Molecular Biology of Cell Division*

Baserga, Renato; Waechter, Dieter E.; Soprano, Kenneth J.; Galanti, Norbel

doi:10.1111/j.1749-6632.1982.tb43421.x

Cited by 30 publications

(11 citation statements)

References 36 publications

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“…It is becoming increasingly evident that DNA replication and cell growth are separable processes (Baserga, 1981;Baserga et al, 1982;Zetterberg et al, 1982) and the present observations support this. Thus, pp60"src can trigger the events leading to DNA replication almost as effectively as serum and PDGF without having the exogenous growth factors' abilities to stimulate total protein synthesis.…”

Section: Discussionsupporting

confidence: 84%

Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Durkin

Whitfield

1984

Journal Cellular Physiology

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NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, are transformed at 36 degrees C, but at 40 degrees C they behave as nontransformed cells because of the inactivation of the abnormally thermolabile pp60v-src product of the virus' transforming src gene. At 40 degrees C, these tsLA23-NRK cells were arrested in G1/G0 by severe serum deprivation. They were induced to enter G1, initiate DNA synthesis 7 or 10 hours later, and then divide as (1) nontransformed cells by adding serum or platelet-derived growth factor (PDGF) at 40 degrees C, or (2) transformed cells by lowering the temperature to a pp60v-src-activating 36 degrees C without adding exogenous growth factor(s). The level of pp60v-src kinase activity rose dramatically in these serum-deprived cells within 30 minutes of lowering the temperature to the permissive 36 degrees C, and it fell just as rapidly when the cells were returned to the restrictive 40 degrees C. As little as a 2-hour exposure to 36 degrees C, with an attendant 2-hour burst of pp60v-src kinase activity, was enough to stimulate serum-deprived tsLA23-NRK cells to transit G1 and initiate DNA replication, but not to divide. Much more prolonged pp60v-src activity was needed for these serum-deprived cells to complete their cycle and divide. The prereplicative development of quiescent tsLA23-NRK cells stimulated by serum or PDGF was accompanied by greatly increased protein synthesis and slightly decreased protein degradation, but the pp60v-src-stimulated cells progressed through G1 and initiated DNA replication without appreciably affecting the protein synthetic machinery of the cell. The cells stimulated by the mitogenic action of pp60v-src, like the cells stimulated by serum, needed to activate early prereplicative genes in order to initiate DNA replication. The needed RNA transcripts induced by serum and pp60v-src were produced with comparable efficiency, although it took longer for pp60v-src-stimulated cells to translate these transcripts and to initiate DNA replication, probably because of their unstimulated protein-synthetic machinery.

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Section: Discussionsupporting

confidence: 84%

Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Durkin

Whitfield

1984

Journal Cellular Physiology

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“…In a previous paper (19), the finding that ap53 inhibited serum-stimulated DNA synthesis only when microinjected at the time of stimulation (±2 h) suggests that the p53 protein may play a role in the exit of cells from the Go phase or quiescence, or resting stage, or whatever name one wishes to use for noncycling cells (3,5,23). This suggestion receives further support from the present finding that microinjected ap53 does not inhibit the flow of cells through G1 phase.…”

Section: Discussionmentioning

confidence: 99%

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Mercer¹,

Avignolo²,

Baserga³

1984

Mol. Cell. Biol.

214

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Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serumstimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from Go to S phase, but has no effect on the progression of these cells from mitosis to the S phase.The p53 protein is a transformation-related protein that is often present in higher amounts in transformed cells than in their normal, untransformed counterparts (7,10,18,25). It has been detected in cells transformed by DNA viruses (16,17), by RNA viruses (25,27), by chemicals and X-rays (10), and in several human tumor cell lines (7). Although present in uninfected embryonal carcinoma cells (17), it is not detectable in 3T3 cells (10) and in several untransformed cell strains (7). The synthesis of the p53 protein is markedly increased in mixed populations of lymphocytes stimulated by concanavalin A (21, 22). Its relationship to actively dividing cells has suggested to a number of investigators that the p53 protein may play a role in the regulation of cell proliferation (6, 11,17,22).In a previous paper (19) we have shown that microinjection of a monoclonal antibody against the p53 protein (ap53) inhibited the entry into S phase of quiescent Swiss 3T3 cells stimulated by serum. The inhibitory effect was observed only when ap53 was microinjected about the time of serum stimulation. In this paper, we extended our studies on the effect of microinjected ap53 on cells from different species, on the accumulation of cellular DNA, and on the progression of Swiss 3T3 cells from mitosis through G, to S phase. MATERIALS AND METHODSCell lines and culture conditions. The Syrian hamster G1-specific, temperature-sensitive mutant cell line ts13 was maintained under culture conditions as previously described in detail (2, 12 cells per 60-mm petri dish in growth medium containing 1% calf serum, followed by 5 to 7 days of incubation at 37°C for Swiss 3T3 and WI-38 or at 34°C for ts13. At this time, the cells were quiescent (see below) and could be used for serum stimulation and microinjection.Microinjection procedure. The glass-capillary microinjection method of Graessmann and Graessmann (14) and the modifications for antibody microinjection have been described previously (12, 19, 20). Briefly, a small circle was etched on a 22-mm2 glass cover slip before the cells were plated for quiescence. All, or nearly all, of the cells within the circle were microinjected, and the cells outside of the circle (treated in exactly the same way except for microinjection) served as background contr...

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“…Since the synthesis of early RNAs was among the crucial events of cell proliferation [19][20][21]. RNA synthesis was monitored in both DMSO treated and untreated quiescent cells stimulated to grow by the addition of 20% FCS.…”

Section: Effect Of Dmso On Rata Synthesismentioning

confidence: 99%

Effect of dimethyl sulfoxide on mouse embryo fibroblasts: inhibition of plasminogen activator inhibitor deposition and interference with early events of serum‐stimulated growth

Nagashunmugam

Srinivas

Shanmugam

1989

Biology of the Cell

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Quiescent and serum-stimulated cultures of Swiss mouse embryo fibroblasts (MEF) showed alterations in cell morphology including an enlargement in size upon treatment with 2% dimethyl sulfoxide (DMSO). Treatment of MEF and monkey kidney epithelial cells (MK2) with 2% DMSO at the early periods of serum-stimulated growth inhibited RNA, protein and DNA synthesis. DMSO treatment of cells at late stages of serum-stimulated growth (MEF after 1 hr and MK2 cells after 3 hr of stimulation) had little effect on DNA and protein synthesis although cell enlargement occurred in these cells. When the [35S]methionine labelled proteins of the control and the DMSO treated cells were analysed by high resolution polyacrylamide gel electrophoresis, no apparent difference was observed in the pattern of intracellular proteins of these cells. In contrast, the extracellular levels of two serum-induced secreted proteins of MEF (Mr 48,000 and 26,000) were dramatically reduced by DMSO treatment. The DMSO sensitive 48 kDa protein was found to be the major component of the extracellular matrix, while the 26 kDa protein was not. The 48 kDa protein was identified as plasminogen activator inhibitor (PAI-1). Densitometric quantitation showed a gradual accumulation of this protein in the matrix of serum-stimulated cells. The deposition of this protein in the matrix was inhibited by DMSO. Flow-cytometric quantitation of indirect immunofluorescence indicated higher intracellular levels of the 48 kDa protein in fetal calf serum (FCS) + DMSO treated cells, suggesting that the low level of this protein in the medium of DMSO treated cells is probably due to lack of transport of this protein from the cells into the medium.

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Molecular Biology of Cell Division*

Cited by 30 publications

References 36 publications

Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Effect of dimethyl sulfoxide on mouse embryo fibroblasts: inhibition of plasminogen activator inhibitor deposition and interference with early events of serum‐stimulated growth

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Molecular Biology of Cell Division*

Cited by 30 publications

References 36 publications

Partial characterization of the mitogenic action of pp60v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Partial characterization of the mitogenic action of pp60v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Effect of dimethyl sulfoxide on mouse embryo fibroblasts: inhibition of plasminogen activator inhibitor deposition and interference with early events of serum‐stimulated growth

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Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus

Partial characterization of the mitogenic action of pp60^v‐src, the oncogenic protein product of the src gene of avian sarcoma virus