scholarly journals Flow cytometric identification of proliferative subpopulations within normal human epidermis and the localization of the primary hyperproliferative population in psoriasis.

1993 ◽  
Vol 178 (4) ◽  
pp. 1271-1281 ◽  
Author(s):  
Z Bata-Csorgo ◽  
C Hammerberg ◽  
J J Voorhees ◽  
K D Cooper
Keyword(s):  
Cell Cycle ◽  
Human Epidermis ◽  
Optical Characteristics ◽  
Small Cells ◽  
Nuclear Antigen ◽  
Quiescent State ◽  
Flow Cytometric ◽  
M Phase ◽  
Normal Human ◽  
Normal Epidermis

In this study we define the proliferative compartments of in vivo human epidermis, using specific antibodies related to cell differentiation (beta 1 and beta 4 integrins and K1/K10 differentiation keratins) and cell cycle (proliferating cell nuclear antigen [PCNA]) in combination with flow cytometric quantitation of the DNA content and optical characteristics of the cells. The beta 1 integrin (CD29) marked both of the potentially proliferative subsets in normal epidermis. One subset of normal epidermis is CD29+ K1/K10-, which was predominantly basal, and found to be comprised of slow cycling, small cells with primitive cytoplasmic organization. The vast majority (95.5%) of these cells were in a quiescent state (G0/early G1) as indicated by their lack of the cyclin, PCNA. The other proliferative subset of normal epidermis was CD29+ K1/K10+, which was suprabasal and occasional basal, highly proliferative, larger in size, and which exhibited a more complex cytoplasmic structure. Because early differentiation (K1/K10 expression) has begun in the CD29+ K1/K10+ subset, it is highly likely that they represent the proliferative population which is capable of transiently amplifying itself before terminal differentiation. Within lesional psoriatic epidermis, similar proliferative cell populations were present as in normal epidermis, and the hyperproliferative defect was localized to the beta 1 and beta 4 integrin+, K1/K10- populations, which in normal epidermis is basally located and quiescent with regard to cell cycle. In psoriatic epidermis, a six- to sevenfold increase in the number of cells in the S/G2+M phase of cell cycle was found among CD29+ K1/K10- cells (p < 0.05). Furthermore, all lesional K1/K10- cells showed high PCNA positivity, indicating that all these cells had been recently induced into cell cycle. By contrast, the proportion of cycling cells among lesional psoriatic CD29+ K1/K10+ keratinocytes was similar to normals. Anti-HLA-DR, CD45, and vimentin antibodies were used to concomitantly track the proliferative states of Langerhans cell, melanocyte, and infiltrating leukocyte populations. In normal epidermis, the cycling fractions (cells in S/G2/M phase) of these cells were similar to the CD29+K1/K10- keratinocytes, whereas in lesional epidermis their cycling pools were increased relative to normal, but not so much as the proliferative fractions of psoriatic CD29+ K1/K10- keratinocytes. These data demonstrate the use of simultaneous analysis of integrin expression, differentiation keratins, cyclin, cell cycle status, and optical characteristics of freshly isolated human epidermal cells. Such analysis allowed the physical identification and quantification of cy cling populations in normal human skin, and has enabled the precise location of the primary epidermal proliferative defect in psoriasis.

Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells

2010 ◽  
Vol 298 (6) ◽  
pp. R1615-R1626 ◽  
Author(s):  
Neil I. Bower ◽  
Ian A. Johnston
Keyword(s):  
Cell Cycle ◽  
Amino Acids ◽  
Amino Acid ◽  
Atlantic Salmon ◽  
Antigen Expression ◽  
Nuclear Antigen ◽  
Quiescent State ◽  
Mrna Levels ◽  
Proliferating Cells

The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon ( Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18°C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression ( R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G1 and S/G2 phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.

Optical characteristics of UV transmission of normal human epidermis

1994 ◽  
Vol 4 (2) ◽  
pp. 65-68 ◽  
Author(s):  
W. Ambach ◽  
M. Blumthaler ◽  
T. Schöpf ◽  
E. Ambach ◽  
W. Rabl ◽  
...  
Keyword(s):  
Human Epidermis ◽  
Optical Characteristics ◽  
Normal Human

scholarly journals The distribution pattern of proliferating cell nuclear antigen in the nuclei of Leishmania donovani

Microbiology ◽  
2009 ◽  
Vol 155 (11) ◽  
pp. 3748-3757 ◽  
Author(s):  
Devanand Kumar ◽  
Neha Minocha ◽  
Kalpana Rajanala ◽  
Swati Saha
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Proliferating Cell Nuclear Antigen ◽  
Cell Cycle Progression ◽  
Leishmania Donovani ◽  
Systemic Disease ◽  
S Phase ◽  
Nuclear Antigen ◽  
M Phase ◽  
Proliferating Cell

DNA replication in eukaryotes is a highly conserved process marked by the licensing of multiple origins, with pre-replication complex assembly in G1 phase, followed by the onset of replication at these origins in S phase. The two strands replicate by different mechanisms, and DNA synthesis is brought about by the activity of the replicative DNA polymerases Pol δ and Pol ϵ. Proliferating cell nuclear antigen (PCNA) augments the processivity of these polymerases by serving as a DNA sliding clamp protein. This study reports the cloning of PCNA from the protozoan Leishmania donovani, which is the causative agent of the systemic disease visceral leishmaniasis. PCNA was demonstrated to be robustly expressed in actively proliferating L. donovani promastigotes. We found that the protein was present primarily in the nucleus throughout the cell cycle, and it was found in both proliferating procyclic and metacyclic promastigotes. However, levels of expression of PCNA varied through cell cycle progression, with maximum expression evident in G1 and S phases. The subnuclear pattern of expression of PCNA differed in different stages of the cell cycle; it formed distinct subnuclear foci in S phase, while it was distributed in a more diffuse pattern in G2/M phase and post-mitotic phase cells. These subnuclear foci are the sites of active DNA replication, suggesting that replication factories exist in Leishmania, as they do in higher eukaryotes, thus opening avenues for investigating other Leishmania proteins that are involved in DNA replication as part of these replication factories.

Cell cycle in alveolar epithelial type II cells: integration of Matrigel and KGF

1997 ◽  
Vol 273 (3) ◽  
pp. L572-L580 ◽  
Author(s):  
S. Buckley ◽  
B. Driscoll ◽  
K. D. Anderson ◽  
D. Warburton
Keyword(s):  
Cell Cycle ◽  
Keratinocyte Growth Factor ◽  
Cell Cycle Control ◽  
Nuclear Antigen ◽  
Quiescent State ◽  
Type Ii Cells ◽  
Type Ii ◽  
Cell Cycle Regulators ◽  
Alveolar Epithelial

The regulation of cell cycle control in alveolar epithelial type II cells (AEC2) in response to peptide growth factors and extracellular matrix signals is not well understood. Herein, we have determined that, in adult rat AEC2 in primary culture on Engelbreth-Holm-Swarm biomatrix (Matrigel) in the presence of keratinocyte growth factor, the expression of key cell cycle control elements, including cyclins A and D and cyclin-dependent kinases (cdk) 1 and 4, is increased and that retinoblastoma protein (pRb) phosphorylation is also increased, with a corresponding decrease in the expression of p53 and the cdk inhibitors (cdkis) p21WAF1/CIP1 and p27KIP-1 compared with cells cultured on plastic. The Matrigel biomatrix-KGF culture conditions were also associated with an enhanced proliferative response, as measured by fluorescent-activated cell sorter analysis, thymidine incorporation into DNA, and proliferating cell nuclear antigen expression. This enhanced proliferation occurred with neither a soluble extract of Matrigel biomatrix nor with other simple biological matrices. We conclude that coordinated induction of key cyclins and cdks, with the concomitant suppression of key negative cell cycle regulators, occurs in AEC2 on Matrigel biomatrix in the presence of KGF. We speculate that the balance between cyclin and cdk activation and cdki suppression in AEC2 serves to integrate the combined influences of biomatrix and KGF signaling on pRb phosphorylation, thereby controlling transit through S phase of the cell cycle. Conversely, AEC2 express high levels of cdkis and p53 at rest in G1 phase. The latter finding may explain the quiescent state of normal adult AEC2 in vivo.

Growth retardation and dyslymphopoiesis accompanied by G2/M arrest in APEX2-null mice

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4097-4103 ◽  
Author(s):  
Yasuhito Ide ◽  
Daisuke Tsuchimoto ◽  
Yohei Tominaga ◽  
Manabu Nakashima ◽  
Takeshi Watanabe ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Proliferating Cell Nuclear Antigen ◽  
Cell Cycle Progression ◽  
Immunoglobulin M ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Cycle Progression ◽  
Class Switch ◽  
M Phase ◽  
Proliferating Cell

Abstract APEX2/APE2 is a secondary mammalian apurinic/apyrimidinic endonuclease that associates with proliferating cell nuclear antigen (PCNA), and the progression of S phase of the cell cycle is accompanied by its expression. To determine the biologic significance of APEX2, we established APEX2-null mice. These mice were about 80% the size of their wild-type littermates and exhibited a moderate dyshematopoiesis and a relatively severe defect in lymphopoiesis. A significant accumulation of both thymocytes and mitogen-stimulated splenocytes in G2/M phase was seen in APEX2-null mice compared with the wild type, indicating that APEX2 is required for proper cell cycle progression of proliferating lymphocytes. Although APEX2-null mice exhibited an attenuated immune response against ovalbumin in comparison with wild-type mice, they produced both antiovalbumin immunoglobulin M (IgM) and IgG, indicating that class switch recombination can occur even in the absence of APEX2. (Blood. 2004;104: 4097-4103)

scholarly journals Clarithromycin Delays Progression of Bronchial Epithelial Cells from G1 Phase to S Phase and Delays Cell Growth via Extracellular Signal-Regulated Protein Kinase Suppression

2006 ◽  
Vol 50 (5) ◽  
pp. 1738-1744 ◽  
Author(s):  
Masaharu Shinkai ◽  
Jun Tamaoki ◽  
Hideo Kobayashi ◽  
Soichiro Kanoh ◽  
Kazuo Motoyoshi ◽  
...  
Keyword(s):  
Cell Growth ◽  
Epithelial Cells ◽  
Bronchial Epithelial Cells ◽  
S Phase ◽  
Macrolide Antibiotics ◽  
Quiescent State ◽  
Bronchial Epithelial ◽  
Extracellular Signal ◽  
M Phase ◽  
Normal Human

ABSTRACT The nonsteroidal anti-inflammatory drugs have been shown to support cytoprotection of cells by shifting cells toward a quiescent state (G0/G1). Extracellular signal-regulated kinase (ERK) is required for cells to pass from G1 phase into S phase, and macrolide antibiotics can inhibit ERK1/2 phosphorylation. However, previous reports suggest that macrolide antibiotics do not affect cell growth in bronchial epithelial cells. Therefore, we studied normal human bronchial epithelial (NHBE) cells to determine whether clarithromycin (CAM) suppresses ERK, delays bronchial epithelial cells from progressing to S phase, and delays cell growth. Exposure to CAM at 10 μg/ml daily over 4 days irreversibly decreased the cell proliferation with and without growth supplements (P < 0.0001). CAM also inhibited ERK1/2 phosphorylation over the first 90 min of exposure (P < 0.05 for 30 min, P < 0.0001 for 60 min, and P < 0.01 for 90 min) and decreased the ratio of phosphorylated ERK1/2 (pERK1/2) to total ERK1/2 (tERK1/2) (P < 0.0001). Incubation with CAM for 48 h increased the proportion of cells in G1 phase (means ± standard deviations) from 63.5% ± 0.9% to 79.1% ± 1.4% (P < 0.0001), decreased that in S phase from 19.8% ± 1.2% to 10.0% ± 2.1% (P < 0.01), and decreased that in G2/M phase from 16.7% ± 0.4% to 11.0% ± 0.8% (P < 0.001). In contrast, the ratio of pMEK1/2 to tMEK1/2 was not altered after exposure to CAM. These results suggest that macrolide antibiotics can delay the progression of NHBE cells from G1 phase to S phase and can slow cell growth, probably through the suppression of ERK1/2.

scholarly journals Tight association of autophagy and cell cycle in leukemia cells

2021 ◽  
Author(s):  
Alena Gschwind ◽  
Christian Marx ◽  
Marie D. Just ◽  
Paula Severin ◽  
Hannah Behring ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Sorting ◽  
G1 Phase ◽  
Cycle Phase ◽  
Cell Populations ◽  
Flux Analysis ◽  
Double Labeling ◽  
Flow Cytometric ◽  
Sorting Technique ◽  
M Phase

Abstract BackgroundAutophagy plays an essential role in maintaining cellular homeostasis and in the response to cellular stress. Autophagy is also involved in cell cycle progression, yet the relationship between these processes is not clearly defined.ResultsIn exploring this relationship, we observed that the inhibition of autophagy impaired the G2/M phase-arresting activity of etoposide but enhanced the G1 phase-arresting activity of palbociclib. We further investigated the connection of basal autophagy and cell cycle by utilizing the autophagosome tracer dye Cyto-ID in two ways. First, we established a double-labeling flow-cytometric procedure with Cyto-ID and the DNA probe DRAQ5, permitting the cell cycle phase-specific determination of autophagy in live cells. This approach demonstrated that different cell cycle phases were associated with different autophagy levels: G1 phase cells had the lowest one and G2/M phase cells had the highest one. Second, we developed a flow-cytometric cell sorting procedure based on Cyto-ID that separates cell populations into fractions with low, medium and high autophagy. Cell cycle analysis of Cyto-ID-sorted cells confirmed that the high autophagy fraction contained a much higher percentage of G2/M phase cells than the low autophagy fraction. Beyond that, Cyto-ID-based cell sorting proved also to be useful for assessing other autophagy-related processes: extracellular flux analysis revealed metabolic differences between the cell populations, with higher autophagy being associated with higher respiration, higher mitochondrial ATP production and higher glycolysis.ConclusionThis work sheds new light on the interrelation of autophagy and cell cycle by establishing a novel cell sorting technique based on Cyto-ID.

scholarly journals Naringenin inhibits human breast cancer cells (MDA-MB-231) by inducing programmed cell death, caspase stimulation, G2/M phase cell cycle arrest and suppresses cancer metastasis

2021 ◽  
Vol 67 (2) ◽  
pp. 8-13
Author(s):  
Zhaozhen Qi ◽  
Shuangxi Kong ◽  
Shunyu Zhao ◽  
Qiu Tang
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Apoptotic Cell ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Cycle Arrest ◽  
Flow Cytometric ◽  
M Phase

The current study was designed to unveil the anticancer effects of naringenin against breast cancer MDA-MB-231 cells. Cytotoxic effects were estimated via MTT viability assay. Clonogenic assay was performed to assess clonogenic potential of MDA-MB-231 cells. Apoptosis was examined via AO/EB staining, quantified via annexin V/PI staining and western blotting was performed to monitor apoptosis allied protein expressions. Cell cycle was analyzed through flow cytometric analysis. Transwell chambers assay was executed for determination of cell migration and cell invasion tendency of MDA-MB-231 breast cancer cells. Results indicated significant anticancer potential of naringenin drug against MDA-MB-231 cells. On evaluation of cell proliferation rate of breast cancer cells by MTT assay, it was observed that naringenin inhibited proliferation rate in dose as well as time dependent manner. AO/EB staining assay revealed potential morphological changes indicating apoptotic cell death. Annexin V/PI staining assay revealed increased apoptotic cell percentage with increased drug doses. The apoptosis inducing potential of naringenin drug was observed to be mediated via caspase activation. Flow cytometric analysis predicted cell cycle arrest at G2/M phase of cell cycle. Further cell migration as well as cell invasion tendency of MDA-MB-231 cells was reduced to minimum upon application of naringenin drug.

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