Electron microscopic localization of acridine orange binding to euchromatin in human neuroblastoma cells.
The purpose of the present study was to examine the distribution pattern of acridine orange (AO) chromatin interaction products (AOCI) in human neuroblastoma IMR-32 cells and to test whether AO labeling is correlated with BrdU incorporation, and immunohistochemical localization of DNA polymerase alpha, and human N-myc-gene product. Effects of aphidicolin, alpha-amanitin, and actinomycin D on visualization of AO binding to euchromatin and on N-myc-gene expression were also examined. About 25% of the cell nuclei in logarithmic growth phase were immunohistochemically demonstrated to be labeled with BrdU after incubation at 37 degrees for 30 min, indicating cells in DNA synthesis. Most of the cell nuclei showed positive immunoreactivity to DNA polymerase alpha, while human N-myc gene product was found in about 60-80% of the cell nuclei. Electron microscopic studies revealed that about 25% of neuroblastoma cells showed characteristic AOCI within cell nuclei. In the presence of aphidicolin, alpha-amanitin, and actinomycin D, positive cells for N-myc gene product decreased markedly. Percentages of AO positive cells and numbers of AOCI per cell nucleus also showed a marked decrease. But northern blot analysis demonstrated that the expression level of N-myc gene was only repressed by the transcriptional inhibitors alpha-amanitin and actinomycin D. However, no repression was caused by aphidicolin. The present and previous studies of the authors suggest that the ultracytochemical AO method may be indicative for conformational changes of chromatin of cells confined to the cell cycle. Inhibitors of RNA and DNA synthesis then may change the conformational state of chromatin.(ABSTRACT TRUNCATED AT 250 WORDS)
Hiroi
M
M
Department of Pathology, Kochi Medical School, Nankoku, Japan.
Moriki
T
T
Taniguchi
T
T
Yamane
T
T
Lehmann
R
R
Hara
H
H
D000165:Acridine Orange / Q000032:analysis / Q000378:metabolism*; D000546:Amanitins / Q000494:pharmacology; D016590:Aphidicolin; D015152:Blotting, Northern; D001973:Bromodeoxyuridine / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002843:Chromatin / Q000378:metabolism*; D004257:DNA Polymerase II / Q000032:analysis / Q000037:antagonists & inhibitors; D004273:DNA, Neoplasm / Q000032:analysis / Q000096:biosynthesis; D003609:Dactinomycin / Q000494:pharmacology; D004171:Diploidy; D004224:Diterpenes / Q000494:pharmacology; D022041:Euchromatin; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D016259:Genes, myc; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D000546:Amanitins; D002843:Chromatin; D004273:DNA, Neoplasm; D004224:Diterpenes; D022041:Euchromatin; D016271:Proto-Oncogene Proteins c-myc; D003609:Dactinomycin; D016590:Aphidicolin; D004257:DNA Polymerase II; D000165:Acridine Orange; D001973:Bromodeoxyuridine