Estimation of PCNA mRNA Stability in cell cycle by a serum‐deprivation method

Liu, Yin‐Chang; Chen, Gung-Shen; Liu, Weili; Wen, Su-Fang

doi:10.1002/jcb.240570408

Cited by 6 publications

(3 citation statements)

References 19 publications

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“…Transition from G0 quiescence to early G1 phase is, in part, mediated and facilitated through mammalian D-type cyclins that are upregulated in the presence of growth factors (21)(22)(23). By removing mitogenic serum factors from cell culture medium, serum deprivation can result in G0 quiescence (24,25). Releasing cells from cell synchrony is achieved by addition of serum to stimulate cell-cycle progression.…”

Section: Influence Of Cell-cycle Progression On the Kinetics Of Apoptmentioning

confidence: 99%

Cell-cycle progression and response of germ cell tumors to cisplatin in vitro

Mueller

Schittenhelm²,

Honecker³

et al. 2006

Int J Oncol

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Testicular germ cell tumors (GCTs) are highly sensitive to cisplatin-based chemotherapy. It has been suggested that the chemosensitivity of GCTs can be partially attributed to the preference of apoptosis induction over a p21-mediated G1/S phase cell-cycle arrest following induction of p53. Since cell-cycle progression can be manipulated by a growing number of targeted agents, a thorough understanding of the impact of cell-cycle progression on drug-induced cell death might help to enhance the efficacy of chemotherapy. The aim of this study was to assess the cell-cycle dependence of cisplatin-induced cell death in an in vitro model of GCTs. Cell-cycle progression and induction of apoptosis were assessed by flow cytometry and Western blot analysis of PARP cleavage in the GCT derived cell lines, NT2 and 2102 EP, and compared with the breast carcinoma cell line MCF-7. Response to treatment was assessed in different phases of the cell cycle after synchronization by serum depletion and contact inhibition. Following cisplatin exposure, unsynchronized cells accumulated in G2/M after 28 h. This arrest was reversible at sublethal cisplatin doses (0.5-4.5 μM for 2 h). At higher concentrations, cells accumulated in G2 and died in G2/Marrest. A 2-h exposure of cells in G2/M with 10 μM cisplatin resulted in a higher apoptotic index 70 h after treatment (74 and 70% for NT2 and 2102 EP, respectively) compared to treatment in G1/S (34 and 38%). Synchronized cells treated in G1 showed PARP cleavage after 48 h following cisplatin exposure, whereas treatment in G2 resulted in PARP cleavage already after 24 h. Cisplatin-induced cell death in GCTs is highly dependent on cell-cycle phase. All crucial events are restricted to the G2/M phase: cisplatin-induced DNA-damage is sensed, the apoptotic process is initiated and eventually executed in this phase of the cell cycle. The cells are most sensitive to cisplatin in this phase of the cell cycle. As far as the development of targeted agents is concerned, inhibition of the cell cycle in G1/S phase is likely to result in a protective effect against cisplatin, whereas agents arresting cells in G2/M may exert a synergistic effect.

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Section: Influence Of Cell-cycle Progression On the Kinetics Of Apoptmentioning

confidence: 99%

Cell-cycle progression and response of germ cell tumors to cisplatin in vitro

Mueller

Schittenhelm²,

Honecker³

et al. 2006

Int J Oncol

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“…Control of the G 0 quiescence-early G 1 transition is, in part, mediated through mammalian Dtype cyclins that are upregulated in the presence of growth factors and facilitate early G 1 progression (42,62,61). Cellcycle exit into G 0 quiescence can thus be achieved by removing serum, which contains mitogenic factors from the cell culture medium (1,39). Cell-cycle synchrony is achieved following the addition of serum back to the medium to stimulate cell-cycle entry into the early G 1 phase.…”

Section: Serum Deprivationmentioning

confidence: 99%

Biological Methods for Cell-Cycle Synchronization of Mammalian Cells

2001

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Understanding the molecular and biochemical basis of cellular growth and division involves the investigation of regulatory events that most often occur in a cell-cycle phase-dependent fashion. Studies examining cell-cycle regulatory mechanisms and progression invariably require cell-cycle synchronization of cell populations. Thus, many methods have been established to synchronize cells at specific phases of the cell cycle. Several of the common methods involve pharmacological agents, which act at various points throughout the cell cycle. Because of adverse cellular perturbations resulting from many of the synchronizing drugs used, other synchrony methods that involve less perturbation of biological systems, such as serum deprivation, contact inhibition, and centrifugal elutriation have a significant advantage. The advantages and disadvantages of these cell synchronization methods are discussed in this review.

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“…In serum starvation, which arrests the cells at G ! phase [28], HL-60 cells were cultured in a serum-free medium for 60 h. For PMA-induced differentiation, HL-60 cells (8i10& cells\ml) were treated with PMA (Gibco BRL) at a final concentration of 33 nM for 36 h, which arrests the cells in G " phase [29]. Within 4 h of PMA treatment, more than 95 % of the cells became attached to the plates.…”

Section: Partial Methylation At Ts Promoter Is Cell Cycle-independentmentioning

confidence: 99%

Multiple factors regulating the expression of human thromboxane synthase gene

Lee

Baek

Shen

1996

Biochemical Journal

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Characterization of the 5.5 kb promoter of human thromboxane synthase (TS) gene revealed a proximal positive regulatory sequence (PPRS, -90 to -25 bp) and several distal repressive elements. The maximal promoter activity was found to reside within the first 285 bp, approximately 75% of which was contributed by the PPRS. The sequence between -365 and -665 bp exerted a strong repressive effect (approximately 55%) on reporter gene expression independent of orientation and position, consistent with properties expected for a silencer. The sequence upstream of -665 bp to -5.5 kb contains mainly repressive elements which further reduce the promoter activity by 30%. The 65 bp PPRS worked in an orientation-independent, but position-dependent, manner and could be further divided into two independent elements, PPRS1 (-90 to -50 bp) and PPRS2 (-50 to -25 bp). While similar nuclear factor(s) from different cell types interact with PPRS2, those interacting with PPRS1 exhibit cell specificity. Internal sequence deletion and oligonucleotide competition established that a binding sequence for NF-E2 in PPRS1 (-60 tgctgattcat -50) was important for enhancing TS promoter activity in HL-60 cells. The presence of NF-E2 mRNA in HL-60 cells was demonstrated by reverse-transcription PCR amplification of the cDNA and Northern blot analysis. A 9-fold transactivation of luciferase (luc) reporter gene expression had been detected when NF-E2 cDNA was co-expressed with a TS promoter/luc construct. Despite the fact that NF-E2 and the cis-elements could alter the efficiency of TS transcription, they were not sufficient for restricting cell-specific TS expression. Analysis of the methylation status at the TS promoter in several human cell lines reveals cell-specific patterns of methylation that might correlate with TS expression. Taken together, these results suggest that the expression of human TS gene is modulated by multiple factors including cis-elements, trans-activator(s), and possibly genomic methylation.

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Estimation of PCNA mRNA Stability in cell cycle by a serum‐deprivation method

Cited by 6 publications

References 19 publications

Cell-cycle progression and response of germ cell tumors to cisplatin in vitro

Cell-cycle progression and response of germ cell tumors to cisplatin in vitro

Biological Methods for Cell-Cycle Synchronization of Mammalian Cells

Multiple factors regulating the expression of human thromboxane synthase gene

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