Effect of isoprenaline on cells in different phases of the mitotic cycle

The effects of isoprenaline on parotid acinar cells in different phases of the mitotic cycle have been investigated. Cells in mitosis at the time of drug administration are not depleted of secretory granules whilst those in other phases are. The drug causes temporary blocks both in metaphase and in the G2 phase. The blocks are prolonged by repeated injections of the drug. Cells continue to undergo DNA synthesis during the period of secretion following the drug. The mitotic delay appears to be specific for the parotid and submaxillary glands.     

Does beta-adrenoceptor activation stimulate Ca2+ mobilization and inositol trisphosphate formation in parotid acinar cells?

The effects of the beta-adrenoceptor agonist, isoprenaline, on Ca2+ mobilization and inositol phosphate formation in parotid acinar cells were examined. Isoprenaline (2 microM) failed to increase cytosolic [Ca2+] in acinar cells, as measured by Fura-2 fluorescence, even in the presence of a phosphodiesterase inhibitor. Likewise, neither the 8-bromo nor the dibutyryl derivatives of cAMP (both at 2 mM concentration) increased [Ca2+]i. However, in confirmation of results previously published, a higher concentration of isoprenaline (200 microM) increased cytosolic [Ca2+]i of rat parotid acinar cells, from 104 +/- 4 nM to 151 +/- 18 nM. The increase in [Ca2+]i in response to isoprenaline, while transient in the absence of extracellular Ca2+, was sustained in Ca2(+)-containing medium. This isoprenaline-stimulated Ca2+ signal was more potently antagonized by phentolamine than by propranolol, suggesting that the higher concentration of isoprenaline activated alpha-adrenoceptors. Furthermore, the Ca2+ signal generated in response to the alpha-adrenoceptor agonist, phenylephrine, also was blocked by the same concentrations of propranolol necessary to block the effects of isoprenaline, suggesting that propranolol may block alpha-adrenoceptors under certain experimental conditions. The high concentration of (-)isoprenaline (200 microM) also increased inositol (1,4,5) trisphosphate and inositol (1,3,4) trisphosphate formation 45% within 30 s. Analogous to the increase in intracellular Ca2+, the formation of inositol phosphates stimulated by isoprenaline was more potently antagonized by the alpha-adrenoceptor antagonist, phentolamine, than by the beta-adrenoceptor antagonist, propranolol, again suggesting that isoprenaline interacts with alpha-adrenoceptors on parotid cells. Thus, the effects of isoprenaline on [Ca2+]i do not appear to be mediated by cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Cycloheximide on Mitosis in Vicia faba Root-meristem Cells

Three different responses of dividing root-meristem cells tocycloheximide have been observed. Cells in anaphase or telophasecan complete these stages and pass into G1; movement of prophaseand metaphase cells into anaphase is reversibly inhibited; entryof G2 cells into mitosis is irreversibly inhibited. While thislast effect is probably a consequence of inhibition of proteinsynthesis by cycloheximide, it is suggested that the reversiblemitotic arrest induced by the drug results from inhibition ofsome other cellular activity, possible related to energy transfer.     

Characteristics of the isoprenaline stimulated proliferative response of rat submaxillary gland.

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using 3H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMWa) waves gave a mean Gis transit time of 21-2 hr, mean Gis +S transit time of 27-0 hr, and mean Gis + S + G2 transit time of 35-8 hr for a single injection of isoprenaline, where Gis is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the Gis phase dropped from 18-1 to 1-3 while the other variances remained unchanged. The estimated proliferative fraction was 0-24 after a single injection of isoprenaline, and 0.31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated 3H-TdR injections, were 0-21 and 0-36 respectively.     

Progression of mouse oocytes from metaphase I to metaphase II is inhibited by fusion with G2 cells

We show that in contrast to metaphase II oocytes, metaphase I oocytes cannot be activated by fusion with the zygote. Fusion of metaphase I oocytes with G2 zygotes was followed by premature chromosome condensation, with 60% of the hybrids becoming arrested at metaphase I, the remainder progressing and arresting at metaphase II. Hybrids of metaphase I oocytes and M-phase zygotes underwent accelerated maturation, but all arrested at metaphase II. In both cases the arrest could be overcome by treatment with the parthenogenetic activators ethanol and cycloheximide. We discuss these findings in relation to the possibility that the metaphase I oocyte contains cytostatic factor activity that is activated by its zygotic partner. Alternatively, the G2 zygote may provide an inhibitor of anaphase, normally never present in the metaphase I oocyte and which is absent from the M-phase zygote.     

Mammalian cell fusion. VI. Regulation of mitosis in binucleate HeLa cells.

In two different cell fusion experiments a synchronized population of HeLa cells, prelabeled with ³H-TdR, was fused with an unlabeled one using inactivated Sendai virus. In the first experiment, HeLa cells in early G2 phase which were exposed to either 4 °C, cycloheximide, actinomycin D or X-irradiation were fused separately with untreated and more advanced G2 cells. A comparison of the rates of mitotic accumulation (in the presence of Colcemid) for the various classes of mono- and binucleate cells revealed that the hybrid (binucleate) cells were intermediate between those of the advanced and the retarded parental types indicating that the chromosome condensing factors of the advanced component were diluted as a result of such fusion. The manner in which the retarding effects of actinomycin D and cycloheximide were reversed in the hybrid cells suggested that proteins had a major role as chromosome condensing factors in the G2 mitotic transition. In the second experiment, when S phase HeLa cells were fused with those in G2, the resulting heterophasic (S/G2) binucleate cells reached mitosis at about the same time as the homophasic (S/S) cells of the lagging parent indicating a complete dominance of the S over the G2 with regard to their progress towards mitosis. However, the addition of Mg²⁺ (2 × 10^?2 M of MgCl₂) to the medium helped the G2 nuclei to enter mitosis asynchronously, which consequently induced premature chromosome condensation (PCC) in the S phase component. These data suggested that in the heterophasic (S/G2) binucleate cells the S phase component caused decondensation of the G2 chromatin thus blocking it from entering into mitosis. This effect which did not appear to be dose-dependent could be neutralized and the G2 nuclei relieved from this repression by an external supply of Mg²⁺ ions.     

Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53

We investigated the effect of Adriamycin on FL-amnion (FL) cells. After treatment with the drug, the cells arrested at G2, but we did not detect an increase in the p21 levels. We established a p53-deficient derivative of these cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, while late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells. Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDC2 kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis. CDC2 kinase activation was inhibited by cycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cyclin A, which normally precedes CDC2 kinase activation in caffeine-treated cells. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint.     

钙离子信号对非细胞体系中小鼠卵母细胞游离生发泡减数分裂启动的影响

本文构建了包括HeLa裂解液和游离小鼠卵母细胞生发泡的实验体系,用于研究Ca^2 及其下游信号对小鼠卵母细胞减数分裂启动的影响。游离的卵母细胞生发泡可以在M期细胞裂解液中发生减数分裂启动,表现为染色质的凝集。进一步的研究表明,Ca^2 信号的存在对G2期细胞裂解液促进减数分裂启动是至关重要的,G2期中期的细胞裂解液只有经Ca^2 诱导后才具有启动生发泡减数分裂的作用,而G2期晚期无论Ca^2 存在与否均诱发减数分裂的启动,但是G2期早期的裂解液无启动减数分裂的作用。卵母细胞的体外培养实验分析也表明,抑制CaM和CaMKⅡ的活性可以阻止GVBD和报制第一极体的释放。免疫沉淀及Western Blotting结果显示,HeLa细胞裂解液中的MPF从G2期中期到M期均存在,且Cdc2亚基的Tyr由磷酸化向去磷酸化转变。结果进一步证明,卵母细胞减数的分裂的启动可能是通过一种Ca^2 ／CaM依赖的途径来推动的。     

Novel response to microtubule perturbation in meiosis

During the mitotic cell cycle, microtubule depolymerization leads to a cell cycle arrest in metaphase, due to activation of the spindle checkpoint. Here, we show that under microtubule-destabilizing conditions, such as low temperature or the presence of the spindle-depolymerizing drug benomyl, meiotic budding yeast cells arrest in G(1) or G(2), instead of metaphase. Cells arrest in G(1) if microtubule perturbation occurs as they enter the meiotic cell cycle and in G(2) if cells are already undergoing premeiotic S phase. Concomitantly, cells down-regulate genes required for cell cycle progression, meiotic differentiation, and spore formation in a highly coordinated manner. Decreased expression of these genes is likely to be responsible for halting both cell cycle progression and meiotic development. Our results point towards the existence of a novel surveillance mechanism of microtubule integrity that may be particularly important during specialized cell cycles when coordination of cell cycle progression with a developmental program is necessary.     

Effects of caffeine and cycloheximide during G2 prophase in control and X-ray-irradiated human lymphocytes

The effect of caffeine and cycloheximide during the G2 phase on frequency of chromosomal aberrations and G2 duration was studied in control and X-ray-irradiated human lymphocytes in vitro. Caffeine treatments alone increase the frequencies of chromatid breakage and decrease the average G2 duration in control and X-ray-irradiated lymphocytes (40 R). Both caffeine effects are reversed by 0.5 micrograms/ml cycloheximide in combination treatments. Cycloheximide treatments alone prolong G2 duration in control as well as in X-ray-irradiated lymphocytes although no improvement in chromosome repairing by this inhibitor of protein synthesis was observed under the conditions of our experiments. We propose that the cycloheximide effect is associated with a low level of mitotic factors, required for the entrance into mitosis, which is maintained at a higher level in caffeine treatment alone. Finally, G2 delay has generally been associated with certain genome damage. The fact that the caffeine and cycloheximide effects on X-irradiated lymphocytes are also present in control lymphocytes (without X-rays) suggests that control of the G2 duration constitutes one of the mechanisms involved in DNA repair operating during the G2 phase.     

钙离子信号对非细胞体系中小鼠卵母细胞游离生发泡减数分裂启动的影响

本文构建了包括HeLa裂解液和游离小鼠卵母细胞生发泡的实验体系,用于研究Ca2+及其下游信号对小鼠卵母细胞减数分裂启动的影响.游离的卵母细胞生发泡可以在M期细胞裂解液中发生减数分裂启动,表现为染色质的凝集.进一步的研究表明,Ca2+信号的存在对G2期细胞裂解液促进减数分裂启动是至关重要的,G2期中期的细胞裂解液只有经Ca2+诱导后才具有启动生发泡减数分裂的作用,而G2期晚期无论Ca2+存在与否均诱发减数分裂的启动,但是G2期早期的裂解液元启动减数分裂的作用.卵母细胞的体外培养实验分析也表明,抑制CaM和CaMKII的活性可以阻止GVBD和抑制第一极体的释放.免疫沉淀及Western Blotting结果显示,HeLa细胞裂解液中的MPF从G2期中期到M期均存在,且Cdc2亚基的Tyr由磷酸化向去磷酸化转变.结果进一步证明,卵母细胞减数分裂的启动可能是通过一种Ca2+/CaM依赖的途径来推动的.     

Absence of a direct role of phospholipid methylation in stimulus-secretion coupling and control of adenylate cyclase in guinea-pig and rat parotid gland.

The present study was undertaken to investigate a possible involvement of phospholipid methyltransferases in the coupling of receptor-mediated stimulation to secretion. Phospholipid methyltransferases were assayed in isolated parotid acini in the presence of carbamoylcholine or isoprenaline. Carbamoylcholine reduced the incorporation of methyl groups into phospholipids, whereas isoprenaline showed no effect. Amylase secretion stimulated either by carbamoylcholine or by isoprenaline could not be affected by inhibitors of methyltransferases (3-deaza-adenosine alone or plus homocysteine thiolactone) under conditions where phospholipid methylation was strongly inhibited. The activity of adenylate cyclase in isolated parotid microsomal membranes was not inhibited or stimulated by S-adenosyl-homocysteine or -methionine respectively. These results indicate that phospholipid methylation does not play an essential role in stimulus-secretion coupling in the parotid gland.     

Characterization of human and rat immortalized clones of parotid acinar cells with respect to specific proteins and their mRNAs,and receptor-linked adenylate cyclase

Summary This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP₁G). The levels ofα-amylase mRNAs detected when usingα-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP₁G cells. In contrast toα-amylase mRNAs levels, theα-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2HP₁G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the sameα-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP₁G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP₁G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E₁ (PGE₁) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP₁G cells, suggesting that the PGE₁ receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.     

Isoprenaline-induced changes in rat parotid and submandibular glands are age- and dosage-dependent.

Neonatal rats treated with chronic injections of isoprenaline (isoproterenol) for 10 days revealed differential induction of proline-rich proteins and glycoprotein synthesis between the parotid and submandibular glands. Biosynthesis of proline-rich proteins (Mr 17000-35000) and a Mr-220000 glycoprotein were detectable by solubilization in 10%-trichloroacetic acid extracts from parotid glands 14 days after birth. The enzyme lactose synthase (UDP-galactose: 2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase) (EC 2.4.1.22) is also induced 4-7-fold in specific activity compared with control neonatal rats, but again only after 14 days post partum, with isoprenaline treatment. This is in accord with the ability of the parotid gland to respond to beta-receptor stimulation and subsequent increases in intracellular cyclic AMP necessary for induction of protein synthesis [Grand, Chong & Ryan (1975) Am. J. Physiol. 228, 608-612]. Induction of the proline-rich proteins and a Mr-190000 glycoprotein in the soluble fraction from the submandibular gland were not detected until 49 days after birth under identical conditions in the same animal. Cyclic AMP in the submandibular gland undergoes increases on beta-receptor stimulation similar to those achieved in the adult animal, 1 day after birth (Grand et al., 1975). This same differential induction between parotid and submandibular gland was obtained with a range of isoprenaline dosages in adult animals. Trichloroacetic acid-soluble proline-rich proteins were isolated from parotid glands at a dosage of 4.0 mg of isoprenaline/kg body wt., but 7.0 mg/kg was required to induce also biosynthesis of these proteins in the submandibular gland. Gland hypertrophy showed the same differential dosage kinetics, based on gland weight, between the two glands; however, hypertrophy could be accomplished at a lower dosage of isoprenaline than that used to induce proline-rich-protein biosynthesis.     

Isoprenaline-induced cell proliferation in mouse salivary glands: the effect of castration

The proliferative response to isoprenaline in the submaxillary and parotid glands of the Balb/c mouse has been studied in the intact male and female, and also in the male castrated one month prior to stimulation. The hyperplastic response of the acinar cells has been monitored by serial measurements of the flash tritiated thymidine labelling index and the mitotic index. Castration caused the atrophy of the granular ducts in the submaxillary gland, and therefore an increased predominance of the acini. At one month after castration the acini occupied an area almost 1.5-fold greater than that of the granular ducts, but this was not as great as in the intact female gland where acini occupied twice the area of the granular ducts. Hyperplasia was induced by a single injection of isoprenaline (0.3 mM/kg body weight). The response of the submaxillary gland in the intact male and intact female was very similar, DNA synthesis commencing 21-24 h after stimulation and mitotic activity first noted after 33-36 h. On the other hand, in the submaxillary gland of the castrated male, DNA synthesis began after only 18-21 h and mitotic activity after only 27-30 h. A metaphase arrest experiment with vincristine confirmed the more prompt response in the castrated animals; between 33-36 h after isoprenaline injection, the rate of entry of cells into mitosis was 4 cells/100 cells/h in the castrated group but only 0.4 cells/100 cells/h in the intact males. Thus castration appears to bestow a unique state of responsiveness upon the submaxillary gland to isoprenaline stimulation. The mechanisms underlying this change are not yet understood, for it is paradoxical that atrophy of a structural component rich in specific protein growth factors can alter the format of isoprenaline-induced hyperplasia in acinar cells that produce secretory glycoproteins.     

Discrepancies among the metaphase, telophase, and the G0/G1 and G2 DNA peaks of heteroploid cell lines

Heteroploid cell populations often show narrow peaks of G0/G1 and G2/M DNA content and broadly distributed chromosome numbers. This was originally explained by the selective metaphase arrest of the cells that have non-modal chromosome numbers. To test whether this explanation applies, we have measured the chromosome number distributions, as well as the G0/G1, G2, metaphase (M), and telophase (T) DNA distributions, of the cell lines WCHE-5, MCa-11, and HL-60. The WCHE-5 cells had narrowly distributed chromosome numbers and G0/G1 G2, M, and T DNA peaks. The MCa-11 and HL-60 cells also had narrowly distributed G0/G1 and G2 DNA peaks, but broadly distributed chromosome numbers and M and T DNA peaks. The widths of the MCa-11 and HL-60 M- and T-cell DNA peaks were similar to those of their chromosome number peaks, suggesting that all cells were completing mitosis, regardless of chromosome number or DNA content. Thus, selective metaphase arrest does not seem to be the cause of the narrow G0/G1 and G2 DNA peaks of heteroploid cell populations.     

Cyclic AMP mediates beta-adrenergic-induced increases in N-linked protein glycosylation in rat parotid acinar cells.

beta-Adrenergic stimulation of rat parotid cells by isoprenaline (isoproterenol) results in 2-3-fold increases in [3H]mannose incorporation into N-linked oligosaccharides. This occurs without perceptible lag and is linear with time for 60 min after agonist addition. Concomitantly, isoprenaline markedly increases cellular cyclic AMP. Examination of individual proteins by sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis reveals that glycosylation changes are primarily associated with four secretory proteins, of approx. Mr 17000, 32000, 38000 and 220000. Beta-Adrenoreceptor activation additionally elicits a slight increase in parotid protein synthesis. The greatest increase in [14C]leucine incorporation is that into another secretory protein (Mr approx. 24000). Exposure of cells to dibutyryl cyclic AMP yields results comparable with those after isoprenaline treatment. Forskolin, which increases parotid-cell cyclic AMP, also causes similar effects. Conversely, dibutyryl cyclic GMP shows no such response. The data are consistent with the notion that beta-adrenergic stimulation of N-linked protein glycosylation in rat parotid cells is mediated by cyclic AMP.     

The Short-Term Effects of A Single Injection of Isoproterenol On Proliferation In the Submandibular Gland, Parotid Gland and Oesophagus In Vivo

Abstract. Mitotic and labelling indices were studied in the submandibular, parotid and oesophageal cells of male mice within the first 6 hr (but particularly within the 1st hr) of a single injection of isoproterenol or saline, using the metaphase arrest agent (vincristine) which was previously tested for efficacy in submandibular gland. There was a significant increase in the metaphase index of the salivary glands over control values 5, 15, 30, 45 and 60 min after isoproterenol. In contrast, there were no significant changes in the metaphase index of basal cells of the oesophagus. There was no significant change in the labelling index in isoproterenol-treated mice in comparison with saline-injected control animals. Possible explanations for the rapid mitotic response in murine salivary glands are considered; a rapid efflux from G₂ into mitosis is thought to be the most likely.     

Quantitative ultrastructural study of nucleolus-organizing regions at some stages of the cell cycle (G0 period, G2 period, mitosis)

The quantitative characteristics of chromosomal nucleolus-organizing regions (NORs) and some other nucleolar components were studied on ultra-thin sections of pig embryo kidney cells (PK cells). It was shown that: 1) nucleoli-per-cell volumes were 3 times smaller in the G0 period than in the G2 period; 2) the number of fibrillar centers (FCs) per cell in the G0 period, the G2 period, and at metaphase was equal to 7, 33.7, and 8, respectively; 3) mean volumes of individual FCs in the G0 period (0.033 +/- 0.005 micron3), G2 period (0.014 +/- 0.001 micron3), and at metaphase (0.025 +/- 0.002 micron3) were significantly different; 4) the total volumes of FCs calculated per haploid set of chromosomes were practically the same in the G0 (0.105 micron3) and G2 (0.107 micron3) periods, but were twice as large as those at metaphase (0.04-0.05 micron3). These data show that partial activation and inactivation of ribosomal genes in interphase PK cells are not accompanied by a considerable change in the total volume of FCs and may be due to the fragmentation and fusion of individual FCs. Complete inactivation of ribosomal genes in mitosis results in a decrease of total volumes of FCs per cell; 5) in G0 and G2 periods the total volume of the dense fibrillar component per nucleolus is practically proportional to the nucleolus volume (r = 0.99); 6) in the G2 period, the nucleolus volume is also proportional to the number of FCs (r = 0.99; 7) the volume of the dense fibrillar component within individual fibrillar complexes is not a constant one.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection by means of cell fusion of macromolecular synthesis involved in the reconstruction of the nuclear envelope in mitosis

Using the cultured Chinese hamster cell line Don, G1 or S or a mixture of late-S/G2 cells were prepared by release from metaphase arrest. Metaphase (M) cells were also obtained by mitotic arrest of log-phase cultures with Colcemid and held in metaphase; such M cells remained untreated with any other compound and were termed standard M cells. When interphase (I) cells were fused at pH 8.0 and 37 degrees C with standard cells in the presence of Colcemid by means of UV-inactivated Sendai virus, binucleate interphase-metaphase (I-M) cells were obtained. In a given I-M cell there occurred within 30 min after fusion either prophasing of the I nucleus or formation of a nuclear envelope (NE) around the chromosomes. About 20% of early G1 cells, 35% of cells at the G1/S boundary, 50% of S cells, and 70% of late S/G2 cells could induce NE formation. If, before fusion, cycloheximide (CHE), an inhibitor of protein synthesis, was present during release from M arrest, the cells entered G1 but not S. About 20% of such early G1 cells, like the untreated early G1 cells, had the capacity to induce NE formation during subsequent fusion. If the cells were blocked in S with 5 mM thymidine (TdR), At least 80% of these cells could induce NE formation during subsequent fusion, but in the presence of both TdR and CHE only 35% could do so. It appeared, therefore, that protein synthesis in interphase was required for NE formation. Experiments with actinomycin D indicated that RNA synthesis was also necessary for acquisition of NE-inducing capacity. About 35% of G1 cells from confluent monolayers had the NE-inducing capacity, but prolonged exposure to CHE reduced their number to 8% . Removal of CHE restored the ability while the cells still remained in G1. This result indicated that continuing protein synthesis in the G1 cell was needed for NE formation subsequent to fusion. The fact that macromolecular synthesis must occur in the I cell before fusion if NE formation was to occur in the fused I-M cell lends further support to evidence adduced earlier that this phenomenon is a normal mitotic event. Prophasing of the I nucleus in I-M cells did not appear to be dependent on macromolecular synthesis in the I cell; earlier results from this laboratory showed, however, that protein synthesis in the prior G2 period of the M cell of the I-M pair was required for prophasing.