CELL CONTACT DEPENDENCE OF SURFACE GALACTOSYLTRANSFERASE ACTIVITY AS A FUNCTION OF THE CELL CYCLE

Mitotic, nonmalignant Balb/c 3T3 cells exhibit endogenous, surface galactosyltransferase activity that does not require intercellular contact throughout the assay period. In this respect, mitotic 3T3 cells resemble malignant Balb/c 3T12 cells which similarly show no contact requirement for optimum transferase activity in any phase of their cell cycle. Previously, it was shown that randomly growing populations of 3T3 cells have lower galactosyltransferase activity when assayed under conditions which decreased cell contact. This led to the conclusion that these normal (3T3) and malignant (3T12) cells differed in that intercellular contact is required for optimum activity of surface galactosyltransferases on the normal cell type. The present data indicate that mitotic 3T3 cells may be capable of expressing enzyme activities exhibited at all times by malignant cells. That is, mitotic 3T3 cells and randomly growing 3T12 cells may readily catalyze galactosyltransferase reactions between enzymes and acceptors on the same cell. Interphase 3T3 cells, on the other hand, might require that enzymes glycosylate acceptors on adjacent cells. A model is proposed that suggests that changes in the spatial arrangement of surface enzymes and acceptors or variations in the fluidity of the cell membrane can account for this contact-related glycosylation.

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Localization of Hematopoietic Stem Cells in Coculture with Mesenchymal Stromal Cells Impacts on Phenotype and Cell Cycle Status

Blood ◽

10.1182/blood.v112.11.4774.4774 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4774-4774

Author(s):

Duohui Jing ◽

Nael Alakel ◽

Fernando Fierro ◽

Katrin Mueller ◽

Martin Bornhaeuser ◽

...

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Cell Division ◽

Stromal Cells ◽

The Other ◽

Cell Contact ◽

Adherent Cells ◽

Hematopoietic Stem ◽

Cell Fractions

Abstract Hematopoietic stem cells (HSC) are defined by their capacity of self-renewal and differentiation. In recent years it became clear that cell to cell contact mediated communication between mesenchymal stromal cells (MSC) and HSC is important for homeostasis of hematopoiesis. MSC play a crucial role in the so called bone marrow niche giving rise to the majority of marrow stromal cell lineages. In vitro we investigated the impact of MSC on CD34 purified HSC expansion and differentiation demonstrating a promoting impact of MSC on adherent HSC in comparison to non adherent HSC in terms of phenotype, migration capacity and clonogenicity. Performing phase contrast microscopy and confocal microscopy we are able to distinguish HSC which are located on the surface of a MSC monolayer (phase-bright cells) and HSC which are covered by MSC monolayer (phase-dim cells). Both HSC fractions and the non-adherent cells were isolated separately by performing serial washing steps. All three fractions were analyzed at fixed time points during the first week of co-culture in term of cell cycle progression, proliferation, maturation and cell division accompanied differentiation. First we performed propidium iodide (PI) staining for cell cycle analysis revealing that the phase-bright cells contained the highest percentage of G2 cells in comparison to the non adherent cells and the phase-dim cells; 13.9 ±1.0% vs 1.3 ±1.2% vs 2.7 ±2.0%, p<0.001. The data indicate the facilitating impact of MSC on HSC in performing mitosis which is however depending on the location of interaction. When HSC are released into supernatant (non adherent cells) or covered by MSC, G2 phase was significantly down-regulated. Next we studied the proliferation capacity of the separate cell fractions. Consistent with the data of cell cycle, cell number of phase-bright faction increased much faster than the other two fractions during the first 4 days suggesting that the MSC surface in vitro is the predominant location of HSC proliferation. Next we investigated the phenotype of HSC. According to FACS analysis results (CD34+CD38-) phase-dim cells revealed a more immature phenotype in comparison to the non adherent cells and the phase-bright cells. During the first four days 80% of phase-dim cells remained CD34+CD38-, while cells of the phase-bright- and the non adherent fraction exhibited a significant more mature phenotype. Performing cell division tracking using CFSE we were able to show that over time number of divisions of phase-dim cells were significantly diminished in comparison to the other two cell fractions in co-cultures. In addition, phase-dim cells started to lose CD34 at the 7th generation, while non-adherent and phase-bright cells already lost CD34 at the 4th generation. These data suggest that “stemness” of HSC was rather preserved in the cell fraction which was covered by MSC monolayer than in the cell fraction on the surface of MSC. In conclusion we demonstrate HSC in distinct locations in vitro showing different behaviors in terms of phenotype and proliferation. It becomes evident that not only the cell to cell contact matters but also the localization of contact. Further experiments are needed to investigate NOD/SCID repopulation potential of the different cell fractions.

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Cell adhesion and cell surface topography in aggregates of 3T3 and SV40-virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy.

The Journal of Cell Biology ◽

10.1083/jcb.76.3.639 ◽

1978 ◽

Vol 76 (3) ◽

pp. 639-651 ◽

Cited By ~ 7

Author(s):

H Gershman ◽

J J Rosen

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Surface Topography ◽

Single Cells ◽

Cell Contact ◽

Intercellular Contact ◽

3T3 Cells ◽

Peripheral Cell ◽

Cellular Processes ◽

Scanning Electron

A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants.

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THE EFFECT OF CELL-TO-CELL CONTACT ON THE SURFACE MORPHOLOGY OF CHINESE HAMSTER OVARY CELLS

The Journal of Cell Biology ◽

10.1083/jcb.57.3.837 ◽

1973 ◽

Vol 57 (3) ◽

pp. 837-844 ◽

Cited By ~ 66

Author(s):

R. W. Rubin ◽

L. P. Everhart

Keyword(s):

Cell Cycle ◽

Chinese Hamster Ovary ◽

Morphological Changes ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Cell Contact ◽

Low Density ◽

Intercellular Contact ◽

Ovary Cells ◽

Transformed Cell Line

In the previous report (Porter et al., in this issue) morphological changes in Chinese hamster ovary (CHO) cells during the cell cycle were described. In this report we describe the role of intercellular contact on these changes. We find that intercellular contact is required for cells to exhibit the morphologies Porter et al. described for S and G2. When cells are synchronized by mitotic selection and plated onto cover slips at very low density such that no intercellular contact occurs, the cells remain in a G1 configuration (rounded and highly blebbed through G1, S, and G2). This G1 morphology is also observed in nonsynchronized log phase cells plated at low densities and allowed to grow for several generations. The addition of conditioned medium from confluent cultures does not induce low density cells to change morphology during the cell cycle. These results indicate that extensive intercellular contact is required for the complete expression of the morphological changes associated with the cell cycle (as described by Porter et al.). It is concluded that although classic contact inhibition of movement and of growth may be absent in this transformed cell line, some contact-dependent response persists.

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Design, Synthesis and In Vitro Anti-Cancer Evaluation of Novel Derivatives of 2-(2-Methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-3- yl)acetamide

Medicinal Chemistry ◽

10.2174/1573406415666190425153717 ◽

2020 ◽

Vol 16 (3) ◽

pp. 340-349

Author(s):

Ebrahim S. Moghadam ◽

Farhad Saravani ◽

Ernest Hamel ◽

Zahra Shahsavari ◽

Mohsen Alipour ◽

...

Keyword(s):

Cell Cycle ◽

Peripheral Neuropathy ◽

Cell Line ◽

Normal Cell ◽

Caspase 3 ◽

Annexin V ◽

Normal Cell Line ◽

Anti Cancer ◽

Mcf 7

Objective: Several anti-tubulin agents were introduced for the cancer treatment so far. Despite successes in the treatment of cancer, these agents cause toxic side effects, including peripheral neuropathy. Comparing anti-tubulin agents, indibulin seemed to cause minimal peripheral neuropathy, but its poor aqueous solubility and other potential clinical problems have led to its remaining in a preclinical stage. Methods: Herein, indibulin analogues were synthesized and evaluated for their in vitro anti-cancer activity using MTT assay (on the MCF-7, T47-D, MDA-MB231 and NIH-3T3 cell lines), annexin V/PI staining assay, cell cycle analysis, anti-tubulin assay and caspase 3/7 activation assay. Results: One of the compounds, 4a, showed good anti-proliferative activity against MCF-7 cells (IC50: 7.5 μM) and low toxicity on a normal cell line (IC50 > 100 μM). All of the tested compounds showed lower cytotoxicity on normal cell line in comparison to reference compound, indibulin. In the annexin V/PI staining assay, induction of apoptosis in the MCF-7 cell line was observed. Cell cycle analysis illustrated an increasing proportion of cells in the sub-G-1 phase, consistent with an increasing proportion of apoptotic cells. No increase in G2/M cells was observed, consistent with the absence of anti-tubulin activity. A caspase 3/7 assay protocol showed that apoptosis induction by more potent compounds was due to activation of caspase 3. Conclusion: Newly synthesized compounds exerted acceptable anticancer activity and further investigation of current scaffold would be beneficial.

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Recombination of Ty elements in yeast can be induced by a double-strand break.

Genetics ◽

10.1093/genetics/140.1.67 ◽

1995 ◽

Vol 140 (1) ◽

pp. 67-77 ◽

Cited By ~ 1

Author(s):

A Parket ◽

O Inbar ◽

M Kupiec

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Strand Break ◽

Double Strand Break ◽

The Other ◽

Direct Repeats ◽

Yeast Saccharomyces Cerevisiae ◽

Low Frequencies ◽

Ty Elements ◽

Main Family

Abstract The Ty retrotransposons are the main family of dispersed repeated sequences in the yeast Saccharomyces cerevisiae. These elements are flanked by a pair of long terminal direct repeats (LTRs). Previous experiments have shown that Ty elements recombine at low frequencies, despite the fact that they are present in 30 copies per genome. This frequency is not highly increased by treatments that cause DNA damage, such as UV irradiation. In this study, we show that it is possible to increase the recombination level of a genetically marked Ty by creating a double-strand break in it. This break is repaired by two competing mechanisms: one of them leaves a single LTR in place of the Ty, and the other is a gene conversion event in which the marked Ty is replaced by an ectopically located one. In a strain in which the marked Ty has only one LTR, the double-strand break is repaired by conversion. We have also measured the efficiency of repair and monitored the progression of the cells through the cell-cycle. We found that in the presence of a double-strand break in the marked Ty, a proportion of the cells is unable to resume growth.

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Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle

Scientific Reports ◽

10.1038/s41598-021-82539-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

John A. Halsall ◽

Simon Andrews ◽

Felix Krueger ◽

Charlotte E. Rutledge ◽

Gabriella Ficz ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Histone Modifications ◽

Expression Patterns ◽

Cell Types ◽

Cell Type ◽

Bar Code ◽

Genes Encoding ◽

Cell Type Specific ◽

Rolling Windows

AbstractChromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10–50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1–5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

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The Consequences of Budding versus Binary Fission on Adaptation and Aging in Primitive Multicellularity

Genes ◽

10.3390/genes12050661 ◽

2021 ◽

Vol 12 (5) ◽

pp. 661

Author(s):

Hanna Isaksson ◽

Peter L. Conlin ◽

Ben Kerr ◽

William C. Ratcliff ◽

Eric Libby

Keyword(s):

Cell Cycle ◽

Growth Rate ◽

Cellular Senescence ◽

Carrying Capacity ◽

The Other ◽

Binary Fission ◽

Fundamental Aspect ◽

Accumulated Damage ◽

Cell Age ◽

Multicellular Organisms

Early multicellular organisms must gain adaptations to outcompete their unicellular ancestors, as well as other multicellular lineages. The tempo and mode of multicellular adaptation is influenced by many factors including the traits of individual cells. We consider how a fundamental aspect of cells, whether they reproduce via binary fission or budding, can affect the rate of adaptation in primitive multicellularity. We use mathematical models to study the spread of beneficial, growth rate mutations in unicellular populations and populations of multicellular filaments reproducing via binary fission or budding. Comparing populations once they reach carrying capacity, we find that the spread of mutations in multicellular budding populations is qualitatively distinct from the other populations and in general slower. Since budding and binary fission distribute age-accumulated damage differently, we consider the effects of cellular senescence. When growth rate decreases with cell age, we find that beneficial mutations can spread significantly faster in a multicellular budding population than its corresponding unicellular population or a population reproducing via binary fission. Our results demonstrate that basic aspects of the cell cycle can give rise to different rates of adaptation in multicellular organisms.

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From i-doc to VR experience: New forms of user engagement in immersive digital documentaries

New Cinemas Journal of Contemporary Film ◽

10.1386/ncin_00003_1 ◽

2020 ◽

Vol 17 (1) ◽

pp. 37-48 ◽

Cited By ~ 1

Author(s):

Florian Mundhenke

Keyword(s):

User Experience ◽

Digital Storytelling ◽

Computer Game ◽

Spatial Arrangement ◽

The Other ◽

User Engagement ◽

Audience Research ◽

Head Mounted Displays ◽

Digital Formats ◽

The One

Audience research proves that possibilities of interaction in i-docs are often not fulfilled by the user, who is not really part of a ‘work in progress’ (as intended by the makers). With the shift and development of new digital formats (360-degree-films, nonfictional VR experiences, AR apps), the question of the possible interactive potential should be addressed once again. Since VR projects are fully immersive (mostly using head-mounted displays), there is no possible distraction from outside on the one hand. On the other hand, there is a shift from the computer game style aesthetic of early i-docs, with their pure spatial arrangement of events, to a more inclusive digital storytelling modality with the user experiencing his own world-building. This will be discussed with taking into consideration the non-fictional VR experience as a mode of actively combining immersion and storytelling for a satisfactory user experience. Afterwards two very different examples of nonfictional VR production will be presented, and their modalities will be briefly touched; the utilized approach and its user response will be discussed. A look at the future of possible developments concludes the essay.

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Differential Mechanisms of Cell Death Induced by HDAC Inhibitor SAHA and MDM2 Inhibitor RG7388 in MCF-7 Cells

Cells ◽

10.3390/cells8010008 ◽

2018 ◽

Vol 8 (1) ◽

pp. 8 ◽

Cited By ~ 5

Author(s):

Umamaheswari Natarajan ◽

Thiagarajan Venkatesan ◽

Vijayaraghavan Radhakrishnan ◽

Shila Samuel ◽

Appu Rathinavelu

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Cell Death ◽

Cancer Cells ◽

Hdac Inhibitor ◽

The Other ◽

Combination Treatments ◽

Cycle Arrest ◽

Anti Cancer ◽

Mcf 7

Gene expression is often altered by epigenetic modifications that can significantly influence the growth ability and progression of cancers. SAHA (Suberoylanilide hydroxamic acid, also known as Vorinostat), a well-known Histone deacetylase (HDAC) inhibitor, can stop cancer growth and metastatic processes through epigenetic alterations. On the other hand, Letrozole is an aromatase inhibitor that can elicit strong anti-cancer effects on breast cancer through direct and indirect mechanisms. A newly developed inhibitor, RG7388 specific for an oncogene-derived protein called MDM2, is in clinical trials for the treatment of various cancers. In this paper, we performed assays to measure the effects of cell cycle arrest resulting from individual drug treatments or combination treatments with SAHA + letrozole and SAHA + RG7388, using the MCF-7 breast cancer cells. When SAHA was used individually, or in combination treatments with RG7388, a significant increase in the cytotoxic effect was obtained. Induction of cell cycle arrest by SAHA in cancer cells was evidenced by elevated p21 protein levels. In addition, SAHA treatment in MCF-7 cells showed significant up-regulation in phospho-RIP3 and MLKL levels. Our results confirmed that cell death caused by SAHA treatment was primarily through the induction of necroptosis. On the other hand, the RG7388 treatment was able to induce apoptosis by elevating BAX levels. It appears that, during combination treatments, with SAHA and RG7388, two parallel pathways might be induced simultaneously, that could lead to increased cancer cell death. SAHA appears to induce cell necroptosis in a p21-dependent manner, and RG7388 seems to induce apoptosis in a p21-independent manner, outlining differential mechanisms of cell death induction. However, further studies are needed to fully understand the intracellular mechanisms that are triggered by these two anti-cancer agents.

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The Novel Vascular Disrupting Agent ANG501 Induces Cell Cycle Arrest and Enhances Endothelial Cell Sensitivity to Radiation

Cancer Growth and Metastasis ◽

10.4137/cgm.s2596 ◽

2009 ◽

Vol 2 ◽

pp. CGM.S2596 ◽

Cited By ~ 2

Author(s):

Shona T. Dougherty ◽

Sean E. Walker ◽

Peter D. Davis ◽

Graeme J. Dougherty

Keyword(s):

Cell Cycle ◽

Endothelial Cells ◽

Normal Cell ◽

Cell Cycle Progression ◽

Stress Proteins ◽

Vascular Disrupting Agent ◽

Heat Shock Stress ◽

Cell Cycling ◽

Vascular Disrupting

The efficacy of approaches in which vascular disrupting agents (VDA) are used in combination with conventional chemotherapy and/or radiation therapy in the treatment of cancer might be improved if there were a better understanding of the cellular and molecular changes induced in normal and malignant cells as a result of VD A exposure. Toward this goal, murine endothelial cells were treated in vitro with ANG501, a novel stilbene VDA developed in our laboratory, and alterations in gene expression determined by genome-wide microarray analysis and subsequently confirmed by Western blot analysis. Among the genes that were shown to be induced upon brief exposure to non-cytotoxic doses of ANG501 were several involved in the control of cell cycle progression and apoptosis, including p21Wafl and the heat shock/stress proteins hsp25, hsp70 and anti-B-crystallin. Reflecting such induction, functional studies confirmed that normal cell cycling is temporarily inhibited following treatment with ANG501 such that the majority of cells accumulate at the radiation-sensitive G2/M phase of the cell cycle at 6 hr. The effects were transient and by 24 hr normal cell cycling had largely resumed. Combination experiments confirmed that endothelial cells treated 6 hr previously with ANG501 were more readily killed by radiation. Importantly, significant effects were evident at clinically relevant radiation doses. Taken together these findings emphasize the need to consider the radiosensitizing activity of VD As when developing therapies in which these promising compounds are used in combination with radiation.

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