scholarly journals Circadian Clock Core Component Bmal1 Dictates Cell Cycle Rhythm of Proliferating Hepatocytes during Liver Regeneration

2021 ◽  
Author(s):  
Huaizhou Jiang ◽  
Veronica Garcia ◽  
Jennifer Abla Yanum ◽  
Joonyong Lee ◽  
Guoli Dai
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Circadian Clock ◽  
Liver Regeneration ◽  
Nuclear Dna ◽  
Core Component ◽  
Activation Patterns ◽  
Redox Sensor ◽  
M Phase ◽  
Late Stages

Following partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on Bmal1+/+ and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, Bmal1+/+ hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/- hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 h, and the third such wave was lost. Following PH, Bmal1+/+ hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/- hepatocytes fully abolished mitotic oscillation. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied by suppressed expression peaks of a group of cell cycle components and regulators, and dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover, Bmal1+/+ hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this phenomenon was largely inhibited in Bmal1hep-/- hepatocytes. In addition, during late stages of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated liver tissue. Collectively, we demonstrated that during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advancement, and largely governs fluctuations in fat metabolism in replicating hepatocytes, and eventually determines the redox state of regenerated livers.

scholarly journals Circadian Clock Core Component Bmal1 Dictates Cell Cycle Rhythm of Proliferating Hepatocytes during Liver Regeneration

2021 ◽  
Author(s):  
Huaizhou Jiang ◽  
Veronica Garcia ◽  
Jennifer Yanum ◽  
Joonyong Lee ◽  
Guoli Dai
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Circadian Clock ◽  
Liver Regeneration ◽  
Nuclear Dna ◽  
Third Wave ◽  
Wild Type ◽  
Core Component ◽  
Activation Patterns ◽  
M Phase

Following partial hepatectomy (PH), the majority of remnant hepatocytes synchronously enter and rhythmically progress through the cell cycle for three major rounds to regain the lost liver mass. Whether and how the circadian clock core component Bmal1 modulates this process remains elusive. We performed PH on wild-type and hepatocyte-specific Bmal1 knockout (Bmal1hep-/-) mice and compared the initiation and progression of the hepatocyte cell cycle. After PH, wild-type hepatocytes exhibited three major waves of nuclear DNA synthesis. In contrast, in Bmal1hep-/- hepatocytes, the first wave of nuclear DNA synthesis was delayed by 12 hours, the third wave of nuclear DNA synthesis was lost. Following PH, wild-type hepatocytes underwent three major waves of mitosis, whereas Bmal1hep-/- hepatocytes fully abolished the oscillation of mitosis. These Bmal1-dependent disruptions in the rhythmicity of hepatocyte cell cycle after PH were accompanied with prevented expression peaks of a group of the cell cycle components and regulators, dysregulated activation patterns of mitogenic signaling molecules c-Met and EGFR. Moreover, wild-type hepatocytes rhythmically accumulated fat as they expanded following PH, whereas this event was largely prohibited in Bmal1hep-/- hepatocytes. In addition, during the late stage of liver regrowth, Bmal1 absence in hepatocytes caused the activation of redox sensor Nrf2, suggesting an oxidative stress state in regenerated livers. Collectively, we demonstrate that, during liver regeneration, Bmal1 partially modulates the oscillation of S-phase progression, fully controls the rhythmicity of M-phase advance, and largely govern the fluctuation of fat metabolism of replicating hepatocytes, and eventually determines the redox state of regenerated livers.

scholarly journals SYNCHRONIZATION OF MITOCHONDRIAL DNA SYNTHESIS IN CHINESE HAMSTER CELLS (LINE CHO) DEPRIVED OF ISOLEUCINE

1973 ◽  
Vol 58 (2) ◽  
pp. 340-345 ◽  
Author(s):  
Kenneth D. Ley ◽  
Marilyn M. Murphy
Keyword(s):  
Cell Cycle ◽  
Mitochondrial Dna ◽  
Dna Synthesis ◽  
Cell Cycle Progression ◽  
Time Course ◽  
Nuclear Dna ◽  
Tritiated Thymidine ◽  
Chinese Hamster ◽  
Chinese Hamster Cells ◽  
In Cells

Mitochondrial DNA (mit-DNA) synthesis was compared in suspension cultures of Chinese hamster cells (line CHO) whose cell cycle events had been synchronized by isoleucine deprivation or mitotic selection. At hourly intervals during cell cycle progression, synchronized cells were exposed to tritiated thymidine ([3H]TdR), homogenized, and nuclei and mitochondria isolated by differential centrifugation. Mit-DNA and nuclear DNA were isolated and incorporation of radioisotope measured as counts per minute ([3H]TdR) per microgram DNA. Mit-DNA synthesis in cells synchronized by mitotic selection began after 4 h and continued for approximately 9 h. This time-course pattern resembled that of nuclear DNA synthesis. In contrast, mit-DNA synthesis in cells synchronized by isoleucine deprivation did not begin until 9–12 h after addition of isoleucine and virtually all [3H]TdR was incorporated during a 3-h interval. We have concluded from these results that mit-DNA synthesis is inhibited in CHO cells which are arrested in G1 because of isoleucine deprivation and that addition of isoleucine stimulates synchronous synthesis of mit-DNA. We believe this method of synchronizing mit-DNA synthesis may be of value in studies of factors which regulate synthesis of mit-DNA.

scholarly journals Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity

2000 ◽  
Author(s):  
Gideon Grafi ◽  
Brian Larkins
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Storage Protein ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Cyclin B ◽  
Regulatory Subunit ◽  
Maize Endosperm ◽  
M Phase

The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

scholarly journals INDEPENDENCE OF CENTRIOLE FORMATION AND DNA SYNTHESIS

1973 ◽  
Vol 57 (2) ◽  
pp. 359-372 ◽  
Author(s):  
J. B. Rattner ◽  
Stephanie G. Phillips
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Temporal Relationship ◽  
Nuclear Dna ◽  
S Phase ◽  
Centriole Duplication ◽  
And Migration ◽  
Cellular Dna ◽  
Arabinosyl Cytosine ◽  
Mitotic Cells

The temporal relationship between cell cycle events and centriole duplication was investigated electron microscopically in L cells synchronized by mechanically selecting mitotic cells. The two mature centrioles which each cell received at telophase migrated together from the side of the telophase nucleus distal to the stem body around to a region of the cytoplasm near the stem body and then into a groovelike indention in the early G1 nucleus, where they were found throughout interphase. Procentrioles appeared in association with each mature centriole at times varying from 4 to 12 h after mitosis. Since S phase was found to begin on the average about 9 h after mitotic selection, it appeared that cells generated procentrioles late in G1 or early in S. During prophase, the two centriolar duplexes migrated to opposite sides of the nucleus and the daughter centrioles elongated to the mature length. To ascertain whether any aspect of centriolar duplication was contingent upon nuclear DNA synthesis, arabinosyl cytosine was added to mitotic cells at a concentration which inhibited cellular DNA synthesis by more than 99%. Though cells were thus prevented from entering S phase, the course of procentriole formation was not detectibly affected. However, cells were inhibited from proceeding to the next mitosis, and the centriolar elongation and migration normally associated with prophase did not occur.

scholarly journals Mcm2 and Mcm3 are constitutive nuclear proteins that exhibit distinct isoforms and bind chromatin during specific cell cycle stages of Saccharomyces cerevisiae.

1997 ◽  
Vol 8 (8) ◽  
pp. 1587-1601 ◽  
Author(s):  
M R Young ◽  
B K Tye
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Synthesis ◽  
S Phase ◽  
Specific Cell ◽  
Replication Origins ◽  
Proliferating Cells ◽  
M Phase ◽  
Mcm Proteins ◽  
And Function

The Mcm2-7 proteins are a family of conserved proteins whose functions are essential for the initiation of DNA synthesis in all eukaryotes. These patients are constitutively present in high abundance in actively proliferating cells. In Saccharomyces cerevisiae, the intracellular concentrations of Mcms are between 100 and 500 times the number of replication origins. However, these proteins are limiting for the initiation of DNA synthesis at replication origins. Our studies indicate that only a small fraction of Mcm2 and Mcm3 tightly associates with chromatin, from late M phase to the beginning of the S phase. The rest of the Mcm2 and Mcm3 proteins are disturbed to both the cytoplasm and nucleoplasm in relatively constant levels throughout the cell cycle. We also show that S. cerevisiae Mcm3 is a phosphoprotein that exists in multiple isoforms and that distinct isoforms of Mcm2 and Mcm3 can be detected at specific stages of the cell cycle. These results suggest that the localization and function of the Mcm proteins are regulated by posttranslational phosphorylation in a manner that is consistent with a role for the Mcm proteins in restricting DNA replication to once per cell cycle.

scholarly journals Nrf2 is essential for timely M phase entry of replicating hepatocytes during liver regeneration

2015 ◽  
Vol 308 (4) ◽  
pp. G262-G268 ◽  
Author(s):  
Yuhong Zou ◽  
Min Hu ◽  
Joonyong Lee ◽  
Shashank Manohar Nambiar ◽  
Veronica Garcia ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Liver Regeneration ◽  
Redox Balance ◽  
Cyclin B1 ◽  
Hepatocyte Proliferation ◽  
Related Factor ◽  
Multiple Time ◽  
M Phase ◽  
Cyclin A2 ◽  
Phase Entry

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates various cellular activities, including redox balance, detoxification, metabolism, autophagy, proliferation, and apoptosis. Several studies have demonstrated that Nrf2 regulates hepatocyte proliferation during liver regeneration. The aim of this study was to investigate how Nrf2 modulates the cell cycle of replicating hepatocytes in regenerating livers. Wild-type and Nrf2 null mice were subjected to 2/3 partial hepatectomy (PH) and killed at multiple time points for various analyses. Nrf2 null mice exhibited delayed liver regrowth, although the lost liver mass was eventually restored 7 days after PH. Nrf2 deficiency did not affect the number of hepatocytes entering the cell cycle but did delay hepatocyte mitosis. Mechanistically, the lack of Nrf2 resulted in increased mRNA and protein levels of hepatic cyclin A2 when the remaining hepatocytes were replicating in response to PH. Moreover, Nrf2 deficiency in regenerating livers caused dysregulation of Wee1, Cdc2, and cyclin B1 mRNA and protein expression, leading to decreased Cdc2 activity. Thus, Nrf2 is required for timely M phase entry of replicating hepatocytes by ensuring proper regulation of cyclin A2 and the Wee1/Cdc2/cyclin B1 pathway during liver regeneration.

scholarly journals Relationship between the timing of DNA replication and the developmental competence in Acanthamoeba castellanii

1988 ◽  
Vol 91 (3) ◽  
pp. 389-399
Author(s):  
H. Jantzen ◽  
I. Schulze ◽  
M. Stohr
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Dna ◽  
Defined Medium ◽  
Reciprocal Relationship ◽  
Developmental Competence ◽  
Replication Phase ◽  
G2 Phase ◽  
D Cells

In Acanthamoeba, two different cell types are known. Trophozoites are generated in the mitotic division cycle, whereas cells committed at late G2 phase of the cell cycle develop into cysts in response to starvation. In this paper we study the role of timing of DNA replication in regulating development. The investigation was performed with cultures growing in a non-defined medium (ND cells) that show a high encystation competence and with cultures that have been growing in a chemically defined medium (D cells) for several years and show a low encystation competence. Bivariate DNA/BrdUrd distributions show that ND cells progress through a cycle in which the short replication phase occurs immediately and exclusively after prior completion of mitosis. These cells arrest at late G2 phase of the cell cycle during the stationary stage. In D cells, DNA replication and mitosis seem to be uncoupled, since replication takes place before as well as after mitosis. These cells arrest within their replication phase during the stationary stage. These findings indicate that D cells do not progress into late G2 phase of the cell cycle and hence do not have the competence for commitment. The alternate timing of DNA replication and the low encystation competence of D cells can be reversed by cultivation of these cells in ND medium. Synchronization experiments reveal that late G2 phase ND cells exhibit a low capacity for BrdUrd incorporation and growth after transfer into D medium, whereas ND cells of earlier phases of the cell cycle show premitotic incorporation of BrdUrd into nuclear DNA and growth. These findings suggest on the one hand that premitotic DNA synthesis is a prerequisite for growth of cells in D medium, and that there is a dependence of the induction of premitotic DNA synthesis on the cell cycle, and on the other hand that a reciprocal relationship exists between the capacity of premitotic DNA synthesis and commitment to differentiation.

scholarly journals Indicators of the cell cycle in the thyroid gland in rats when applying infusion of 0.9% solution of NaCl, lactoprotein with sorbitol or HAES-LX 5%

2019 ◽  
Vol 25 (1) ◽  
pp. 62-67
Author(s):  
O.I. Tiron ◽  
O.L. Appelhans ◽  
I.V. Gunas ◽  
I.L. Chereshniuk
Keyword(s):  
Cell Cycle ◽  
Thyroid Gland ◽  
Dna Synthesis ◽  
Dna Fragmentation ◽  
Dna Content ◽  
Male Rats ◽  
Nacl Solution ◽  
Thyroid Cells ◽  
M Phase ◽  
Medical University

The thyroid gland is an important organ that is involved in the regulation of homeostasis and adaptation in various pathological conditions. However, the question of the study of the proliferative activity of thyroid cells by flow cytometry is still poorly understood. Purpose of study: to investigate the indices of the cell cycle and DNA fragmentation of thyroid cells in rats against the background of infusion of 0.9% NaCl solution, lactoprotein with sorbitol or HAES-LX 5%. Experimental studies were performed on 90 white male rats weighing 160-180 g. Infusion of 0.9% NaCl solution, lactoprotein with sorbitol or HAES-LX 5% was performed in the inferior vena cava after its catheterization in aseptic conditions through the femoral vein. The infusions were performed once a day for the first 7 days. Trunk catheterization and decapitation of animals (after 1, 3, 7, 14, 21, and 30 days) were performed under propofol anesthesia (60 mg/kg i/v). Within the framework of the agreement on scientific cooperation between the Research Center of National Pirogov Memorial Medical University, Vinnytsya and the Department of Histology, Cytology and Embryology of the Odessa National Medical University (from 01/01/2018), DNA content in the nuclei of thyroid cells of rats was determined by flow DNA cytometry. Cell cycle analysis was performed using the software FloMax (Partec, Germany) in full digital accordance with the mathematical model, which determined: G0G1 – the percentage of cells of the phase G0G1 to all cells of the cell cycle (DNA content = 2c); S – the percentage of the phase of DNA synthesis to all cells of the cell cycle (DNA content > 2c and < 4c); G2+M – the percentage ratio of the G2+M phase to all cells in the cell cycle (DNA = 4c). Determination of DNA fragmentation (SUB-G0G1, apoptosis) was performed by isolating the RN2 region on DNA histograms before the G0G1 peak, indicating nuclei of cells with a DNA content < 2c. The statistical processing of the obtained results was carried out in the license package “STATISTICA 6.1” using nonparametric estimation methods. The data obtained showed a virtually identical pattern of rat cell cycle and DNA fragmentation of the thyroid gland cells at all study times against the use of 0.9% NaCl solution, lactoprotein with sorbitol or HAES-LX 5%. Thyroid cells in rats are predominantly in the inactive phase of DNA synthesis (G0G1) (90.32% – 91.88%), significantly fewer cells are in the G2+M phase (7.56% – 9.17%), and there is a small percentage of cells in the S-phase (DNA synthesis) (0.52% – 0.67%) and the SUB-G0G1 interval (DNA fragmentation, apoptosis) (2.23% – 2.81%). We can state that the activity of the main part of the thyroid gland is rather low without pathological irritation.

scholarly journals Silymarin Accelerates Liver Regeneration after Partial Hepatectomy

2015 ◽  
Vol 2015 ◽  
pp. 1-14 ◽  
Author(s):  
Jia-Ping Wu ◽  
Chin-Chuan Tsai ◽  
Yu-Lan Yeh ◽  
Yueh-Min Lin ◽  
Chien-Chung Lin ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Liver Regeneration ◽  
Partial Hepatectomy ◽  
Tissue Injury ◽  
Hepatic Regeneration ◽  
Sprague Dawley ◽  
Rt Pcr ◽  
Sprague Dawley Rats ◽  
M Phase ◽  
G2 Phase

Partial hepatectomy (PHx) is a liver regeneration physiological response induced to maintain homeostasis. Liver regeneration evolved presumably to protect wild animals from catastrophic liver loss caused by toxins or tissue injury. Silymarin (Sm) ability to stimulate liver regeneration has been an object of curiosity for many years. Silymarin has been investigated for use as an antioxidant and anticarcinogen. However, its use as a supportive treatment for liver damage is elusive. In this study, we fed silymarin (Sm, 25 mg/kg) to male Sprague-Dawley rats for 7 weeks. Surgical 2/3 PHx was then conducted on the rats at 6 hrs, 24 hrs, and 72 hrs. Western blot and RT-PCR were conducted to detect the cell cycle activities and silymarin effects on hepatic regeneration. The results showed that silymarin enhanced liver regeneration by accelerating the cell cycle in PHx liver. Silymarin led to increased G1 phase (cyclin D1/pRb), S phase (cyclin E/E2F), G2 phase (cyclin B), and M phase (cyclin A) protein and mRNA at 6 hrs, 24 hrs, and 72 hrs PHx. HGF, TGFα, and TGFβ1 growth factor expressions were also enhanced. We suggest that silymarin plays a crucial role in accelerated liver regeneration after PHx.

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