal antibody. Antibody-drug conjugates were made with five structurally different morpholinodoxorubicin derivatives including oxime, phenylhydrazone, (sulfonylphenyl)hydrazone, and acylhydrazone moieties. Hydrolytic stability of the antibody conjugates directly correlated with their in vitro cytotoxicity against melanoma cells. Derivatives or conjugates with the greatest hydrolytic stability showed the least cytotoxicity.
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PMID:Antibody conjugates with morpholinodoxorubicin and acid-cleavable linkers. 171 77

Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates. Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA. Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA. After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine. This indicated that plasminogen was activated on the cell surface. The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA. The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media. The media were found to inhibit u-PA but not t-PA. This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity. These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin. Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion.
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PMID:Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion. 171 33

In order to identify the regions of recombinant (r) tissue plasminogen activator (tPA) that mediate its kinetically relevant interaction with r-plasminogen activator inhibitor-1 (rPAI-1), we have determined the second-order association rate (k1) constants of domain-altered variants of tPA with rPAI-1, at 10 degrees C. With two-chain, wild-type recombinant tPA (tcwt-rtPA), obtained by expression of the human cDNA for tPA in five different cell systems (viz. insect cells, human kidney 293 cells, Chinese hamster ovary cells, human melanoma cells, and mouse C127 cells), the average k1 was 1.45 x 10(7) M-1 s-1 (range, 1.34 10(7) M-1 s-1-1.68 x 10(7) M-1 s-1). Since this value was not significantly different for the different tcwt-rtPA preparations, it appears as though the nature of the glycosylation of tPA plays little role in its initial interaction with PAI-1. The k1 determined for tcwt-rtPA was slightly higher than that of 0.87 x 10(7) M-1 s-1, obtained for a similar inhibition of human urokinase by rPAI-1. The k1 value obtained for single-chain (sc) wt-rtPA was approximately 6-fold lower than that of the two-chain molecules, results consistent with previous conclusions on this matter. The k1 value for tcwt-rtPA was not influenced by the presence of epsilon-aminocaproic acid, suggesting that the lysine-binding site associated with the kringle 2 (K2) region of tPA does not modulate the rate of its initial interaction with rPAI-1. Removal of the K2 domain from tPA, by recombinant DNA technology, results in a protein, F-E-K1-P (tc-r delta K2-tPA), containing only the finger (F), growth factor (E), kringle 1 (K1), and serine protease (P) domains. This variant protein was more rapidly inhibited by rPAI-1 (k1 = 3.00 x 10(7) M-1 s-1) than its wild-type counterparts. Deletion of both the K1 and K2 domains resulted in a variant molecule, F-E-P (tc-r delta K1 delta K2-tPA), that was slightly more rapidly inhibited by rPAI-1 (k1 = 2.01 x 10(7) M-1 s-1).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural determinants of the noncatalytic chain of tissue-type plasminogen activator that modulate its association rate with plasminogen activator inhibitor-1. 211 57

Antibody conjugates were prepared by coupling F(ab')2 or Fab' fragments of an antibody specific for the human high molecular weight-melanoma associated antigen to Fab' fragments of an antibody specific for indium-diethylenetriaminepentaacetate complexes. Monovalent and bivalent haptens were synthesized by reacting the dipeptide tyrosyl-lysine with diethylenetriaminepentaacetic cyclic anhydride. In vitro, the antibody conjugate mediated binding of the 111In-labeled haptens to melanoma cells. In vivo, it allowed specific localization of the haptens in A375 tumors. The bivalent hapten exhibited much higher efficiency at targeting 111In onto cells, both in vitro and in vivo. Antibody conjugate and hapten doses (2 micrograms and 1 pmol, respectively) and the delay between antibody conjugate and tracer injections (24 h) were adjusted to maximize tumor uptake (4% injected dose/g) and tumor to normal tissue contrast (greater than 3) obtained 3 h after injection of the 111In-labeled bivalent hapten. This two-step technique, when compared to direct targeting of 111In-labeled F(ab')2 fragments, provided lower localization of injected activity into the tumor (x 0.25), but higher tumor/tissue ratios, especially with respect to liver (x 7), spleen (x 8), and kidneys (x 10). In addition, high contrast images were obtained within 3 hours, instead of days. Thus, antibody conjugate-mediated targeting of small bivalent haptens, labeled with short half-life isotopes, is proposed as a general method for improving tumor radioimmunolocalization.
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PMID:Targeting of indium 111-labeled bivalent hapten to human melanoma mediated by bispecific monoclonal antibody conjugates: imaging of tumors hosted in nude mice. 233 41

The two coupling agents SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate) and SATA (N-succinimidyl-S-acetylthioacetate) were compared in their efficiency and feasibility to couple monoclonal antibodies (Abs) via thioether linkage to liposomes functionalized by various lipophilic maleimide compounds like N-(3-maleimidopropionyl)-N2-palmitoyl-L-lysine methyl ester (MP-PL), N-(3-maleimidopropionyl)phosphatidylethanolamide (MP-PE), N6-(6-maleimidocaproyl)-N2-palmitoyl-L-lysine methyl ester (EMC-PL), and N-(6-maleimidocaproyl)phosphatidylethanolamine (EMC-PE). The composition of the liposomes was soy phosphatidylcholine (SPC), cholesterol, maleimide compounds and alpha-tocopherol (1:0.2:0.02:0.01, mol parts), plus N4-oleylcytosine arabinoside (NOAC) as cytostatic prodrug (0.2 mol parts) and a new, lipophilic and highly fluorescent dye N,N'-bis(1-hexylhfetyl)-3,4:9,10-perylenebis(dicarboximid ) (BHPD, 0.006 mol parts). From the maleimide derivatives MP-PL was the most effective in terms of preservation of the coupling activity in dependence of liposome storage. The coupling of the monoclonal A B8-24.3 (mouse IgG2b, MHC class I, anti H-2kb) and IB16-6 (rat IgG2a, anti B16 mouse melanoma) to the drug carrying liposomes was more effective and easier to accomplish with SATA as compared to SPDP. Coupling rates of 60-65% were obtained with SATA at molar ratios of 12 SATA:1 Ab:40 maleimide spacer groups on the surface of one liposome. The highest coupling rates with SPDP were obtained at the ratio of 24 SPDP:1 Ab:40 liposomal maleimide groups, with an Ab binding efficiency of only 20-25%. The optimal in vitro binding conditions to specific target cells (EL4 for B8-24.3-liposomes and B16-F10 for IB16-6-liposomes) were determined by cytofluorometric measurement of the liposomal BHPD fluorescence with SATA linked Abs. Optimal immunoliposome binding to specific epitopes on the target cells was achieved with 1-2 Ab molecules coupled to one liposome, with immunoliposome concentrations of 20-130 nM and with a small incubation volume of 0.3-0.4 ml. The specificity of the binding of B8-24.3-liposomes to EL4 target cells was visualized by scanning electron microscopy. Antibody mediated endocytic uptake of immunoliposomes could be demonstrated by transmission electron microscopy.
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PMID:Comparative studies of the preparation of immunoliposomes with the use of two bifunctional coupling agents and investigation of in vitro immunoliposome-target cell binding by cytofluorometry and electron microscopy. 237 82

Incubation of C3H/Hen thymocytes in the presence of recombinant human tumor necrosis factor alpha (TNF) and interleukin-2 (IL-2) augmented the generation of antibody-dependent cellular cytotoxicity (ADCC) when compared to cells cultured in TNF or IL-2 alone. This effect was optimal when 100-200 units/ml IL-2 was used together with 10(3)-10(4) units/ml TNF. TNF alone at any concentration could not mediate the induction of ADCC. Similar to the results obtained in vitro, TNF, when given alone, had no effect on the generation of ADCC in vivo. The addition, however, of TNF to IL-2, given at 10,000 and 20,000 but not 40,000 units, enhanced the IL-2-induced ADCC on a per-cell basis. Furthermore, TNF enhanced the total ADCC activity in various organs including the liver, spleen and thymus as a result of an increase in the number of mononuclear cells isolated from these organs. The increase in total ADCC activity was optimal when 110,000-220,000 units (5-10 micrograms) TNF were employed together with IL-2. The combined treatment with TNF and IL-2 also increased the intracellular benzyloxycarbonyl-1-L-lysine-thiobenzyl-ester esterase content in cells isolated from the livers of mice treated with these cytokines. On the basis of these results we treated mice bearing a single B16 melanoma nodule with TNF and TNF + IL-2 given with or without anti-B16 monoclonal antibody. We found that TNF administration augmented the anti-tumor effect of specific anti-B16 antibodies, and the addition of IL-2 further increased this anti-tumor effect.
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PMID:Effect of recombinant human tumor necrosis factor alpha on the induction of antibody-dependent cellular cytotoxicity in the treatment of established B16 melanoma liver nodules. 237 20

The heat response of five human tumor biopsies has been examined using the fluorescent probe dansyl lysine and multiparameter flow cytometry. Dansyl lysine has previously been shown to possess specificity for heat killed mammalian cells. The human tumors tested included a cervical squamous cell carcinoma, malignant melanoma, colon adenocarcinoma, ovarian carcinoma, and a mesothelioma. The samples were excised, mechanically disrupted into single cell suspensions and heated in vitro for various lengths of time at 45 degrees C. The cells were returned to 37 degrees C incubation for 12 to 15 hours prior to staining with dansyl lysine. The fraction of cells staining dansyl lysine was quantitated by flow cytometry after gating on high forward angle light scatter and 90 degrees C light scatter. This gate excluded much of the normal cell contamination within the tumor sample. The data show that the heat response of human tumor biopsies varied significantly, with cervical carcinoma and malignant melanoma being the most resistant and the mesothelioma and ovarian carcinoma the most heat sensitive. Finally, evidence is presented for the expression of thermotolerance in ovarian carcinoma and mesothelioma biopsies pre-heated in vitro. Dansyl lysine appears to be useful in measuring the intrinsic cellular heat sensitivity of human tumors and in determining the kinetics of decay of thermotolerance following an initial heat exposure.
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PMID:A predictive assay for human tumor cellular response to hyperthermia using dansyl lysine staining and flow cytometry. 244 73


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