Binding of Tritiated Actinomycin and Cell Differentiation

Brachet, Jean; Hulin, Nicole

doi:10.1038/222481a0

Cited by 53 publications

(23 citation statements)

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“…Measurements of AMD binding during spermiogenesis (Brachet and Hulin, 1969 ;Darzynkiewicz, Gledhill, and Ringertz, 1969), in phytohemagglutinin-transformed lymphocytes (Darzynkiewicz, Bolund, and Ringertz, 1969) prednisolonetreated thymocytes (Darzynkiewicz and Andersson, 1971), and in heterochromatin vs. euchromatin within the same nucleus (Berlowitz et al ., 1969) have all indicated reduced ligand binding in condensed chromatin . While a comparison of random and mitotic HeLa cells would have led to the same conclusion, the use of cells synchronized in S and G 2 reveals that AMD binding decreases progressively over this interval and is not temporally coordinated with chromosome coiling per se .…”

Section: Amd Binding Capacity Of Dna Purified From Synchronized Cellsmentioning

confidence: 99%

“…A number of studies have indicated that the binding of actinomycin to DNA of living or fixed cells is significantly influenced by the intranuclear organization of the chromatin (Darzynkiewicz, Bolund, and Ringertz, 1969 ;Brachet and Hulin, 1969 ;Berlowitz, et al ., 1969 ;Bolund, 1970) ; in general, highly condensed chromatin binds considerably less actinomycin than the same amount of DNA disposed in a more diffuse configuration . We have employed actinomycin-3H to study the organization of chromatin over the cell division cycle in synchronized HeLa cells and have found significant changes in binding during interphase, when there is no cytological evidence of chromosome condensation .…”

Section: Introductionmentioning

confidence: 99%

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Chromatin Structure and the Cell Division Cycle

Pederson

Robbins

1972

The Journal of Cell Biology

102

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Measurements of actinomycin-3H binding in synchronized HeLa cells reveal that the binding capacity of chromatin decreases progressively during the S phase despite a doubling of nuclear DNA content, reaches a minimal level during G 3 and mitosis, and then increases gradually throughout the subsequent G, interval . Since this pattern was evident in experiments with living cells, ethanol-fixed cells, and isolated nuclei, but not with purified DNA, the actinomycin binding profile may reflect changes in the degree of association between DNA and chromosomal proteins at different stages of the cell cycle .

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Section: Amd Binding Capacity Of Dna Purified From Synchronized Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Chromatin Structure and the Cell Division Cycle

Pederson

Robbins

1972

The Journal of Cell Biology

102

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“…We confirm this observation (Table 1) In other cell systems, when the nuclear genome is activated, the extent to which actinomycin D binds to the nucleus increases (18), and when the nuclear genome is suppressed, the extent to which actinomycin D binds to the nucleus falls (19- 23). Unmasking of DNA in nuclear chromatin is thought to permit binding of actinomycin D to newly accessible DNA sites during genome activation, and masking of DNA is held responsible for decreased actinomycin D binding to nuclear DNA during genome suppression (18)(19)(20)(21)(22)(23)(24) (1974) .--.--.-.…”

Section: Discussionmentioning

confidence: 99%

Chick-Erythrocyte Nucleus Reactivation in Heterokaryons: Suppression by Inhibitors of Proteolytic Enzymes

Darżynkiewicz

Chelmicka-Szorc

Arnason

1974

Proc. Natl. Acad. Sci. U.S.A.

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Reactivation of chick-erythrocyte nuclei in heterokaryons (obtained by Sendai virus-induced fusion of chick erythrocytes with HeLa cells) is suppressed by specific inhibitors of trypsin and trypsin-like enzymes.N-a-tosyl-L-lysyl-chloromethane and N-a-tosyl-L-arginine methylester inhibit erythrocyte nuclear enlargement and suppress RNA and DNA synthesis in nuclei of erythrocytes and HeLa cells in heterokaryons at concentrations that only minimally influence individual HeLa cells or HeLa homokaryons.Although other unknown mechanisms of action cannot be formally excluded, the data are interpreted as fitting best with an intracellular site of action of the protease inhibitors studied, and as suggesting a role for cellular proteases in reactivation of chick-erythrocyte nuclei in heterokaryons.The mature chick erythrocyte contains a dormant nucleus, the replicative and transcriptional activities of its nuclear DNA having ceased during the latter stages of erythropoiesis. The nuclear chromatin of this end-stage cell is condensed and nucleoli are absent (1, 2). When chick erythrocytes are fused with active cells (e.g., HeLa cells or fibroblasts) to form heterokaryons, the erythrocyte nucleus is reactivated (3, 4). Shortly after cell fusion, the volume and dry mass of the erythrocyte nucleus increase, its tightly packed chromatin ("nuclear bodies") disperses, and RNA synthesis resumes. DNA replication begins 1-2 days after fusion, and nucleoli appear a day or two later (3-8).The basis for reactivation of the dormant erythrocyte nucleus in heterokaryons is not understood. During Sendai virus-induced cell fusion the erythrocyte lyses and erythrocyte nuclei enter the cytoplasm of their partner cells without appreciable quantities of erythrocyte cytoplasm (3-9). For this reason it is believed that factors present in partner cell cytoplasm reactivate erythrocyte nuclei (5, 6, 10). We report here that certain inhibitors of proteolytic enzymes suppress reactivation of erythrocyte nuclei in heterokaryons. In addition, HeLa nuclei, ordinarily resistant to protease inhibitors, become sensitive to them when paired with chick-erythrocyte nuclei in heterokaryons, suggesting that they are sensitized by factors released by the erythrocyte nucleus. The results implicate cellular proteases in reactivation of chick-erythrocyte nuclei in heterokaryons.Abbreviations: TLCK, N-a-tosyl-L-lysyl-chloromethane (trivial name: L-1-tosylamide-S2leucylchloromethylketone); TAME, Na-tosyl-L-arginine methylester; TPCK, N-a-tosyl-L-phenylalanylchloromethane (trivial name: L-1-tosylamide-2-phenyl-chloromethylketone); PheMeSF, phenyl-methyl sulfonyl fluoride; Me2SO, dimethylsulfoxide. MATERIALS AND METHODSChick erythrocytes were obtained from 15-to 16-day-old embryos as described (7,11). HeLa cell cultures were maintained in Eagle's medium containing 10% fetal bovine serum and antibiotics (penicillin 100 u/ml; streptomycin 100 ,ug/ ml). Heterokaryons were prepared by fusion of chick erythrocytes with HeLa cells using UV-inactivated Sendai virus (80...

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“…In this connection, the binding of actinomycin to chromatin, which is an index of the amount of free DNA, is closely related to genetic activity, or at any rate to the physicochemical characteristics of the DNA-protein complex [12][13][14][15]. The autoradiographic method can be usefully employed with such highly heterogeneous materials in order to obtain a valid comparison between normal and leukaemic cells [16].…”

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confidence: 99%

Myeloid Cell Actinomycin Binding in Human Myeloid Leukaemia

Pileri¹,

Masera²,

Garbarino³

et al. 1974

Acta Haematol

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Decreased actinomycin D binding was observed in the marrow cells of some patients with chronic myeloid leukaemia. Blast uptake was still reduced in 2 cases of blast crisis and in acute myeloblastic leukaemia. However, the differentiating cells in blast crisis, i.e., those that had passed the blast stage, had exactly the same actinomycin D binding values as normal myeloid cells. After 0.5 mg/kg methotrexate, increased actinomycin D binding on the part of the entire leukaemic population was observed in one case of acute myeloblastic leukaemia. These findings suggest differences in chromatin behaviour and genetic activity in myeloid cells in the different stages of myeloid leukaemia.

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Binding of Tritiated Actinomycin and Cell Differentiation

Cited by 53 publications

References 6 publications

Chromatin Structure and the Cell Division Cycle

Chromatin Structure and the Cell Division Cycle

Chick-Erythrocyte Nucleus Reactivation in Heterokaryons: Suppression by Inhibitors of Proteolytic Enzymes

Myeloid Cell Actinomycin Binding in Human Myeloid Leukaemia

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