Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

Mercer, W E; Avignolo, Carlo; Galanti, Norbel; Rose, Kathleen M.; Hyland, Julia K.; Jacob, Samson T.; Baserga, Renato

doi:10.1016/0014-4827(84)90707-9

Cited by 42 publications

(20 citation statements)

References 24 publications

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“…We next sought to answer a question left ambiguous by previous studies, namely, is blockage of RNA polymerase II in the absence of DNA damage sufficient to induce p53 accumulation? To specifically inhibit RNA polymerase II transcription without initially inducing DNA damage, we microinjected into the nuclei of human fibroblasts anti-RNA polymerase II antibodies, which has been shown to specifically inhibit transcription within 3 h of microinjection (24). Four different antibodies recognizing RNA polymerase II at different stages of transcription were used ( Fig.…”

Section: Phosphorylation Of the Ser-15 Site Of P53 Can Occur Independmentioning

confidence: 99%

RPA and ATR link transcriptional stress to p53

Derheimer

O’Hagan

Krueger

et al. 2007

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

112

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The mechanisms by which DNA-damaging agents trigger the induction of the stress response protein p53 are poorly understood but may involve alterations of chromatin structure or blockage of either transcription or replication. Here we show that transcriptionblocking agents can induce phosphorylation of the Ser-15 site of p53 in a replication-independent manner. Furthermore, microinjection of anti-RNA polymerase II antibodies into the nuclei of cells showed that blockage of transcription is sufficient for p53 accumulation even in the absence of DNA damage. This induction of p53 occurs by two independent mechanisms. First, accumulation of p53 is linked to diminished nuclear export of mRNA; and second, inhibition specifically of elongating RNA polymerase II complexes results in the phosphorylation of the Ser-15 site of p53 in a replication protein A (RPA)-and ATM and Rad3-related (ATR)-dependent manner. We propose that this transcription-based stress response involving RPA, ATR, and p53 has evolved as a DNA damage-sensing mechanism to safeguard cells against DNA damage-induced mutagenesis.antibody microinjection ͉ DNA damage response ͉ RNA polymerase II ͉ nuclear export ͉ phosphorylation T he mechanisms by which DNA lesions are detected and how damage-response pathways are activated in cells are not well understood (1, 2). The stress-response kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) link DNA damage to p53 activation by phosphorylating the Ser-15 site of p53 (3-6). Although the ATM kinase is activated after exposure to ionizing radiation (7), the ATR kinase responds to agents that interfere with replication such as UV light and hydroxyurea (6). The tumor suppressor p53 is induced in response to a variety of different agents in all phases of the cell cycle (8,9). By acting as a transcription factor, p53 can induce the expression of gene products involved in DNA repair, cell cycle arrest, or apoptosis (10, 11). It can also regulate DNA repair and apoptosis by transcription-independent mechanisms (12). Under normal conditions, the p53 protein is rapidly targeted for nuclear export and degradation in a process regulated by the MDM2 protein (13). After cellular stress, the MDM2-mediated negative regulation of p53 is abrogated, and p53 proteins accumulate in the cell nucleus. This nuclear accumulation can be accomplished by (i) DNA damage-induced phosphorylation of p53 and MDM2, leading to the interference of the interaction between MDM2 and p53 (14); (ii) inactivation of proteasomes (15); or (iii) inhibition of the nuclear export machinery (13,16).DNA lesions that block RNA polymerase II induce the recruitment of transcription-coupled repair (TCR) and chromatin remodeling factors to recover RNA synthesis (17)(18)(19). Cells defective in TCR induce p53 and apoptosis at much lower doses than cells with proficient TCR, suggesting that lesions in the transcribed strand of active genes trigger these responses (9,(20)(21)(22). Although these studies have shown a correlation between blockage of ...

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Section: Phosphorylation Of the Ser-15 Site Of P53 Can Occur Independmentioning

confidence: 99%

RPA and ATR link transcriptional stress to p53

Derheimer

O’Hagan

Krueger

et al. 2007

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

112

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“…DNA synthesis was analyzed by standard autoradiography after labeling with 0.5 ,uCi of [3H]thymidine (6.7 ,uCi/mmol) per ml obtained from New England Nuclear. Computer-operated microfluorimetry was used to determine the relative amount of DNA in individual cells stained with acridine orange (Polysciences) as described (26). Green fluorescence intensity was determined using a barrier filter in the range of 540-560 nm.…”

mentioning

confidence: 99%

Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.

Mercer

Shields

Amin

et al. 1990

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

440

243

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To investigate the effect that human wild-type p53 (wt-p53) expression has on cell proliferation we constructed a recombinant plasmid, pM47, in which wt-p53 cDNA is under transcriptional control of the hormone-inducible mouse mammary tumor virus promoter linked to the dominant biochemical selection marker gene Eco gpt. The pM47 plasmid was introduced into T98G cells derived from a human glioblastoma multiforme tumor, and a stable clonal cell line, GM47.23, was derived that conditionally expressed wt-p53 following exposure to dexamethasone. We show that induction of wt-p53 expression in exponentially growing cells inhibits cell cycle progression and that the inhibitory effect is reversible upon removal of the inducer or infection with simian virus 40. Moreover, when growth-arrested cells are stimulated to proliferate, induction of wt-p53 expression inhibits Go/Gj progression into S phase and the cells accumulate with a DNA content equivalent to cells arrested in the Go/Gj phase of the cell cycle. Taken together, these studies suggest that wt-p53 may play a negative role in growth regulation.

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“…The two kinds of antibodies used, i.e. experimentally raised antibodies to rat RNA polymerase I and human RNA polymerase I auto antibodies have previously been shown to neutralize RNA polymerase I activity in vitro and to reduce or even suppress rRNA synthesis upon rnicroinjection into cultured mammalian cells and Xenopus oocytes (Rose et al 1981 ;Mercer et al 1984;Schlegel et al 1985 ;Reimer et al 1987 a). In addition, recent microinjection experiments have demonstrated that these antibodies also inhibit the postmitotic reformation of nucleoli when microinjected into mitotic PtK 2 cells (Benavente et al 1987).…”

Section: Discussionmentioning

confidence: 99%

“…These morphological changes were first detectable at about 1-2 h after antibody injection. Based on 3H-uridine incorporation studies, it has been shown that inhibition ofnucleolar RNA synthesis by microinjection of RNA polymerase I antibodies reaches a maximum at this time point and is subsequently maintained for several hours (Mercer et al 1984). By morphological and immunological criteria the experimentally induced extranucleolar bodies resembled fragments of the DFC of normal nucleoli as they contained the protein fibrillarin which is a specific marker of this nucleolar structure (Ochs et al 1985;Reimer et al 1987b).…”

Section: Discussionmentioning

confidence: 99%

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Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells

Benavente

Reimer

Rose³

et al. 1988

Chromosoma

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Abstract. After microinjection of antibodies against RNA polymerase I into the nuclei of cultured rat kangaroo (PtK z ) and rat (RVF-SMC) cells alterations in nucleolar structure and composition were observed . These were detected by electron microscopy and double-label immunofluorescence microscopy using antibodies to proteins representative of the three major components of the nucleolus. The microinjected antibodies produced a progressive loss of the material of the dense fibrillar component (DFC) from the nucleoli which, at 4 h after injection, were transformed into bodies with purely granular component (GC) structure with attached fibrillar centers (FCs). Concomitantly, numerous extranucleolar aggregates appeared in the nucleoplasm which morphologically resembled fragments of the DFC and contained a protein (fibrillarin) diagnostic for this nucleolar structure. These observations indicate that the topological distribution of the material constituting the DFC can be experimentally influenced in interphase cells, apparently by modulating the transcriptional activity of the rRNA genes. These effects are different from nucleolar lesions induced by inhibitory drugs such as actinomycin D-dependent "nucleolar segregation". The structural alterations induced by antibodies to RNA polymerase I resemble, however, the initial events of nucleolar disintegration during mitotic prophase.

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Cellular DNA replication is independent of the synthesis or accumulation of ribosomal RNA

Cited by 42 publications

References 24 publications

RPA and ATR link transcriptional stress to p53

RPA and ATR link transcriptional stress to p53

Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.

Nucleolar changes after microinjection of antibodies to RNA polymerase I into the nucleus of mammalian cells

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