ABSTRACT
The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), encoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- or heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific functions. The nm23-H1 gene is implicated in tumor progression and metastasis, and the nm23-H2 gene product is a transcription factor for c-myc. To determine if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluorescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231) using highly specific polyclonal and monoclonal antibodies. Interphasic cells exhibited a granular and filamentous cytoplasmic staining, particularly intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with microtubules, such as taxol and colchicine. Confirming published biochemical data, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B was shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are similarly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in accord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures which are not intermediate filament aggregates.
Subject(s)
Cytoskeleton/metabolism , Monomeric GTP-Binding Proteins , Nucleoside-Diphosphate Kinase/metabolism , Transcription Factors/metabolism , Animals , Antibody Specificity , Breast Neoplasms , Cell Division , Cell Nucleus , Female , Fluorescent Antibody Technique, Indirect , Humans , Interphase , Microtubules/metabolism , Mitosis , NM23 Nucleoside Diphosphate Kinases , Nucleoside-Diphosphate Kinase/immunology , Rabbits , Transcription Factors/immunology , Tumor Cells, Cultured , Vimentin/analysisABSTRACT
We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27-31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23-H5 gene is located on chromosome 5q23-31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.
Subject(s)
Histones/genetics , Monomeric GTP-Binding Proteins , Spermatozoa/metabolism , Testis/metabolism , Transcription Factors/genetics , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 5 , Cloning, Molecular , DNA Primers , DNA, Complementary , Humans , In Situ Hybridization , Male , Molecular Sequence Data , NM23 Nucleoside Diphosphate Kinases , Nucleoside-Diphosphate Kinase/metabolism , Polymerase Chain Reaction , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Testis/cytologyABSTRACT
We evaluated (i) the stability of a mixture of the three anti-cancer agents used for the treatment of leukemia, namely etoposide, cytarabine and daunorubicine, in 5% glucose, and (ii) its compatibility towards various materials during an infusion protocol as performed for therapeutic purposes in hospital practice. Etoposide and cytarabine were assayed by high-performance liquid chromatography with a C18 type column and UV detection. Daunorubicine was assayed by visible spectrophotometry. The stability study showed all three anti-cancer drugs to be stable in 5% glucose solution, both alone and mixed. Best conservation was obtained by keeping bottles containing the mixture in the dark at room temperature. During the infusion protocol used in clinical practice, etoposide, cytarabine and daunorubicine were stable and compatible with the various materials present in the infusion sets and extension tubing (polyvinyl chloride, polyethylene) and catheters (silicone). Observed variations in concentration did not exceed 10% of initial concentrations of each drug, though we would advocate changing infusion sets and extension tubing daily.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/chemistry , Cytarabine/administration & dosage , Daunorubicin/administration & dosage , Drug Incompatibility , Drug Stability , Etoposide/administration & dosage , Infusions, IntravenousABSTRACT
A rapid, accurate and selective method for the determination of isosorbide dinitrate and its 2- and 5-isosorbide mononitrate metabolites in 1.0 ml of human plasma has been developed. Before chromatographic quantitation by gas-liquid chromatography with electron-capture detection, the compounds are subjected to solid-phase extraction, using ENVI 18 cartridges (Supelco). The intra-day and inter-day coefficients of variation are less than 10%, except the inter-day coefficient of variation for the assay of 5-isosorbide dinitrate which is less than 15%. Limits of quantitation are 10, 10 and 20 ng/ml for isosorbide dinitrate, 2-isosorbide mononitrate and 5-isosorbide mononitrate, respectively. Recoveries are in excess of 90% for isosorbide dinitrate and 70% for its two metabolites.
