ABSTRACT
In this work, we used colon carcinoma cell-line HCT116 to study the involvement of the 86-kDa subunit (Ku86) of DNA-protein kinase (DNA-PK) in human tumoural cell proliferation. We transfected these cells with a 639-bp cDNA encoding a Ku86 portion inserted into pcDNA3.1 vector in the antisense orientation. After selection by neomycin, we obtained more than 300 resistant colonies. In the Y'A5 colony that we chose as total population, we showed by PCR and RT-PCR that pcDNA3/Ku86 antisense was integrated in genomic DNA and that transcript was present. After cloning, we selected two clones, A20 and A23, which contained significatively reduced level of Ku86 protein. These two clones displayed a reduced DNA-PK activity from 44% to 71% and a slower growth than control cells. These results suggest that the HCT116 cell-line is a useful tool to investigate the role of Ku86 in the regulation of human tumoural cell growth.
Subject(s)
Antigens, Nuclear , Colonic Neoplasms/pathology , DNA Helicases , DNA, Antisense/genetics , DNA-Binding Proteins/metabolism , Nuclear Proteins/metabolism , Blotting, Western , Cell Division/drug effects , Cloning, Molecular , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , DNA-Binding Proteins/genetics , Drug Resistance, Neoplasm , Humans , Ku Autoantigen , Neomycin/pharmacology , Nuclear Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transfection , Tumor Cells, CulturedABSTRACT
DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylates, in the presence of double-stranded DNA, several transcription-, replication- and repair -factors. Its interaction with the DNA-binding regulatory component Ku (p86-/p70-Ku) is required for stabilization and activity. We have previously shown that p86-Ku behaves as a specific receptor for the growth inhibitory tetradecapeptide, somatostatin. In this work, we investigate a possible regulation by somatostatin analogs, of DNA-PK activity in the human gastric tumoral HGT1/clone6 cell-line. We demonstrate that a 48 h-preincubation of cells with octreotide or RC-160, stimulates DNA-PK activity by 8 and 10 fold with ED50s of 1 and 0.1 nM, respectively. These stimulations appearing only after 3 h were inhibited by cycloheximide. They were not observed in a cell clone which was transfected by a cDNA encoding p86-Ku antisense. This study demonstrates the existence of a new somatostatin signaling pathway involving the stimulation of DNA-PK activity.
Subject(s)
Antigens, Nuclear , Antineoplastic Agents/pharmacology , DNA Helicases , Octreotide/pharmacology , Protein Serine-Threonine Kinases/metabolism , Somatostatin/analogs & derivatives , Stomach Neoplasms/enzymology , Catalysis , Clone Cells , DNA, Complementary/genetics , DNA, Complementary/metabolism , DNA-Activated Protein Kinase , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Dose-Response Relationship, Drug , Humans , Kinetics , Ku Autoantigen , Nuclear Proteins/biosynthesis , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oligodeoxyribonucleotides, Antisense/genetics , Oligodeoxyribonucleotides, Antisense/metabolism , Somatostatin/pharmacology , Stomach Neoplasms/pathology , Transfection , Tumor Cells, CulturedABSTRACT
In Xenopus laevis, three nucleoside diphosphate kinase (NDPK) monomers have been described (NDPK X1, X2 and X3) (Ouatas et al., 1997). In eucaryotes, this kinase is known as a hetero- or homohexamer. Here, we examine the distribution of the enzyme and its different subunit mRNAs during oogenesis and early embryogenesis of Xenopus laevis, respectively by immunohistofluorescence and whole-mount in situ hybridization. These analyses show that NDPKs and their mRNAs are differentially distributed throughout the oocyte and early embryos with a high level of transcription in somites and brain. We emphasize two points. First, each mRNA displays a distinct subcellular localization in somites, suggesting a complex regulation of NDPK genes both at the transcriptional and translational level and a possible involvement of NDPK X2 homohexamers in the dorsal muscle differentiation. Second, in oocytes and early embryos, the proteins are mainly localized in the nucleus, suggesting a new mechanism for their nuclear import, since they do not possess any known nuclear import sequences.
