Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells

Gutowski, Janice K.; Cohen, Stanley

doi:10.1016/0008-8749(83)90328-3

Cited by 26 publications

(24 citation statements)

References 20 publications

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“…Assays were performed in triplicate in 96-well microtiter plates (Falcon) as described (7). Nuclei (2 x 105 nuclei) were cultured with reaction buffer [0.25 M sucrose/25 mM Hepes, pH 7.8/2% (wt/vol) dextran (Sigma)] and cytoplasmic extract.…”

Section: Methodsmentioning

confidence: 99%

“…Using cell-free systems, a number of investigators have shown that cytoplasmic extracts from proliferating cells contain a protein(s) that can initiate nuclear DNA synthesis (2)(3)(4)(5)(6)(7)(8)(9). We have reported such cytoplasmic intermediate(s) in spontaneously proliferating and mitogen-activated lymphoid cells (7,8). This factor, which we have called activator of DNA replication (ADR), is a heat-labile protein of Mr >100,000.…”

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confidence: 99%

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Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Wong¹,

Gutowski²,

Katz³

et al. 1987

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

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Cytoplasmic extracts from spontaneously proliferating and mitogen-activated lymphoid cells contain a protein factor called ADR (activator of DNA replication) that induces DNA synthesis in isolated quiescent nuclei. ADRcontaining preparations have proteolytic activity, as indicated by their ability to degrade fibrin in a plasminogen-independent and plasminogen-dependent manner. In addition, aprotinin, a nonspecific protease inhibitor, abrogates ADR-induced DNA synthesis in a dose-dependent fashion. Preincubation studies demonstrated that the effect of aprotinin is not due to its suppressive effects on the nuclei themselves. Other protease inhibitors such as leupeptin, p-aminobenzamidine, and N-atosyllysine chloromethyl ketone are also inhibitory, but soybean trypsin inhibitor is without effect. ADR activity can be removed from active extracts by adsorption with aprotininconjugated agarose beads and can be recovered by elution with an acetate buffer (pH 5). These findings are consistent with the interpretation that the initiation of DNA synthesis in resting nuclei may be protease dependent and, further, that the cytoplasmic stimulatory factor we have called ADR may be a protease itself.It has been known for many years that nuclear DNA synthesis is under cytoplasmic control (1). Using cell-free systems, a number of investigators have shown that cytoplasmic extracts from proliferating cells contain a protein(s) that can initiate nuclear DNA synthesis (2-9). We have reported such cytoplasmic intermediate(s) in spontaneously proliferating and mitogen-activated lymphoid cells (7,8). This factor, which we have called activator of DNA replication (ADR), is a heat-labile protein of Mr >100,000. It is not detectable in resting cells. Although it activates isolated quiescent nuclei, it has no effect on intact cells. ADR is not species specific. Furthermore, ADR appears to mediate an intracellular mitogenic signal in the interleukin 2 (IL-2) T-cell activation pathway (9). Das (6) has described a cytoplasmic intermediate in epidermal growth factor-stimulated 3T3 fibroblasts that shares many functional and physicochemical properties with ADR. In addition, Benbow and Ford (2) reported a similar cytoplasmic stimulatory factor in early embryonic tissue. The description of ADR-like proteins in a number of cell types and experimental systems suggests a significant role for these factors in cellular proliferation and differentiation.To our knowledge, no information is available as to the biochemical nature of ADR or the ADR-like factors in other cell types (2-6). We now report that ADR preparations have proteolytic activity and that the functional activity of ADR can be significantly inhibited by aprotinin and a number of other protease inhibitors. The inhibition of ADR-induced DNA synthesis was not due to any direct effects of aprotinin on the isolated resting nuclei. Rather, the protease inhibitor appears to interact with ADR itself. MATERIALS AND METHODSCell Cultures and Preparation of Cytoplasmic Extracts. MOLT-4, a sp...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Wong¹,

Gutowski²,

Katz³

et al. 1987

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

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“…These preparations were not contaminated by intact cells or as determined by visual examination (phase contrast and electron microscopy). In any event, ADR has no effect on intact cells (8), so that even if intact cells remained in the nuclear preparations, they could rot have influenced the results.…”

Section: Introductionmentioning

confidence: 98%

“…that is capable of inducing DNA synthesis in isolated quiescent nuclei (8,9). This factor is not released from the cell, has no effect on intact cells, and is not detectable in the cytoplasm of resting PBL (8). These findings suggest that ADR may serve as a cytoplasmic "second signal" for PHA-or IL-2-induced lymphoproliferation.…”

Section: Introductionmentioning

confidence: 99%

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Impaired nuclear responsiveness to cytoplasmic signals in lymphocytes from elderly humans with depressed proliferative responses.

Gutowski¹,

Innes²,

Weksler³

et al. 1986

J. Clin. Invest.

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Lymphocytes from many elderly individuals exhibit depressed proliferative responses to the plant phytohemagglutinin (PHA). We have previously reported that this proliferative defect is not due to a failure to generate a cytoplasmic activator of DNA replication (ADR). In the present study, we tested the DNA synthetic response of nuclei derived from aged cells to an exogenous source of ADR. We found that nuclei from aged lymphocytes exhibiting low PHA responses were impaired in their ability to synthesize DNA in response to ADR, compared with nuclei from younger adult donors. In contrast, those aged cells maintaining intact PHA responses provided nuclei that were also unimpaired in their response to ADR. The relationship between PHA responsiveness of the intact cells and ADR responsiveness of nuclei derived from these was a linear one. These results suggest that the depressed cellular reactivity of aged lymphocytes to PHA (when seen) may be due to a failure of these nuclei to respond to cytoplasmic stimulatory signals induced by the mitogen.

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Intracellular signalling of initiation of DNA synthesis: Effect of cell‐free extracts from anti‐receptor‐stimulated B lymphocytes on spleen cell nuclei

Ullah

Kern

1987

J of Cellular Biochemistry

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To study the relatively late intracellular signals involved in the proliferative response of B lymphocytes to antibodies specific for surface membrane immunoglobulins, extracts from antibody activated cells were mixed with Xenopus laevis splenic nuclei, and the incorporation of thymidine 5'-triphosphate into DNA was assessed. The slight incorporation observed with either nuclei or extract alone was markedly enhanced upon mixing the two entities when the extract was derived from cells cultured with but not without anti-receptor antibody. The appearance of active extract correlated well with the culture requirements necessary for the induction of B lymphocyte proliferation and, as revealed by time course studies, the active component arises relatively late in the activation process. Moreover, the appearance of active extracts is independent of DNA synthesis but is dependent on protein synthesis as judged from studies with metabolic inhibitors. Appropriate homogenization of activated cells yielded nuclei and cytoplasm with 85% of the activity confined to nuclei. In addition, purified active extracts exhibited DNA binding although the active component was readily distinguishable from polymerase alpha by chromatographic techniques. It is tentatively concluded that the active component represents either some replication protein other than polymerase or some earlier signal necessary to induce the formation or utilization of replicating proteins.

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Induction of DNA synthesis in isolated nuclei by cytoplasmic factors from spontaneously proliferating and mitogen-activated lymphoid cells

Cited by 26 publications

References 20 publications

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Induction of DNA synthesis in isolated nuclei by cytoplasmic factors: inhibition by protease inhibitors.

Impaired nuclear responsiveness to cytoplasmic signals in lymphocytes from elderly humans with depressed proliferative responses.

Intracellular signalling of initiation of DNA synthesis: Effect of cell‐free extracts from anti‐receptor‐stimulated B lymphocytes on spleen cell nuclei

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