scholarly journals Long-Term Exposure to Zidovudine Delays Cell Cycle Progression, Induces Apoptosis, and Decreases Telomerase Activity in Human Hepatocytes

2009 ◽  
Vol 111 (1) ◽  
pp. 120-130 ◽  
Author(s):  
Jia-Long Fang ◽  
Frederick A. Beland
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Telomerase Activity ◽  
Human Hepatocytes ◽  
Cycle Progression

The Underlying Mechanism of Telomere Maintenance in Human Bone Marrow-Derived Mesenchymal Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4252-4252
Author(s):  
He Huang ◽  
Jingyuan Li ◽  
Jianping Lan ◽  
Yanmin Zhao ◽  
Xiaoyu Lai
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Telomere Length ◽  
Hela Cells ◽  
Cell Cycle Progression ◽  
Telomerase Activity ◽  
Telomeric Repeat ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Cycle Progression

Abstract Objective: Human bone marrow-derived mesenchymal stem cells(MSCs) are thought to be promising tools in cell and gene therapy. Unfortunately, the low frequency of MSCs in bone marrow and rapid aging in in vitro expansion, which profoundly compromise their proliferative capacity, give rise to a huge hindrance for their clinical use. Previous study indicated that MSCs would undergo quick telomere shortening as well as reduced replicative capacity during in vitro expansion. These findings suggested that MSCs’ telomere loss might be associated with their decreased proliferative and differentiative potentials. However, the mechanisms by which MSCs maintain their telomere homeostasis have not yet been fully addressed to date. In the present study, we compared the telomere length, the distribution pattern of telomeric repeat binding factor 1(TRF1) between MSCs and other telomerase-positive cells or telomerase-negative cells, detected extrachromosomal telomeric repeat DNA (ECTR DNA) in MSCs and the variation of telomerase activity during cell cycle progression in order to unveil the mystery of telomere regulation in MSCs. METHODS: MSCs were isolated from healthy human bone marrow (n=34) by the plastic adherence protocols and identified by flow cytometry with markers of CD14, CD45, CD44, HLA-DR, CD34, CD29 and CD166. Telomere length and ECTR DNA were detected with Southern hybridization. The TRF1 distribution were probed with immunofluorescence staining. Telomeric repeat amplification protocol (TRAP ) and/or semi-quantitive Western blot assay were performed to determine the telomerase activity in MSCs, MSCs-derived adipocytes and telomerase levels during cell cycle progression. MSCs were synchronized by serum starvation and Aphidicolin treatment for the aforementioned assay. RESULTS: The mean telomere restriction fragment (mTRF) in MSCs was 8.0 kbp( range, 2.7 kbp-18.0 kbp), similar to telomerase-positive HeLa cells 6.0 kbp (range, 2.7 kbp-8.6 kbp) and 293T cells 5.0 kbp(range, 2.7 kbp-8.6 kbp); while the mTRF in telomerase-negative cells WI-38–2RA was 21.2 kb (range 2.0 kbp->21.2 kbp). The results indicated that telomere length in MSCs and HeLa cells were shorter and relatively more homogeneous than WI-38–2RA cells. TRF1 did not coincide with promyelocytic leukemia (PML) nuclear body in MSCs and HeLa cells while it exclusively did in WI-38–2RA cells. ECTR DNA was negative in MSCs and HeLa cells but positive in WI-38–2RA cells. Detected by TRAP, telomerase activity in MSCs(n=34) was negative with relative telomerase activity (RTA) of 1.44%±0.77%, but it was positive in MSCs-derived adipocytes (n=3) with RTA of 11.80±2.52%(P<0.001). Moreover, a cell cycle-dependent expression profile of telomerase was found in MSCs when they were synchronized by serum starvation and Aphidicolin treatment. Untreated MSCs expressed extremely low level of telomerase probed by Western blot with the 2C4 mAb, but the telomerase level had significantly increased when these cells were trapped in S phase. CONCLUSION: Since MSCs possessed similar features to telomerase-positive cells in telomere length, TRF1 localization pattern and ECTR DNA which were distinct from telomerase-negative ALT cells, and they had increased telomerase activity following differentiation into adipocytes and entrance into S phase, We postulated that the telomere in MSCs was maintained by telomerase pathway other than ALT pathway. The telomerase expression level of MSCs was tightly regulated with cell cycle progression.

scholarly journals CDK1-mediated CENP-C phosphorylation modulates CENP-A binding and mitotic kinetochore localization

2019 ◽  
Vol 218 (12) ◽  
pp. 4042-4062 ◽  
Author(s):  
Reito Watanabe ◽  
Masatoshi Hara ◽  
Ei-ichi Okumura ◽  
Solène Hervé ◽  
Daniele Fachinetti ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Interaction Network ◽  
Binding Motif ◽  
Cycle Progression ◽  
Kinetochore Assembly ◽  
Centromere Proteins

The kinetochore is essential for faithful chromosome segregation during mitosis. To form a functional kinetochore, constitutive centromere-associated network (CCAN) proteins are assembled on the centromere chromatin that contains the centromere-specific histone CENP-A. CENP-C, a CCAN protein, directly interacts with the CENP-A nucleosome to nucleate the kinetochore structure. As CENP-C is a hub protein for kinetochore assembly, it is critical to address how the CENP-A–CENP-C interaction is regulated during cell cycle progression. To address this question, we investigated the CENP-C C-terminal region, including a conserved CENP-A–binding motif, in both chicken and human cells and found that CDK1-mediated phosphorylation of CENP-C facilitates its binding to CENP-A in vitro and in vivo. We observed that CENP-A binding is involved in CENP-C kinetochore localization during mitosis. We also demonstrate that the CENP-A–CENP-C interaction is critical for long-term viability in human RPE-1 cells. These results provide deeper insights into protein-interaction network plasticity in centromere proteins during cell cycle progression.

Key Roles of the Raf-1 and Akt in Drug Resistance, Cell Cycle Progression and the Prevention of Apoptosis in Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1356-1356
Author(s):  
Steven L. Abrams ◽  
Linda S. Steelman ◽  
John G. Shelton ◽  
William H. Chappell ◽  
Ellis W.T. Wong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Drug Resistance ◽  
Cell Cycle Progression ◽  
Mtor Inhibitors ◽  
Drug Resistant ◽  
Cycle Progression ◽  
Doxorubicin Treatment ◽  
Induced Apoptosis ◽  
Resistant Cells

Abstract The Raf/MEK/ERK and PI3K/Akt kinase cascades are pivotal in transmitting signals from membrane receptors to transcription factors which regulate growth, apoptosis and chemotherapeutic drug resistance. Previously we determined that activated Raf-1 or Akt expression by themselves, did not relieve cytokine-dependence of FL5.12 hematopoietic cells. However, when activated forms were introduced and activated in the same cell, cytokine-dependence was relieved. This was determined with conditionally active forms of Raf (Raf-1:AR) and Akt (Akt:ER) that are activated upon testosterone (binds AR) and tamoxifen (binds ER) treatment respectively. Upon hormone deprivation for 24 hrs, FL/Akt:ER+Raf-1:AR cells exited the cell cycle and underwent apoptosis. The effects of Raf, Akt and cytokines on cell cycle progression, prevention of apoptosis, downstream signal transduction pathways and chemotherapeutic drug resistance were examined. Activation of Raf or Akt individually suppressed but did not totally inhibit apoptosis, while their simultaneous activation prevented apoptosis. Activation of Raf was associated with cell cycle progression and prevention of caspase 3 activation. In fact, Raf activation by itself resulted in a more potent induction of cell cycle progression than when Raf and Akt were both activated but Akt activation was required for the long term growth of the cells. Treatment with either MEK or mTOR inhibitors suppressed proliferation and induced apoptosis and their combined treatment had a synergistic effect. Parental FL5.12 cells normally undergo G1 arrest after doxorubicin treatment and G2/M arrest after paclitaxel treament for 24 to 48 hrs. The FL/Akt:ER+Raf-1:AR cells are more resistant to these drugs and did not display as dramatic G1 or G2/M arrests after either doxorubicin or paclitaxel treatment respectively. Stable drug resistant FL/Akt:ER+Raf-1:AR cells were obtained by culturing the cells under limiting dilution conditions in the presence of 10 to 50 nM doxorubicin. These cells also displayed resistance to paclitaxel. Activation of Raf in the drug resistant cells increased the IC50 for doxorubicin 80-fold as compared to when it was not activated or when just Akt was activated. In contrast, activation of Raf in the unselected FL/Akt:ER+Raf-1:AR cells increased the IC50 for doxorubicin 10-fold. The drug resistant and drug sensitive cells displayed activation of p53 phosphorylation at S15 in response to doxorubicin indicating that the drug resistant cells did not become drug resistant by an altered p53 response. Both drug sensitive and resistant cells induced ERK upon Raf activation or doxorubicin treatment. Higher levels of the MRP-1 drug pump and p27Kip1 CDK inhibitor were detected in the drug resistant cells in response to Raf-1 activation than in the drug sensitive cells. The effects of MEK and mTOR inhibitors on the sensitivity to doxorubicin and paclitaxel were examined to determine the efficacy of combining classical chemotherapy with targeted therapy. MEK inhibitors efficiently induced apoptosis in the presence of doxorubicin or paclitaxel while the mTOR inhibitor rapamycin was less effective. Thus the Raf/MEK/ERK pathway is associated with cell cycle progression, prevention of caspase 3 activation and subsequent apoptosis and increase in MRP-1 and p27Kip1 expression in drug resistant cells. The Akt pathway is necessary for long term proliferation in the presence and absence of doxorubicin.

Cdkn3 Knockout Mice Develop Hematopoietic Malignancies

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1537-1537 ◽  
Author(s):  
Zejin Sun ◽  
Donna Cerabona ◽  
Ying He ◽  
Grzegorz Nalepa
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cell Cycle Progression ◽  
Conditional Knockout ◽  
Cycle Progression ◽  
Mitotic Kinase ◽  
Risk Of Death

Abstract Cyclin dependent kinase inhibitor 3 (CDKN3) is a dual-specificity cell cycle regulatory phosphatase. In interphase, CDKN3 prevents premature G1/S transition by dephosphorylating interphase cyclin-dependent kinases (CDKs) to prevent premature inactivation of the RB pathway. During cell division, CDKN3 dephosphorylates the key mitotic kinase CDK1 at threonine-161 to extinguish CDK1 activity at the exit from mitosis. CDKN3 knockdown in cultured cells impairs the spindle assembly checkpoint (SAC), accelerates cell cycle progression and causes chromosomal instability, suggesting that it may function as a tumor suppressor. However, since CDKN3 has been reported as overexpressed in some malignancies and mutated or silenced in others, it is unclear whether it functions as an oncogene or a tumor suppressor. To understand the in vivo role of CDKN3 in carcinogenesis, we generated the first Cdkn3 conditional knockout mouse model. We found that Cdkn3-/- mice were viable, non-dysmorphic and born at expected Mendelian ratios, indicating that this gene is dispensable for normal embryonic development. In agreement with the postulated role of this phosphatase in cell cycle progression and regulation of CDKs, we found that Cdkn3-/- cells had increased CDK1, CDK2 and CDK4 activity; increased inhibitory phosphorylation of Rb; increased DNA replication and proliferation; and impaired SAC. Increased CDK activity and accelerated cell cycle progression caused genomic instability reflected by increased frequency of in vivo micronucleation during hematopoiesis as well as higher frequency of aneuploidy and multinucleation and accumulation of supernumerary centrosomes in Cdkn3-/- cells cultured ex vivo. Cdkn3-/- mice had increased myeloid colony-forming units in progenitor assays. Long-term observation of Cdkn3-/- mice revealed an increased risk of death from a variety of hematopoietic (leukemia and lymphoma) and non-hematopoietic (lung, prostate and ovarian) malignancies. Our findings establish Cdkn3 as an in vivo tumor suppressor in bone marrow and a variety of other tissues. In the long term, Cdkn3-/- mice will serve as a tool to dissect the function of this phosphatase in cell cycle control in more detail, and may prove useful in preclinical studies of chemotherapy of CDK-hyperactive, genomically unstable leukemia and lymphoma. Disclosures No relevant conflicts of interest to declare.

scholarly journals Fungal zygote survival and ploidy maintenance rely on cell cycle-dependent and independent mating blocks

2020 ◽  
Author(s):  
Aleksandar Vještica ◽  
Melvin Bérard ◽  
Gaowen Liu ◽  
Laura Merlini ◽  
Pedro Junior Nkosi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Cycle Progression ◽  
Mating Behaviors ◽  
Cycle Dependent ◽  
Necessary And Sufficient ◽  
Meiotic Cycle

AbstractTo ensure genome stability, sexually reproducing organisms require that mating brings together exactly two haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins post-fusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least two mechanisms where the zygotic fate imposed by Mei2 and the cell cycle re-entry triggered by Mei3 synergize to prevent zygotic mating.

scholarly journals A stochastic model of adult neurogenesis coupling cell cycle progression and differentiation

2019 ◽  
Author(s):  
Anna Stopka ◽  
Marcelo Boareto
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Stochastic Model ◽  
Cell Cycle Progression ◽  
Population Level ◽  
Tissue Homeostasis ◽  
Adult Brain ◽  
Differentiation Markers ◽  
Cycle Progression

Long-term tissue homeostasis requires a precise balance between stem cell self-renewal and the generation of differentiated progeny. Recently, it has been shown that in the adult murine brain, neural stem cells (NSCs) divide mostly symmetrically. This finding suggests that the required balance for tissue homeostasis is accomplished at the population level. However, it remains unclear how this balance is enabled. Furthermore, there is experimental evidence that proneural differentiation factors not only promote differentiation, but also cell cycle progression, suggesting a link between the two processes in NSCs. To study the effect of such a link on NSC dynamics, we developed a stochastic model in which stem cells have an intrinsic probability to progress through cell cycle and to differentiate. Our results show that increasing heterogeneity in differentiation probabilities leads to a decreased probability of long-term tissue homeostasis, and that this effect can be compensated when cell cycle progression and differentiation are positively coupled. Using single-cell RNA-Seq profiling of adult NSCs, we found a positive correlation in the expression levels of cell cycle and differentiation markers. Our findings suggest that a coupling between cell cycle progression and differentiation on the cellular level is part of the process that maintains tissue homeostasis in the adult brain.

scholarly journals Cell cycle-dependent and independent mating blocks ensure fungal zygote survival and ploidy maintenance

PLoS Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
pp. e3001067
Author(s):  
Aleksandar Vještica ◽  
Melvin Bérard ◽  
Gaowen Liu ◽  
Laura Merlini ◽  
Pedro Junior Nkosi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Genome Stability ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Cell Cycle Reentry ◽  
Mating Behaviors ◽  
Cycle Dependent ◽  
Necessary And Sufficient ◽  
Meiotic Cycle

To ensure genome stability, sexually reproducing organisms require that mating brings together exactly 2 haploid gametes and that meiosis occurs only in diploid zygotes. In the fission yeast Schizosaccharomyces pombe, fertilization triggers the Mei3-Pat1-Mei2 signaling cascade, which represses subsequent mating and initiates meiosis. Here, we establish a degron system to specifically degrade proteins postfusion and demonstrate that mating blocks not only safeguard zygote ploidy but also prevent lysis caused by aberrant fusion attempts. Using long-term imaging and flow-cytometry approaches, we identify previously unrecognized and independent roles for Mei3 and Mei2 in zygotes. We show that Mei3 promotes premeiotic S-phase independently of Mei2 and that cell cycle progression is both necessary and sufficient to reduce zygotic mating behaviors. Mei2 not only imposes the meiotic program and promotes the meiotic cycle, but also blocks mating behaviors independently of Mei3 and cell cycle progression. Thus, we find that fungi preserve zygote ploidy and survival by at least 2 mechanisms where the zygotic fate imposed by Mei2 and the cell cycle reentry triggered by Mei3 synergize to prevent zygotic mating.

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