ABSTRACT
Hepatic tissue concentrations of FK506 have been correlated with early acute rejection following liver transplantation. Asialoglycoproteins (AGP) reputedly bind FK506 in blood. AGP are removed from the circulation by the liver via the AGP receptor (AGPr), which resides on hepatocytes. This study was undertaken to determine if the AGP-AGPr mechanism enhances the delivery of FK506 to hepatocytes. Human orosomucoid (OM) was used as a representative AGP. asialoOM (aOM) was prepared by desialation of OM. Fresh rat hepatocytes were isolated by collagenase digestion. Tritium labeled FK506 (FK) was used to identify and quantitate FK506. Quantitation of FK in serum and culture media was by direct counting. FK in animal tissues used a method developed in our laboratory for the purpose. AGPr on resting hepatocytes was demonstrated by flow cytometry using FITC-orosomucoid and FITC-BSA controls. AGPr were enhanced by 2 g glucose/L. Two serum FK-binding fractions, 44 kD and 15 kD, were identified by gel filtration. Exogenous OM avidly bound FK and displaced FK activity from the 15 kD fraction. Serum (1%) and the 44 kD fraction enhanced the uptake of FK by hepatocytes, while serum depleted of OM-aOM by affinity chromatography was only 72.5% as effective as control serum; aOM enhanced the uptake of FK by hepatocytes to a degree similar to that of control serum but OM did not significantly affect the uptake of FK. Cold FK506 blocked the uptake and was dose dependent; cold CsA had no effect. Affinity extraction of OM from serum to which FK had previously been added removed 28.4% of FK activity. Following i.v. infusion, the kidney had the highest and liver the lowest tissue concentration of FK at 1 hr and 3 hr. In contrast, after oral administration the liver had the highest concentrations of the other tissues tested. The AGP-AGPr mechanism plays a significant role in the delivery of FK506 to hepatocytes and is likely responsible for the differences in bioavailability observed after oral and i.v. administration. Factors governing the AGP-AGPr mechanism are germane to understanding both the efficacy and toxicity of FK506 and the development optimal therapeutic strategies.
Subject(s)
Asialoglycoproteins/metabolism , Immunosuppressive Agents/pharmacokinetics , Liver/immunology , Orosomucoid/metabolism , Receptors, Cell Surface/metabolism , Tacrolimus/pharmacokinetics , Administration, Oral , Animals , Asialoglycoprotein Receptor , Carrier Proteins/blood , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cells, Cultured , DNA-Binding Proteins/blood , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Flow Cytometry , Heat-Shock Proteins/blood , Heat-Shock Proteins/isolation & purification , Heat-Shock Proteins/metabolism , Humans , Immunosuppressive Agents/administration & dosage , Liver/metabolism , Rats , Tacrolimus/administration & dosage , Tacrolimus Binding Proteins , Tissue DistributionABSTRACT
Gene amplification allows transformed cells to overexpress specific genes and gain a survival advantage. For this reason, cloning and characterization of amplified genes can improve our understanding of the biology of transformed cells. The techniques of in-gel renaturation and chromosome microdissection can enrich for amplified DNA sequences, but both are labor intensive and have other drawbacks. We have developed an alternative strategy of enriching for amplified DNA sequences that involves two-directional agarose gel electrophoresis of extrachromosomal circular DNA. Extrachromosomal circles can be detected with repetitive DNA probes and can be used to produce DNA probes suitable for fluorescence in situ hybridization for location of genomic origin. The ability to enrich for amplified DNA without specialized equipment or transformed cell metaphases should prove useful in the search for new genes which are important in tumor cell progression.
Subject(s)
DNA, Circular/isolation & purification , DNA, Neoplasm/isolation & purification , Base Sequence , Chloroquine , Chromosomes, Human , DNA Primers , DNA Probes , Electrophoresis, Agar Gel/methods , Electrophoresis, Gel, Two-Dimensional , Humans , In Situ Hybridization, Fluorescence , KB Cells , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
Cytogenetically visible gene amplification structures can consist of arrays of amplicons presumably formed by secondary "rearrangements" following amplicon formation. The structural evolution of gene amplification sites in tumor cells suggests that complex secondary structures may have some selective advantage in the tumor cell environment. Although secondary amplicon rearrangements are a hallmark of the gene amplification process, little is known about the mechanics of this process. COLO320 neuroendocrine tumor cells carry two different types of amplified MYC oncogene sequences, one type with an intact MYC gene and the other with a rearranged "chimeric" MYC gene. We have studied various clonal subpopulations of COLO320 cells and identified regions within and downstream of the MYC locus that are unique to each amplicon type. Using double-label fluorescence in situ hybridization with DNA probes unique to each amplicon type, we have observed that both chromosomal and extrachromosomal MYC amplicon arrays in COLO320 cells frequently consist of heterogeneous mixtures of each MYC amplicon type. Our results suggest that the two MYC amplicon types of COLO320 cells were formed simultaneously but independently, and that double minute chromosomes observed in COLO320 cells were formed by intermolecular homologous recombination secondary to amplicon formation.
Subject(s)
Gene Amplification , Genes, myc , Recombination, Genetic , Tumor Cells, Cultured , Adenocarcinoma/genetics , Adenocarcinoma/pathology , DNA Probes , DNA, Neoplasm/genetics , Extrachromosomal Inheritance , Gene Rearrangement , Humans , In Situ Hybridization, Fluorescence , Sigmoid Neoplasms/genetics , Sigmoid Neoplasms/pathologyABSTRACT
Preparation of high molecular weight DNA from resected tumor tissues suitable for pulsed-field gel electrophoresis (PFGE) can be complicated by the presence of nonviable cells and lymphocytes. We have developed a simple procedure to reduce the level of degraded DNA in PFGE DNA samples prepared from resected tumor tissues. The procedure employs a single, three component Percoll step gradient centrifugation and can be performed on several tumor samples simultaneously. Analyses of DNAs from 15 tumor specimens (7 solid tumors and 8 aspirated fluids) demonstrate that the technique enriches the integrity of PFGE DNA samples. Morphologic evaluation of 9 specimens suggested that both cellular debris and contaminating normal lymphocytes are removed from starting cell populations during the enrichment procedure. Fractionation of cells also reduced cell clumping, allowing for the formation of more uniform PFGE DNA samples.
Subject(s)
DNA, Neoplasm/isolation & purification , Electrophoresis, Gel, Pulsed-Field/methods , Centrifugation, Density Gradient , Evaluation Studies as Topic , Humans , Molecular Weight , Neoplasms/chemistry , Neoplasms/pathology , Povidone , Silicon DioxideABSTRACT
It was found that the acid protease of Fusarium culmorum can hydrolyze various proteins of plant origin including polygalacturonase inhibitor from bean (BPI) and soybean trypsin inhibitor (STI). The highest hydrolysis extent of BPI and STI by the enzyme was only 5% and 3% respectively. The partially hydrolyzed BPI lost its inhibition ability to fungal polyglacturonases. Similarly, the partially hydrolyzed STI lost its inhibition ability to trypsin and fungal alkaline protease. The F. culmorum acid protease showed broad substrate specificity towards synthetic dipeptides.
