Differential Inhibition of Protein Synthesis in the Nucleus and Cytoplasm of HeLa Cells in the Presence of Actinomycin, Puromycin and Hydroxyurea

Abstract

A comparison has been made between the synthesis of histones, other nuclear proteins and cytoplasmic proteins after treatment of HeLa cells with substances known to alter their metabolic state : actinomycin, puromycin and hydroxyurea. The use of sensitive methods involving amino acid analysis and measurement of specific radioactivity has revealed differential responses to the action of the different inhibitors. Analysis of these findings as a function of the effects of these inhibitors on other metabolic pathways, suggests that control mechanisms or basic mode of synthesis of some of the histones may differ from that of the other proteins, and that DNA and histone synthesis are probably coordinated in some way yet to be elucidated.Since the hypothesis of genetic repression by histones was advanced [l], many authors have sought to characterize their synthesis [2]. The present investigation has been carried out in an attempt to determine whether histones are synthesized in exactly the same way as the other proteins of the cell or whether some biosynthetic parameter (either a feature of control or a basic mechanism) distinguishes them. Their site of synthesis may be nuclear [3--51, nucleolar [6, 71, or cytoplasmic [8]; experimental evidence a t present available does not indicate clearly which of these possibilities is correct. Nor is it yet known whether the synthesis of all the histones is concomitant with that of DNA [lo-151 or whether some histones are synthesized outside the "S" period[16]. Differential rates of turnover, during different stages of the cell cycle, have been observed for different histone fractions [17]. The way in which such biosynthetic mechanisms are controlled and coordinated remains to be discovered. I n studying the effects of actinomycin D, hydroxyurea and puromycin on the synthesis of histones, residual nuclear proteins and cytoplasmic proteins, we have hoped to throw some light upon these problems. I n some experiments, in order to detect differential mechanisms, F1 (lysine-rich) histones were distinguished from other histones. Parallel experiments also established the effects of similar concentrations of the inhibitors on DNA synthesis. MATERIALS AND METHODSHeLa cells were grown a t 37" as monolayers in Puromycin, at concentrations ranging from 1.5 to 25 pg/ml, or hydroxyurea (76pg/ml) were administered a t the same time as the radioisotope, for a period of I to 1.5 hours. I n the case of actinomycin (1 pg/ml), the antibiotic was administered for an initial "pretreatment" period of 3 hours prior to the addition to the medium of labeled amino acids or thymidine. When protein synthesis was to be analysed, HeLa cells were exposed to ~-[4,5-~H]lysine 5 pC/ml (specific activity 510 mC/mmole) or to L3H]pro1ine 5 pC/ml (specific activity 324 mC/mmole). For studies of DNA synthesis, [6-3H]thymidine 2 pC/ml (specific activity 3.0 C/mmole) was added. Radioisotopes were all obtained from the Radiochemical Centre (Amersham, Buckinghamshire, Great Britain). Preparation of Protein FractionsThe inc...

Amphibian Lens Histones and Their Relation to the Cell Cycle

“…Our results agree with those in the literature which suggest that histone synthesis does continue at a reduced level in the absence of concurrent DNA synthesis (15,16,29,35,43). This is in disagreement with Bloch and coworkers, and Flamm and Birnstiel who found histone synthesis continuing at the normal rate in the presence of DNA synthetic blocking agents (6,14).…”

Syntheses of DNA and Chromosomal Proteins in the Rat Thymus Following Whole-body X-irradiation

“…Studies using diverse systems have implied an intimate relationship between histone biosynthesis and DNA replication (Elgin andWeintraub 1975, Johnson, Douvas andBonner 1974) . A number of investigations have indicated, however, that the interlinking between DNA and histone synthesis may not always be obligatory (Gurley and Hardin 1968, Sadgopal and Bonner 1969, Yarbro 1967, Zampetti-Bosseler, Malpoix and Fievez 1969, Ontko and Moorehead 1964 . Although non-histone proteins of chromatin are intimately associated with DNA, it has been found that there is no strict correspondence between synthesis of DNA and turnover of non-histone proteins (Holbrook, Evans and Irvin 1962, Rovera and Baserga 1971, Stein and Baserga 1970 .…”