Cell proliferation in the gastrulating chick embryo: a study using BrdU incorporation and PCNA localization

Development ◽  
1993 ◽  
Vol 118 (2) ◽  
pp. 389-399 ◽  
Author(s):  
E.J. Sanders ◽  
M. Varedi ◽  
A.S. French
Keyword(s):  
Cell Proliferation ◽  
Chick Embryo ◽  
Cell Density ◽  
Generation Time ◽  
Primitive Streak ◽  
S Phase ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Similar Proportion ◽  
Differential Growth

Cell proliferation in the gastrulating chick embryo was assessed using two independent techniques which mark cells in S phase of the mitotic cycle: nuclear incorporation of bromodeoxyuridine (BrdU) detected immunocytochemically and immunolocalization of proliferating cell nuclear antigen (PCNA). Computer-reconstructed maps were produced showing the distribution of labelled nuclei in the primitive streak and the cell layers. These distributions were also normalized to take into account regional differences in cell density across the embryo. Results from a 2 hour pulse of BrdU indicated that although cells at caudal levels of the primitive streak showed the highest incorporation, this region showed a similar proportion of labelled cells to the surrounding caudal regions of the epiblast and mesoderm when normalized for cell density. The entire caudal third of the embryo showed the highest proportion of cells in S phase. Cells of Hensen's node showed a relatively low rate of incorporation and, although the chordamesoderm cells showed many labelled nuclei, this appeared to be a reflection of a high cell density in this region. Combining this result with results from a 4 hour pulse of BrdU permitted mapping of cell generation time across the entire embryo. Generation times ranged from a low value of approximately 2 hours at caudal levels of both the epiblast and mesoderm, to an upper value of approximately 10 hours in the rostral regions of the primitive streak, in the mid-lateral levels of the epiblast and in the chordamesoderm rostral to Hensen's node. Cells at caudal regions of the primitive streak showed a generation time of approximately 5 hours. Taking into account that cells are generally considered to be continuously moving through the primitive streak, we conclude that cell division, as judged by generation time, is greatly reduced during transit through this region, despite the presence there of cells in S phase and M phase. Immunocytochemical localization of PCNA-positive nuclei gave generally similar distributions to those obtained with BrdU incorporation, confirming that this endogenous molecule is a useful S-phase marker during early embryogenesis. Mid-levels and caudal levels of the primitive streak showed the highest numbers of positive nuclei, and the highest proportion of labelling after cell density was accounted for. As with BrdU incorporation, the highest proportions of PCNA-positive nuclei were found towards the caudal regions of the epiblast and mesoderm. These results suggest that the differential growth of the caudal region of the embryo at this time is a direct consequence of elevated levels of cell proliferation in this region.(ABSTRACT TRUNCATED AT 400 WORDS)

The relationship between cell proliferation and the transcription of the nuclear oncogenes c-myc, c-myb and c-ets-1 during feather morphogenesis in the chick embryo

Development ◽  
1991 ◽  
Vol 111 (3) ◽  
pp. 699-713 ◽  
Author(s):  
X. Desbiens ◽  
C. Queva ◽  
T. Jaffredo ◽  
D. Stehelin ◽  
B. Vandenbunder
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Spatial Distribution ◽  
Chick Embryo ◽  
Expression Patterns ◽  
S Phase ◽  
Skin Morphogenesis ◽  
Dermal Cells ◽  
The Relationship

We have described the expression of three nuclear protooncogenes, c-myc, c-myb and c-ets-1 during feather morphogenesis in the chick embryo. In parallel with the expression patterns obtained by in situ hybridization, we have mapped the spatial distribution of S-phase cells by monitoring the incorporation of 5-bromodeoxyuridine. We do not detect c-myc or c-myb transcripts during the early stages when S-phase cells are scattered in the dermis and in the epidermis. Rather c-ets-1 transcripts are abundant in the dermal cells which divide and accumulate under the uniform epidermis. At the onset of the formation of the feather bud, cells within each rudiment cease DNA replicative activities and c-myc transcripts are detected both in the epidermis and in the underlying dermis. This expression precedes the reentry into the S phase. The transcription of c-myb, which has been previously tightly linked to hemopoietic cells is also detected in the developing skin. This expression is essentially located in proliferating epidermal cells on and after the beginning of feather outgrowth. As feather outgrowth proceeds, the distribution of c-myc and c-myb transcripts is restricted to the highly proliferating epidermis. In contrast c-ets-1 transcripts are never detected in the epidermis. During the later stages of skin morphogenesis, the transcription of c-ets-1 is restricted to the endothelial cells of blood vessels, as previously described. We suggest that the differential expression of these nuclear oncogenes reflects the activation of different mitotic controlling pathways during the development of the skin.

scholarly journals The histone methyltransferase SET8 is required for S-phase progression

2007 ◽  
Vol 179 (7) ◽  
pp. 1337-1345 ◽  
Author(s):  
Stine Jørgensen ◽  
Ingegerd Elvers ◽  
Morten Beck Trelle ◽  
Tobias Menzel ◽  
Morten Eskildsen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Dna Damage ◽  
Posttranslational Modifications ◽  
Mammalian Cells ◽  
Protein A ◽  
Strand Break ◽  
Histone Methyltransferase ◽  
S Phase ◽  
Nuclear Antigen ◽  
And Function

Chromatin structure and function is influenced by histone posttranslational modifications. SET8 (also known as PR-Set7 and SETD8) is a histone methyltransferase that monomethylates histonfe H4-K20. However, a function for SET8 in mammalian cell proliferation has not been determined. We show that small interfering RNA inhibition of SET8 expression leads to decreased cell proliferation and accumulation of cells in S phase. This is accompanied by DNA double-strand break (DSB) induction and recruitment of the DNA repair proteins replication protein A, Rad51, and 53BP1 to damaged regions. SET8 depletion causes DNA damage specifically during replication, which induces a Chk1-mediated S-phase checkpoint. Furthermore, we find that SET8 interacts with proliferating cell nuclear antigen through a conserved motif, and SET8 is required for DNA replication fork progression. Finally, codepletion of Rad51, an important homologous recombination repair protein, abrogates the DNA damage after SET8 depletion. Overall, we show that SET8 is essential for genomic stability in mammalian cells and that decreased expression of SET8 results in DNA damage and Chk1-dependent S-phase arrest.

scholarly journals In Situ Analysis of Cellular Proliferation in Canine, Feline and Equine Tumors by Immunohistochemistry: A Comparison of Bromodeoxyuridine, Proliferating Cell Nuclear Antigen, and Interchromatin-Associated Antigen Immunostaining Techniques

1994 ◽  
Vol 6 (4) ◽  
pp. 453-457 ◽  
Author(s):  
Alain Pierre Théon ◽  
Loretta Metzger ◽  
Stephen Griffey
Keyword(s):  
Cell Proliferation ◽  
Proliferating Cell Nuclear Antigen ◽  
Cellular Proliferation ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Immunohistochemical Detection ◽  
Proliferating Cells ◽  
Proliferating Cell

Cell proliferation in canine, feline, and equine tumors was evaluated using immunohistochemical detection of in vitro 5–bromodeoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen (PCNA), and interchromatin-associated antigen (p105). Ten tumors in each species were analyzed. The tumor proliferative fraction (PF) was defined as the percentage of labeled nuclei for 5,000 tumor nuclei counted. Immunoreactivity was observed with all techniques in all species. A good correlation was observed between the proliferative fractions measured with the BrdU (PFBrdU) and PCNA (PFPCNA) techniques ( rs = 0.523, P = 0.0026). There was no correlation between the PFs measured with the BrdU (PFBrdU) and p105 (PFP105) techniques. Using the median values obtained from the different approaches as cutoff points to define slowly and rapidly proliferating tumors, there was an 80% agreement ( P = 0.009) between PFBrdU and PFPCNA and no agreement between PFBrdU and PFP105 The results of this study indicate that both BrdU and PCNA labeling methods can be used reliably for identifying proliferating cells in animal tumors. In addition, PCNA could be used to replace the BrdU method to assess tumor proliferative fraction because it does not require pretreatment of tissues.

scholarly journals Molecular mechanism of green microalgae, Dunaliella salina, involved in attenuating balloon injury-induced neointimal formation

2010 ◽  
Vol 104 (3) ◽  
pp. 326-335 ◽  
Author(s):  
Ming-Jyh Sheu ◽  
Hsu-Chen Cheng ◽  
Yi-Chung Chien ◽  
Pei-Yu Chou ◽  
Guang-Jhong Huang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Molecular Mechanism ◽  
Dunaliella Salina ◽  
Flow Cytometric Analysis ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Dependent Manner ◽  
Balloon Injury ◽  
Neointimal Formation

The pathological mechanism of restenosis is primarily attributed to excessive proliferation of vascular smooth muscle cells (VSMC). The preventive effects of ethanol extract of Dunaliella salina (EDS) on balloon injury-induced neointimal formation were investigated. To explore its molecular mechanism in regulating cell proliferation, we first showed that EDS markedly reduced the human aortic smooth muscle cell proliferation via the inhibition of 5′-bromo-2′-deoxyuridine (BrdU) incorporation at 40 and 80 μg/ml. This was further supported by the G0/G1-phase arrest using a flow cytometric analysis. In an in vivo study, EDS at 40 and 80 μg/ml was previously administered to the Sprague–Dawley rats and found that the thickness of neointima, and the ratio of neointima:media were also reduced. EDS inhibited VSMC proliferation in a dose-dependent manner following stimulation of VSMC cultures with 15 % fetal bovine serum (FBS). Suppressed by EDS were 15 % FBS-stimulated intracellular Raf, phosphorylated extracellular signal-regulated kinases (p-Erk) involved in cell-cycle arrest and proliferating cell nuclear antigen. Phosphorylated focal adhesion kinase (p-FAK) was also suppressed by EDS. Also active caspase-9, caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) protein expression levels were increased by administration with EDS; the apoptotic pathway may play an important role in the regulatory effects of EDS on cell growth. These observations provide a mechanism of EDS in attenuating cell proliferation, thus as a potential intervention for restenosis.

scholarly journals An essential role of p27 downregulation in fenvalerate-induced cell growth in human uterine leiomyoma and smooth muscle cells

2012 ◽  
Vol 303 (8) ◽  
pp. E1025-E1035 ◽  
Author(s):  
Xiaohua Gao ◽  
Linda Yu ◽  
Lysandra Castro ◽  
Charles J. Tucker ◽  
Alicia B. Moore ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Uterine Leiomyoma ◽  
Muscle Cells ◽  
S Phase ◽  
Total Cell ◽  
Brdu Incorporation ◽  
Mrna Levels ◽  
Cell Numbers

Previously, we reported that fenvalerate (Fen) promotes proliferation of human uterine leiomyoma (UtLM) cells by enhancing progression of cells from G0-G1to S phase through molecular mechanisms independent of estrogen receptor-α and -β. The cyclin-dependent kinase (CDK) inhibitor p27, which blocks G1to S phase transitions and is an important regulator of CDK2, is often decreased in hormonally regulated diseases, including uterine leiomyomas. Therefore, we were interested in whether Fen could regulate the expression of p27 and whether p27 might play a role in Fen-induced cell proliferation. Expression of p27 in Fen-treated UtLM and uterine smooth muscle cells (UtSMCs) was examined. We found that p27 mRNA was significantly downregulated and that protein levels were decreased in both cell types treated with 10 μM Fen for 24 h compared with respective controls. Overexpression of p27 in UtLM cells and UtSMCs using an adenovirus doxycycline (Dox)-regulated Tet-off system abrogated the proliferative effects of Fen, as evidenced by decreased total cell numbers and BrdU incorporation. Fen treatment increased CDK2 mRNA expression levels; however, overexpression of p27 also abolished this effect. In contrast, Dox treatment dramatically restored the above muted responses. Finally, we utilized siRNA to knock down p27 expression. After transfection, mRNA levels of p27 were downregulated in UtLM cells and UtSMCs and total cell numbers and BrdU incorporation increased significantly compared with nontransfected cells. Fen treatment in the presence of p27 silencing enhanced the increased cell counts and BrdU labeling in UtLM cells and UtSMCs. Taken together, these results indicate that p27 downregulation is critical for Fen-induced cell proliferation.

scholarly journals Cell proliferation and gill morphology in anoxic crucian carp

2005 ◽  
Vol 289 (4) ◽  
pp. R1196-R1201 ◽  
Author(s):  
Jørund Sollid ◽  
Aina Kjernsli ◽  
Paula M. De Angelis ◽  
Åsmund K. Røhr ◽  
Göran E. Nilsson
Keyword(s):  
Cell Proliferation ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Crucian Carp ◽  
Fold Increase ◽  
S Phase ◽  
Nuclear Antigen ◽  
Tyrosyl Radical ◽  
Radical Generation ◽  
Proliferating Cell

Is DNA replication/cell proliferation in vertebrates possible during anoxia? The oxygen dependence of ribonucleotide reductase (RNR) could lead to a stop in DNA synthesis, thereby making anoxic DNA replication impossible. We have studied this question in an anoxia-tolerant vertebrate, the crucian carp ( Carassius carassius), by examining 5′-bromo-2′-deoxyuridine incorporation and proliferating cell nuclear antigen levels in the gills, intestinal crypts, and liver. We exposed crucian carp to 1 and 7 days of anoxia followed by 7 days of reoxygenation. There was a reduced incidence of S-phase cells (from 12.2 to 5.0%) in gills during anoxia, which coincided with a concomitant increase of G0 cells. Anoxia also decreased the number of S-phase cells in intestine (from 8.1 to 1.8%). No change in the fraction of S-phase cells (∼1%) in liver was found. Thus new S-phase cells after 7 days of anoxia were present in all tissues, revealing a considerable rate of DNA synthesis. Subsequently, the oxygen-dependent subunit of crucian carp RNR (RNRR2) was cloned. We found no differences in amino acids involved in radical generation and availability of the iron center compared with mouse, which could have explained reduced oxygen dependence. Furthermore, the amount of RNRR2 mRNA in gills did not decrease throughout anoxia exposure. These results indicate that crucian carp is able to sustain some cell proliferation in anoxia, possibly because RNRR2 retains its tyrosyl radical in anoxia, and that the replication machinery is still maintained. Although hypoxia triggers a 7.5-fold increase of respiratory surface area in crucian carp, this response was not triggered in anoxia.

scholarly journals Phenobarbital-induced hepatocellular proliferation: anti-bromodeoxyuridine and anti-proliferating cell nuclear antigen immunocytochemistry.

1993 ◽  
Vol 41 (1) ◽  
pp. 21-27 ◽  
Author(s):  
H B Jones ◽  
N A Clarke ◽  
N C Barrass
Keyword(s):  
Cell Proliferation ◽  
Proliferating Cell Nuclear Antigen ◽  
Lateral Lobe ◽  
Nuclear Antigen ◽  
Brdu Incorporation ◽  
Advantages And Disadvantages ◽  
Immunocytochemical Detection ◽  
Left And Right ◽  
Proliferating Cell ◽  
Left Lateral Lobe

We report modifications to immunocytochemical detection procedures for proliferating cell nuclear antigen (PCNA) which permit its identification in liver samples previously fixed for BrdU immunocytochemistry. Both methods have been used for the assessment of phenobarbital-induced cell proliferation in rat liver. The difficulties associated with the hitherto unsuccessful application of PCNA immunocytochemical methods to tissues fixed in formalin for BrdU visualization were overcome by epitope unmasking with acid hydrolysis, extension of primary antiserum (PC10) incubation, and employment of streptavidin-ABC-HRP. BrdU delivery via osmotic minipumps for 48 hr before euthanasia, followed by fixation in cold formalin for 14 days, yielded reliable and reproducible hepatocellular labeling and a peak of cell proliferation in all lobes on Day 3 (i.e., labeling during Days 1-3) of dosing with 80 mg/kg/day phenobarbital. Labeling indices (LI) of both control and phenobarbital-treated liver were lower in the left and right median lobes as compared with the lateral lobes. In sections of the left lateral lobe from the same liver, PCNA immunocytochemistry revealed a peak of proliferative activity (about one third of the maximum LI generated by BrdU incorporation) on Day 1. These findings, together with the advantages and disadvantages of both techniques, are discussed in the context of their applications to different investigative requirements.

scholarly journals Evaluation of proliferating cell nuclear antigen (PCNA) as an endogenous marker of cell proliferation in rat liver: a dual-stain comparison with 5-bromo-2'-deoxyuridine.

1993 ◽  
Vol 41 (1) ◽  
pp. 1-6 ◽  
Author(s):  
K M Connolly ◽  
M S Bogdanffy
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Rat Liver ◽  
Proliferating Cell Nuclear Antigen ◽  
Simultaneous Detection ◽  
S Phase ◽  
Phase Fraction ◽  
Nuclear Antigen ◽  
Proliferating Cell ◽  
Formalin Fixed

Proliferating cell nuclear antigen (PCNA) was evaluated as a marker of cell proliferation in formalin-fixed rat liver tissue through a comparative study with the thymidine analogue 5-bromo-2'-deoxyuridine (BrdU). The comparison was conducted through the introduction of a dual immunohistochemical procedure that allows the simultaneous detection of the two antigens. The results of this study suggest that although statistically similar indexes for each can be achieved, what has been reported to be the "S-phase fraction" of PCNA-labeled nuclei is significantly different from the population of cells marked by BrdU. The data also suggest that the reason for this difference is that the "S-phase fraction" of PCNA-labeled nuclei is the population of cells in late G1- and early S-phases. BrdU, by comparison, is incorporated into cells only during DNA synthesis. Therefore, although BrdU and PCNA labeling techniques may both be effective for evaluating cell proliferation rates, it must be recognized that labeling indices derived from each are not entirely synonymous. The method presented here for the simultaneous labeling of PCNA and BrdU antigens may have utility in studies of cell cycle perturbations.

scholarly journals Knockout of the Transducin-Like Enhancer of Split 6 Gene Affects the Proliferation and Cell Cycle Process of Mouse Spermatogonia

2020 ◽  
Vol 21 (16) ◽  
pp. 5827
Author(s):  
Meiying Feng ◽  
Yinshan Bai ◽  
Yun Chen ◽  
Kai Wang
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Developmental Process ◽  
Gene Knockout ◽  
Scientific Basis ◽  
S Phase ◽  
Nuclear Antigen ◽  
Pcr Analysis ◽  
Spermatogonia Cell ◽  
Enhancer Of Split

Tle6 (Transducin-like enhancer of split 6) is a member of the Tle co-repressor superfamily, which is expressed in various tissues of invertebrates and vertebrates and participates in the developmental process. However, the current research has only found that the TLE6 mutation is related to infertility, and the key regulatory mechanism of TLE6 remains to be explored. In this study, we combined Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 and the Tet-on system to construct mouse spermatogonia cell lines that induced TLE6 protein knockout (KO), and studied the effect of Tle6 on mouse spermatogonia proliferation and the cell cycle. The results showed that, after drug induction, the Tle6 gene in mouse spermatogonia was successfully knocked out at the genome and protein levels, and the Tle6 gene knockout efficiency was confirmed to be 87.5% with gene-cloning technology. At the same time, we also found that the mouse spermatogonia proliferated slowly after the Tle6 knockout. Using flow cytometry, we found that the cells did not undergo significant apoptosis, and the number of cells in the S phase decreased. After real-time quantity PCR (qRT-PCR) analysis, we found that the expression of cell-proliferation-related genes, CCAAT enhancer-binding protein α(C/ebp α), granulocyte-colony stimulating factor(G-csf), cyclin-dependent kinases 4(Cdk 4), Cyclin E, proliferating cell nuclear antigen(Pcna), and S-phase kinase-associated protein 2 (Skp2) was significantly reduced, which further affected cell growth. In summary, Tle6 can regulate spermatogonia cell proliferation and the cell cycle and provide a scientific basis for studying the role of TLE6 on spermatogenesis.

scholarly journals Umbelliferone Ameliorates Benign Prostatic Hyperplasia by Inhibiting Cell Proliferation and G1/S Phase Cell Cycle Progression through Regulation of STAT3/E2F1 Axis

2021 ◽  
Vol 22 (16) ◽  
pp. 9019
Author(s):  
Hyo-Jung Kim ◽  
Bo-Ram Jin ◽  
Hyo-Jin An
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Benign Prostatic Hyperplasia ◽  
Cell Cycle Progression ◽  
Underlying Mechanism ◽  
Prostatic Hyperplasia ◽  
S Phase ◽  
Nuclear Antigen ◽  
Phase Cell ◽  
Proliferative Effect

Umbelliferone (UMB), also known as 7-hydroxycoumarin, is a derivative of coumarin, which is widely found in many plants such as carrots, coriander, and garden angelica. Although many studies have already revealed the various pharmacological properties of UMB, its effect on benign prostatic hyperplasia (BPH) remains unclear. Therefore, the present study aimed to elucidate the underlying mechanism of the anti-proliferative effect of UMB in a human benign prostatic hyperplasia cell line (BPH-1), as well as its ameliorative effect on BPH in testosterone propionate (TP)-induced rats. The results showed that UMB exerts an anti-proliferative effect in BPH-1 cells by modulating the signal transducer and activator of transcription 3 (STAT3)/E2F transcription factor 1 (E2F1) axis. UMB treatment not only inhibited androgen/androgen receptor (AR) signaling-related markers, but also downregulated the overexpression of G1/S phase cell cycle-related markers. In TP-induced rats, UMB administration demonstrated an anti-BPH effect by significantly reducing prostate size, weight, and epithelial thickness. In addition, UMB suppressed cell proliferation by reducing the expression of proliferating cell nuclear antigen (PCNA) and p-STAT3 (Tyr 705) in prostate tissue following TP injection. These findings suggest that UMB has pharmacological effects against BPH.

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