No abstract

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...

Highly purified pregnant mares serum gonadotropin (PMSG) was nearly as potent as ovine luteinizing hormone (LH) and human follicle stimulating hormone (hFSH) when bioassayed in vitro in systems known to respond primarily to LH or FSH. An analogue of human chorionic gonadotropin treated with neuraminidase, galactosidase, beta-N-acetylglucosaminidase, and mannosidase (hCG) inhibited the stimulatory effects of hCG, LH, and PMGS on cAMP accumulation in rat Leydig cells but did not inhibit the stimulatory effects of FSH or PMSG on cAMP accumulation in ovarian granulosa cells obtained from immature rats fed diethylstillbestrol. Thus PMSG appeared to form functional complexes with both LH and FSH receptors and may be unique among mammalian gonadotropins. Treatment of PMGS with neuraminidase increased its potency nearly tenfold in vitro apparently by increasing its affinity for both LH and FSH receptors. Although the kinetics of PMSG binding were not investigated with radiolabeled materials, indirect functional binding studies are described that suggest that hCG more rapidly forms stable hormone-receptor complexes than PMSG, asialo-PMSG, FSH, and LH when all hormones are incubated under the same conditions.

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.

We The failure of cytoplasmic extracts prepared from resting cells to exhibit cytoplasmic stimulatory activity may be due to (i) a lack of ADR in quiescent cells, (it) the presence of ADR in an inactive or precursor form, or (iii) the presence of inhibitory signals. There is evidence that, in fact, resting cells may contain inhibitors of DNA synthesis (8-15). In the present study, therefore, we investigated the possibility that unstimulated PBL contain a factor that can inhibit nuclear activation by ADR. We found that resting PBL contain a heat-stable protein 2 50,000 daltons that can suppress the induction of DNA synthesis in quiescent nuclei by ADR derived from both normal and neoplastic cells. MATERIALS AND METHODSCell Cultures. Human PBL, obtained by Ficoll/Hypaque density gradient centrifugation, were cultured in flasks at 106 lymphocytes per ml in RPMI 1640 medium (M. A.Bioproducts, Walkersville, MD) containing 10% human AB serum, 100 units of penicillin and 100 Ag of streptomycin per ml, and 2 mM L-glutamine (GIBCO), with or without phytohemagglutinin (PHA) at 10 ,ug/ml (Sigma). Unless otherwise specified, cytoplasmic extracts were prepared from the PHA-stimulated cells and the control unstimulated cells after 66-72 hr of culture.Suspension cultures of MOLT 4 cells were grown and maintained as reported (2). Extracts were routinely prepared from cultures grown to a density of approximately 106 cells per ml.Macrophage Depletion. Macrophages were depleted from PBL preparations by passage over glass bead columns at 370C in the presence of fetal bovine serum as described (16). Cell recovery from the columns ranged from 40%o to 70% of the starting populations. To assess the purity of these preparations, samples were stained for myeloperoxidase. By this method, the unfractionated PBL contained 12-18% macrophages, whereas the column-purified, depleted preparations contained c 1% macrophages.