Revisiting retinoblastoma protein phosphorylation during the mammalian cell cycle

Cooper, Saul; Shayman, James A.

doi:10.1007/pl00000883

Cited by 43 publications

(31 citation statements)

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“…Because the cyclin E/CDK complex is a major regulator of pRb protein at the late G1 phase of the cell cycle [28], and the serine 807/811 residue of pRb is the target of cyclin E/CDK2 phosphorylation [29], we performed additional experiments to determine the level and phosphorylation status of pRb in RA-treated THP-1 cells. Protein extracts were subjected to Western blot analysis using detection antibodies specific for pRb protein and for the S807/811-phosphorylated form of pRb.…”

Section: Regulation Of Cell Cycle-related Gene Expression By Ra In Thmentioning

confidence: 99%

“…Having observed thus far that RA can regulate the expression and function of cyclin E, p27, pRb, and E2F, all of which are important cell cycle regulators for the G1 to S phase transition [28], we next tested whether cell cycle progression is inhibited. Cells were treated as in previous experiments, as indicated in Fig.…”

Section: Ra Induces the Arrest Of Thp-1 Cells In The G1 Phase Of The mentioning

confidence: 99%

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Retinoic acid regulates cell cycle progression and cell differentiation in human monocytic THP-1 cells

Chen

Ross

2004

Experimental Cell Research

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All-trans-retinoic acid (RA), a natural metabolite of retinol, carries out most of the biological activities of vitamin A and is required for normal growth, cell differentiation, and immune functions. In the present studies, THP-1 human monocytes were used to investigate the mechanisms by which RA may regulate progression through the G1/S phase of the cell cycle. Physiological concentrations of all-trans-RA reduced the levels of cyclin E mRNA by 6 h and reduced cyclin E protein in a dose-and time-dependent manner. Similar reductions were observed for the retinoic acid receptor RAR and RXR proteins. Concomitantly, RA increased the level of the cyclin-dependent kinase inhibitor p27 (Kip-1). The levels of retinoblastoma mRNA and protein (pRb) were also increased, while the proportion of hyperphosphorylated (phosphoserine 807/811) pRb was markedly reduced. Overall, RA increased the functionality of pRb as an inhibitor of cell cycle progression. Furthermore, RA reduced the binding activity of the transcription factor E2F to its core DNA element. Retinoic acid-induced changes in cell cyclerelated proteins occurred in 4-6 h, including reduced cyclin E expression in bromodeoxyuridine (BrdU)-labeled cells, before the onset of cell differentiation as indicated by an increase in the percentage of G1 phase cells and a reduction in S phase cells at 24 h. The expression of CD11b, a cell surface marker of macrophage-like differentiation was increased by RA, as was phagocytic activity. The multiple effects of RA on cell cycle progression may help to explain its welldocumented ability to induce the differentiation of THP-1 cells, and thereby to enhance macrophage-like immune functions.

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Section: Regulation Of Cell Cycle-related Gene Expression By Ra In Thmentioning

confidence: 99%

Section: Ra Induces the Arrest Of Thp-1 Cells In The G1 Phase Of The mentioning

confidence: 99%

Retinoic acid regulates cell cycle progression and cell differentiation in human monocytic THP-1 cells

Chen

Ross

2004

Experimental Cell Research

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“…That a significant number of patterns was attributed to the G 1 phase of the human cell cycle led to this reexamination of the microarray data. This view of the G 1 phase has been extensively reviewed and applied to a number of experimental results (3,5,(9)(10)(11)(12)(13)(14)(15)(16).…”

Section: )mentioning

confidence: 99%

“…Although the analysis presented here is only circumstantially related to the problem of whether or not there are a number of G 1 -phase events, previous publications have dealt with such matters as the existence of G 1 -less cells (14), the statistical variation of interdivision times and the transition-probability model (12), the existence of G(0) (10,17,18), the nature of G 1 -phase arrest (3,5,18), the problem of synchronization (5), the expression of c-myc protein during the division cycle (13), the proposed G 1 -phase-specific phosphorylation of Rb protein (9,15), the effect of cyclins on the length of the G 1 phase (11), and the relationship of the G 1 phase to cell differentiation (16). This alternative view has been the subject of two reviews (3,15).…”

Section: )mentioning

confidence: 99%

Analysis of cell-cycle-specific gene expression in human cells as determined by microarrays and double-thymidine block synchronization

Shedden

Cooper

2002

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

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Microarray analysis of gene expression patterns for thousands of human genes has led to the proposal that a large number of genes are expressed in a cell-cycle-specific manner. The identification of cyclically expressed genes was based on Affymetrix microarray analysis of gene expression after double-thymidine block synchronization. A statistical reanalysis of the original data leads to three principal findings. (i) Randomized data exhibit periodic patterns of similar or greater strength than the experimental data. This finding suggests that all apparent cyclicities in the expression measurements may arise from chance fluctuations. (ii) The presence of cyclicity and the timing of peak cyclicity in a given gene are not reproduced in two replicate experiments. This fact suggests there is an uncontrolled source of experimental variation that is stronger than the innate variation of gene expression in cells over time. (iii) The amplitude of peak expression in the second cycle is not consistently smaller than the corresponding amplitude in the first cycle. This finding places doubt on the assumption that the cells are actually synchronized. We propose that the microarray results do not support the proposal that there are numerous cell-cyclespecifically expressed genes in human cells.Affymetrix ͉ G1 phase M icroarray technology has led to the proposal that there are a large number of mammalian genes (Ϸ700) that have cell-cycle-specific expression patterns (1). The original microarray data presented to support the existence of cyclic gene expression in human cells is now reexamined with a statistical approach. We find there is internal evidence implying that the original microarray data do not support the proposed patterns of gene expression.Analysis of human gene expression during the division cycle (1) was performed by synchronizing primary human foreskin cells with a double-thymidine block. The mRNA was isolated from cells at different times after synchronization (i.e., at what are presumed to be different times during two successive division cycles). mRNA samples were isolated from cells every 2 h for 24 h, covering two cell cycles. The isolated mRNA was labeled with a fluorescent marker and hybridized to microarrays containing probes for 7,129 genes.The primary result of the original authors was the identification of 387 cell-cycle-regulated genes (1). From a larger set of 40,000 transcripts, it was noted that 731 transcripts were assigned to cell-cycle-regulated expression clusters (1); the smaller number relates to those that were assigned to different cell-cycle phases with a smaller Affymetrix (Santa Clara, CA) chip. The putatively cyclic genes were identified by searching among the expression patterns for those that fit, according to a particular threshold, a sine wave pattern over the presumed two cell cycles. The computer pattern search (1) classified as cell-cycle-specific those genes that exhibited a Pearson correlation coefficient of at least 0.7 relative to at least one member of a family of shifted sin...

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“…The experimental and theoretical support for this view of cell cycle control has been reviewed. [4][5][6][7] Instead of envisioning specific G 1 -phase controls, this alternative model proposes that control of cell-cycle events is based on a continuous accumulation occurring in all phases of the cell cycle. The variable G 1 phase is a consequence of variation in growth rate throughout the cell cycle.…”

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confidence: 99%

How the Change from FLM to FACS Affected Our Understanding of the G1-Phase of the Cell Cycle

Cooper

2003

Cell Cycle

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The frequency of labeled mitoses (FLM) method for analyzing cell-cycle phases necessitates a determination of cell-cycle interdivision times and the absolute lengths of the cell-cycle phases. The change to flow sorting (FACS) analysis, a simpler, less labor intensive, and more rapid method, eliminated determinations of absolute phase times, yielding only percents of cells exhibiting particular DNA contents. Without an interdivision time value, conversion of these fractions into absolute phase lengths is not possible. This change in methodology has led to an alteration in how the cell cycle is viewed. The FLM method allowed the conclusion that G 1 -phase variability resulted from constancy of S and G 2 phase lengths. In contrast, with FACS analysis, slow growing cells exhibiting a large fraction of cells with a G 1 -phase amount of DNA appeared to be "arrested in G 1 phase". The loss of absolute phase length determinations has therefore led to the proposals of G 1 -phase arrest, G 1 -phase controls, restriction points, and G 0 phase. It is suggested that these G 1 -phase controls and phenomena require a critical reevaluation in the light of an alternative cell-cycle model that does not require or postulate such G 1 -phase controls.After the original identification of cell-cycle phases by Howard and Pelc in the early 1950s, the next two decades brought forth a large number of measurements of cell-cycle phase lengths using the method of frequency of labeled mitoses (FLM). FLM is essentially the method use by Howard and Pelc. 1,2 The FLM method is illustrated in Figure 1. Because this method is not now in common use, it is of interest to explain the method and the type of results obtained by this method. For FLM, growing cells are pulse-labeled with tritiated thymidine. After removal of label, growing cells are sampled at intervals and subjected to autoradiography. The fraction of mitotic cells that are radioactive is determined. As shown in Figure 1, there is an initial period with no labeled mitoses. This period gives the length, in absolute time, of the G 2 phase. Then there is a rise in labeled mitoses, a plateau, and a fall in labeled mitoses. The time interval of these labeled mitoses indicates the S-phase length. The G 1 phase length is determined from the G 1 + G 2 period-the period between the decrease in labeled mitoses and the subsequent increase in labeled mitoses-by subtracting the initially determined G 2 phase time. The total interdivision time (IDT) is determined by the time between the rise times of labeled mitoses.The major generalization to come out of a large number of FLM analyses in the

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Revisiting retinoblastoma protein phosphorylation during the mammalian cell cycle

Cited by 43 publications

References 60 publications

Retinoic acid regulates cell cycle progression and cell differentiation in human monocytic THP-1 cells

Retinoic acid regulates cell cycle progression and cell differentiation in human monocytic THP-1 cells

Analysis of cell-cycle-specific gene expression in human cells as determined by microarrays and double-thymidine block synchronization

How the Change from FLM to FACS Affected Our Understanding of the G1-Phase of the Cell Cycle

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