Simple, quick and efficient site-directed antibody immobilization in a cartridge.

A method is described for the simple, quick and efficient attachment of antibody within a cartridge for use as an immunoaffinity chromatography column. Antibodies are immobilized via their Fc regions through the use of periodate-oxidized carbohydrate functionalities of the immunoglobulin G. The method allows for the in situ coupling of the immunoglobulin G without prior removal of the oxidizing periodate solution. The entire procedure can be completed in 50 minutes. This method is especially useful for quick determinations of a particular monoclonal antibody's functionality or avidity towards a specific antigen. It may also be used in place of a conventional immunoaffinity column for the rapid isolation of small amounts of an antigen. This method will reduce the lengthy process of preparing an immunoaffinity column from several days to less than an hour.

Cellular pharmacology of 5-fluorouracil in a human colon adenocarcinoma cell line selected for thymidine kinase deficiency.

A human colon adenocarcinoma cell line (GC3TK-) was selected for thymidine kinase (TK) deficiency from cloned parental cells (GC3C1) by exposure to 5-bromodeoxyuridine (BrdUrd). The cellular pharmacology of 5-fluorouracil (FUra) and the influence of physiological concentrations of thymidine (dThd; 0.1 to 1 microM) on FUra cytotoxicity during brief exposure in both cell lines were examined. The uptake of FUra during a 1-hr drug exposure, its metabolism to ribo- and deoxyribonucleotides, incorporation into RNA, and inhibition of thymidylate synthase were similar in GC3C1 and GC3TK- cells as were the IC50 values for FUra (26 and 23 microM respectively). TK deficiency did not reduce the intracellular concentrations of FdUMP generated from FUra. In GC3C1, at FUra concentrations up to 100 microM, cytotoxicity was prevented by co-administration of dThd (0.1 to 20 microM). The relationship between cell survival and thymidylate synthase inhibition was close under these conditions. At higher drug concentrations, less dThd protection was observed, and none was detected in GC3TK- cells. Thus, the metabolism of FUra did not appear to be altered substantially in GC3C1 cells selected for TK deficiency. Also in these cells, at concentrations of FUra less than 100 microM, FUra cytotoxicity appeared to be mediated via the inhibition of thymidylate synthase.

Chemotherapeutic characterization in mice of 2-amino-9-beta-D-ribofuranosylpurine-6-sulfinamide (sulfinosine), a novel purine nucleoside with unique antitumor properties.

In preclinical investigations performed in mice, 2-amino-9-beta-D-ribofuranosyl purine-6-sulfinamide (sulfinosine), a novel derivative of 6-thioguanosine (6TGR), was active against six solid tumors and four strains of experimental leukemia. Sulfinosine penetrated the central nervous system more readily than did 6TGR and, when given repeatedly, was much more effective in the treatment of L1210 leukemia, being curative for some mice. Other findings of major interest to us were the different dosing characteristics of sulfinosine and 6TGR, the divergent efficiencies of the two drugs in generating cellular resistance, and the activity of sulfinosine against experimental leukemias refractory to 6TGR and other experimental or clinically used chemotherapeutic agents. The chemotherapeutic characterization of sulfinosine that evolved from these studies suggests that this agent may have unique properties that deserve clinical consideration. Both the dosing characteristics of the drug and its pronounced activity against thiopurine-resistant experimental leukemia favor the possibility that sulfinosine could be used to advantage in the treatment of human leukemia unresponsive to 6-mercaptopurine or 6-thioguanine.

Biochemical mechanisms in colon xenografts: thymidylate synthase as a target for therapy.

Growth of human adenocarcinomas of the colon and rectum in immunoincompetent mice has allowed for a greater understanding of the interaction of 5-fluorouracil, its metabolism, and mechanism(s) of cytotoxicity under conditions of tumor growth in situ. Conversely, this agent has proven to be a useful tool in defining metabolic characteristics in human colon adenocarcinomas. Analysis of tumor sensitivity to 5-fluorouracil (FUra),5-fluorouridine (FUrd) and 5-fluoro-2'-deoxyuridine (FdUrd) suggests that growth inhibition in vivo is related to a DNA-directed event. Resistance, de novo appears to be a consequence of relatively transient inhibition of the target enzyme thymidylate synthase (dTMP-synthase), which may be a consequence of low concentrations of 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) or its polyglutamate forms within tumor cells in situ. In order to study the relationship between inhibition of dTMP-synthase and growth inhibition, mutant cells deficient in their ability to salvage dThd have been selected, and grown as xenografts. Data suggest that transient inhibition of dTMP-synthase and not dThd salvage is responsible for resistance de novo, and that prolonged inhibition of dTMP-synthase would be a lethal event in vivo. This would predict that a cell lacking dTMP-synthase activity would not be tumorigenic. This has been tested directly by selecting clones of GC3 colon adenocarcinoma cells deficient in dTMP-synthase (TS-) activity. Preliminary data indicate that each of 3 TS- clones is tumorigenic in athymic nude mice. The importance of dTMP-synthase as a target for drug development is discussed with respect to these findings.

Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenograft.

CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of less than 3 microM. The interaction of polyglutamates of [6R]CH2-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 microM) were greater than 200-fold and [6R]CH2-H4PteGlu2 (18.2 microM) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 microM) at stabilizing ternary complexes for a T1/2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 microM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 x 10(-8) M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 x 10(-9) M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 x 10(6) M-1 min-1 for 10 or 20 microM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 microM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n greater than or equal to 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

Characterization of the pools of 5,10-methylenetetrahydrofolates and tetrahydrofolates in xenografts of human colon adenocarcinoma.

The method for measuring polyglutamate forms of CH2-H4PteGlu and H4PteGlu, by entrapment in ternary complexes with [6-3H]5-fluoro-2'-deoxyuridylate and Lactobacillus casei thymidylate synthase has been characterized. Results demonstrated that (a) the relationship between concentration of CH2-H4PteGlu and complex isolated on nondenaturing gels was dependent upon the number of glutamyl residues, and an alternative method for data analysis has been presented, (b) the relationship was linear over a 100-fold change in concentration, (c) formation of isolatable complex was time dependent, (d) noncovalent complexes formed with PteGlu2-5 could be isolated only at concentrations considerably higher than those required for CH2-H4PteGlu1-6, and (e) endogenous deoxyuridylate would be unlikely to interfere significantly with the assay. The distribution of polyglutamates of CH2-H4PteGlu and the combined pools of CH2-H4PteGlu plus H4PteGlu were subsequently examined in three human colon adenocarcinoma xenografts. In each tumor, the pentaglutamate of CH2-H4PteGlu and H4PteGlu was the most prominent species, followed by the hexaglutamate, constituting 68 to 92% of the CH2-H4PteGlu pool, and greater than 93% of the combined pools. A small percentage of di-, tri-, and tetraglutamates were also detected. Using a catalytic assay, the combined pool of CH2-H4PteGlu and H4PteGlu was estimated in the range of 0.5 to 2.7 microM in cell water, and for CH2-H4PteGlu, from 185 nM to 1.7 microM. Using thymidylate synthase purified from colon adenocarcinoma HxVRC5, CH2-H4PteGlu5 (where the subscript digit attached to the glutamate portion equals the number of glutamate residues) stabilized the covalent ternary complex at greater than 200-fold lower concentration in comparison to CH2-H4PteGlu1. Data indicated that in each colon tumor, the concentrations of CH2-H4PteGlun or CH2-H4PteGlun plus H4PteGlun were suboptimal for the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase, and would predict for relatively transient inhibition of thymidylate synthase after treatment with 5-fluorouracil. These data support therapeutic modulation to increase the concentration of CH2-H4PteGlun in the treatment of colon adenocarcinomas with 5-fluorouracil.

Characteristics of thymidylate synthase purified from a human colon adenocarcinoma.

Thymidylate synthase has been purified greater than 4000-fold from a human colon adenocarcinoma maintained as a xenograft in immune-deprived mice. In this disease, the enzyme is an important target for the cytotoxic action of 5-fluorouracil, which is influenced by the reduced folate substrate CH2-H4PteGlu. Due to the importance of this interaction, and the existence in cells of folate species as polyglutamyl forms, the interaction of folylpolyglutamates with thymidylate synthase was examined. Polyglutamates of PteGlu were used as inhibitors, and the interaction of CH2-H4PteGlu polyglutamates as substrates or in an inhibitory ternary complex were also examined. Using PteGlu1-7, Ki values were determined. A maximal 125-fold decrease in Ki was observed between PteGlu1 and PteGlu4; further addition of up to three glutamyl residues did not result in an additional decrease in Ki. Despite the increased binding affinity of folypolyglutamates for this enzyme, no change in the Km values for either dUMP (3.6 microM) or CH2-H4PteGlu (4.3 microM) were detected when polyglutamates of [6R]CH2-H4PteGlu were used as substrates. Product inhibition studies demonstrated competitive inhibition between dTMP and dUMP in the presence of CH2-H4PteGlu5. In addition, CH2-H4PteGlu4 stabilized an inhibitory ternary complex formed between FdUMP, thymidylate synthase, and CH2-H4PteGlu4. Thus the data do not support a change in the order of substrate binding and product release upon polyglutamylation of CH2-H4PteGlu reported for non-human mammalian enzyme. This is the first study to characterize kinetically thymidylate synthase from a human colon adenocarcinoma.

Determination of thymidylate synthase activity in colon tumor tissues after treatment with 5-fluorouracil.

The formation and isolation of [6-3H]FdUMP-thymidylate synthase-5,10-methylenetetrahydrofolate covalent complex have been examined in tumor cytosols incubated with albumin-dextran coated charcoal used to remove endogenous nucleotide. Charcoal suspension (10% charcoal, 0.5% albumin, 0.05% dextran) absorbed greater than 98% of dUMP added to cytosols, but it reduced by 42-87% covalent complex isolated from subsequent incubation with [6-3H]FdUMP and cofactor using cytosols from different tumors. Initial treatment of ternary complex with charcoal suspension did not cause a decrease in stability of covalent complex during subsequent incubation (37 degrees), but complex separated from free ligand by 10% charcoal suspension was not stable to further treatment with 4% charcoal suspension. Treatment of tumor cytosols with 10% charcoal suspension, to remove nucleotide, did not decrease the rate at which enzyme catalyzed the release of 3H2O from [5-3H]dUMP, or release active enzyme from the ternary complex. Based on these observations, a sensitive procedure for determining thymidylate synthase activity has been developed in which unbound nucleotides (dUMP, FdUMP) are removed prior to assay of enzyme activity. The procedure is suitable for assay of small samples of tissue or of tissues with a low (or inhibited) level of thymidylate synthase activity.

1,25-Dihydroxyvitamin D3 receptor replenishment in the chicken intestine.

1,25-Dihydroxyvitamin D3 intestinal receptor replenishment was examined in rachitic chickens after hormone administration. A single injection of 1,25-dihydroxyvitamin D3 caused an increase in the level of occupied receptors with a concomitant decrease in the amount of unoccupied receptors. Maximum occupancy occurred 1 h after hormone injection. The metabolic inhibitor of protein synthesis, cycloheximide, was employed to obtain additional information concerning the fate of 1,25-dihydroxyvitamin D3 receptor complexes. Cycloheximide, at a dose that effectively blocked protein synthesis, had no effect on the time-course or the magnitude of replenishment of nuclear receptors. Additionally, repletion with vitamin D3 or administration of several injections of 1,25-dihydroxyvitamin D3 did not lead to a lag in replenishment time or a significant decrease in total receptor levels. These findings demonstrate that recycling of receptors plays an important functional role for the replenishment of unoccupied 1,25-dihydroxyvitamin D3 intestinal receptors.

Binding of 5-fluorodeoxyuridylate to thymidylate synthase in human colon adenocarcinoma xenografts.

The formation and stability of the covalent ternary complex formed between thymidylate synthase (E.C. 2.1.1.45), 5-fluoro 2'-deoxyuridylate (FdUMP) and 5,10-methylenetetrahydrofolate (CH2-H4PteGlu) has been examined in cytosols derived from xenografts of human colon adenocarcinomas. The rate of association (ka) for FdUMP was low being between 3.4 +/- 0.9 and 10.2 +/- 2.6 X 10(6) M-1 min-1, with the lowest ka value being determined in cytosols from a tumor (HxELC2) which has demonstrated some sensitivity to 5-fluoropyrimidines. Relative to reported ka values for human leukemic cells, the rate of association of FdUMP was 20- to 59-fold lower. This difference is not a consequence of FdUMP catabolism, or metabolism of CH2-H4PteGlu. In cytosols the apparent Km values for dUMP (3.6-4.2 microM) and and [6RS]- CH2-H4PteGlu (25-26.7 microM) were similar to reported values for human enzyme. Data derived from cytosols were similar to those derived using affinity purified enzyme from HxVRC5 colon adenocarcinoma xenografts. The net dissociation of [6-3H] FdUMP from the covalent ternary complex was 31-33 min in the absence of added CH2-H4PteGlu, and the rate of dissociation was dependent upon the concentration of cofactor. The concentration of [6RS]-CH2-H4PteGlu required to stabilize ternary complex derived from HxELC2 cytosols was slightly lower than that required for the same degree of stabilization of complex formed in cytosols from resistant tumors (HxGC3,HxVRC5). Addition of 5-CHO-H4PteGlu, 5-CH3-H4PteGlu, H2PteGlu, and PteGlu did not stabilize the covalent complex, but H4PteGlu substituted for CH2-H4PteGlu.

An exchange assay for quantitating 1,25-dihydroxyvitamin D3 receptors.

Steroid hormone receptors, including those for vitamin D3, contain reactive sulfhydryl group(s) essential for hormone binding. On this basis, several compounds that are capable of interacting with sulfhydryl groups were tested for their ability to dissociate 1,25-dihydroxyvitamin D3 from chicken intestinal receptors. At concentrations resulting in 50% displacement of specifically bound cytoplasmic 1,25-dihydroxyvitamin D3, the order of potency for these displacing reagents is mersalyl acid greater than sodium thiocyanate approximately equal to p-hydroxymercuribenzoate approximately equal to mercuric chloride much greater than 5,5'-dithiobis-(2-nitrobenzoic acid). Hormone displacement by mersalyl acid and p-hydroxymercuribenzoate was completely reversible upon dithiothreitol addition. In contrast limited rebinding of 1,25-dihydroxyvitamin D3 occurred after, 5,5-dithiobis-(2-nitrobenzoic acid) and mercuric chloride treatment. Furthermore, at least for mersalyl acid treatment, hormone displacement and subsequent regeneration of sterol binding did not seem to alter the integrity of the receptor as evidenced by sucrose gradient analysis, DNA-cellulose and phosphocellulose chromatography. Additionally, treatment of the nuclear 1,25-dihydroxyvitamin D3 receptor did not significantly affect the apparent equilibrium dissociation constant for hormone binding (Kd = 2.7 X 10(-10) M; Kd = 1.7 X 10(-10) M, for control and mersalyl acid treated receptor, respectively). Finally, a method has been developed for measurement of occupied 1,25-dihydroxyvitamin D3 receptors. The unfilled receptors were quantitated in the cytoplasm or chromatin extracted fraction by incubation with radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 3-6 h without interference from previously filled sites. The total receptor measurement is carried out by incubation of cytosol or nuclear extract with 0.5-1.0 mM mersalyl acid for 1.0 h. Exchange of radioactive sterol for the bound nonradioactive sterol is accomplished by incubation with 1-2 mM dithiothreitol and 1.0 nM radioactive 1,25-dihydroxyvitamin D3 at 0-4 degrees C for 16 h. Subtracting the unfilled sites from total sites results in a measurement of filled sites. With this exchange assay, estimates of total receptor in untreated and 1,25-dihydroxyvitamin D3 treated chicks (estimated 2 h after injection of doses 0.3-300 nmol) were not significantly different (66.2 +/- 4.5 versus 64.4 +/- 10.4 to 69.0 +/- 5.9 pg/mg protein, respectively). Furthermore, quantitation of total receptor by direct or exchange assay obtained from in vitro incubations of intestinal slices with radioactive and nonradioactive 1,25-dihydroxyvitamin D3 were not