S-phase Inhibition of Cell Cycle Progression by a Novel Class of Pyridopyrimidine Tyrosine Kinase Inhibitors

ABSTRACT The cortactin oncoprotein is frequently overexpressed in head and neck squamous cell carcinoma (HNSCC), often due to amplification of the encoding gene (CTTN). While cortactin overexpression enhances invasive potential, recent research indicates that it also promotes cell proliferation, but how cortactin regulates the cell cycle machinery is unclear. In this article we report that stable short hairpin RNA-mediated cortactin knockdown in the 11q13-amplified cell line FaDu led to increased expression of the Cip/Kip cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/Cip1, p27Kip1, and p57Kip2 and inhibition of S-phase entry. These effects were associated with increased binding of p21WAF1/Cip1 and p27Kip1 to cyclin D1- and E1-containing complexes and decreased retinoblastoma protein phosphorylation. Cortactin regulated expression of p21WAF1/Cip1 and p27Kip1 at the transcriptional and posttranscriptional levels, respectively. The direct roles of p21WAF1/Cip1, p27Kip1, and p57Kip2 downstream of cortactin were confirmed by the transient knockdown of each CDKI by specific small interfering RNAs, which led to partial rescue of cell cycle progression. Interestingly, FaDu cells with reduced cortactin levels also exhibited a significant diminution in RhoA expression and activity, together with decreased expression of Skp2, a critical component of the SCF ubiquitin ligase that targets p27Kip1 and p57Kip2 for degradation. Transient knockdown of RhoA in FaDu cells decreased expression of Skp2, enhanced the level of Cip/Kip CDKIs, and attenuated S-phase entry. These findings identify a novel mechanism for regulation of proliferation in 11q13-amplified HNSCC cells, in which overexpressed cortactin acts via RhoA to decrease expression of Cip/Kip CDKIs, and highlight Skp2 as a downstream effector for RhoA in this process.

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A novel class of pyridopyrimidine tyrosine kinase inhibitors blocks cancer cells in the S-phase of the cell cycle

European Journal of Cancer ◽

10.1016/s0959-8049(02)80805-x ◽

2002 ◽

Vol 38 ◽

pp. S50

Keyword(s):

Cell Cycle ◽

Tyrosine Kinase ◽

Cancer Cells ◽

Tyrosine Kinase Inhibitors ◽

Kinase Inhibitors ◽

S Phase

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Endothelial cells isolated from caveolin-2 knockout mice display higher proliferation rate and cell cycle progression relative to their wild-type counterparts

AJP Cell Physiology ◽

10.1152/ajpcell.00401.2009 ◽

2010 ◽

Vol 298 (3) ◽

pp. C693-C701 ◽

Cited By ~ 18

Author(s):

Leike Xie ◽

Philippe G. Frank ◽

Michael P. Lisanti ◽

Grzegorz Sowa

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Inhibitors ◽

Fold Increase ◽

S Phase ◽

Lower Percentage ◽

Wild Type ◽

Cycle Progression ◽

Molecular Events

The goal of this study was to determine whether caveolin-2 (Cav-2) is capable of controlling endothelial cell (EC) proliferation in vitro. To realize this goal, we have directly compared proliferation rates and cell cycle-associated signaling proteins between lung ECs isolated from wild-type (WT) and Cav-2 knockout (KO) mice. Using three independent proliferation assays, we have determined that Cav-2 KO ECs proliferate by ca. 2-fold faster than their WT counterparts. Cell cycle analysis by flow cytometry of propidium iodide-stained cells showed a relatively higher percentage of Cav-2 KO ECs in S and G2/M and lower percentage in Go/G1 phases of cell cycle relative to their WT counterparts. Furthermore, an over 2-fold increase in the percentage of S phase-associated Cav-2 KO relative to WT ECs was independently determined with bromodeoxyuridine incorporation assay. Mechanistically, the increase in proliferation/cell cycle progression of Cav-2 KO ECs correlated well with elevated expression levels of predominantly S phase- and G2/M phase-associated cyclin A and B1, respectively. Further mechanistic analysis of molecular events controlling cell cycle progression revealed increased level of hyperphosphorylated (inactive) form of G1 to S phase transition inhibitor, the retinoblastoma protein in hyperproliferating Cav-2 KO ECs. Conversely, the expression level of the two cyclin-dependent kinase inhibitors p16INK4 and p27Kip1 was reduced in Cav-2 KO ECs. Finally, increased phosphorylation (activation) of proproliferative extracellular signal-regulated kinase 1/2 was observed in hyperproliferating Cav-2 KO ECs. Overall, our data suggest that Cav-2 negatively regulates lung EC proliferation and cell cycle progression.

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The Meiosis-Specific Crs1 Cyclin Is Required for Efficient S-Phase Progression and Stable Nuclear Architecture

International Journal of Molecular Sciences ◽

10.3390/ijms22115483 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5483

Author(s):

Luisa F. Bustamante-Jaramillo ◽

Celia Ramos ◽

Cristina Martín-Castellanos

Keyword(s):

Cell Cycle ◽

Translational Control ◽

Cell Cycle Progression ◽

Nuclear Architecture ◽

Strand Break ◽

Spindle Pole ◽

S Phase ◽

Cycle Progression ◽

Single Wave ◽

Phase Progression

Cyclins and CDKs (Cyclin Dependent Kinases) are key players in the biology of eukaryotic cells, representing hubs for the orchestration of physiological conditions with cell cycle progression. Furthermore, as in the case of meiosis, cyclins and CDKs have acquired novel functions unrelated to this primal role in driving the division cycle. Meiosis is a specialized developmental program that ensures proper propagation of the genetic information to the next generation by the production of gametes with accurate chromosome content, and meiosis-specific cyclins are widespread in evolution. We have explored the diversification of CDK functions studying the meiosis-specific Crs1 cyclin in fission yeast. In addition to the reported role in DSB (Double Strand Break) formation, this cyclin is required for meiotic S-phase progression, a canonical role, and to maintain the architecture of the meiotic chromosomes. Crs1 localizes at the SPB (Spindle Pole Body) and is required to stabilize the cluster of telomeres at this location (bouquet configuration), as well as for normal SPB motion. In addition, Crs1 exhibits CDK(Cdc2)-dependent kinase activity in a biphasic manner during meiosis, in contrast to a single wave of protein expression, suggesting a post-translational control of its activity. Thus, Crs1 displays multiple functions, acting both in cell cycle progression and in several key meiosis-specific events.

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Spirulina Crude Protein Promotes the Migration and Proliferation in IEC-6 Cells by Activating EGFR/MAPK Signaling Pathway

Marine Drugs ◽

10.3390/md17040205 ◽

2019 ◽

Vol 17 (4) ◽

pp. 205

Author(s):

Su-Jin Jeong ◽

Jeong-Wook Choi ◽

Min-Kyeong Lee ◽

Youn-Hee Choi ◽

Taek-Jeong Nam

Keyword(s):

Cell Cycle ◽

Epithelial Cells ◽

Signaling Pathway ◽

Crude Protein ◽

Cell Cycle Progression ◽

Intestinal Epithelial Cells ◽

Mapk Signaling ◽

S Phase ◽

Intestinal Epithelial ◽

Cycle Progression

Spirulina is a type of filamentous blue-green microalgae known to be rich in nutrients and to have pharmacological effects, but the effect of spirulina on the small intestine epithelium is not well understood. Therefore, this study aims to investigate the proliferative effects of spirulina crude protein (SPCP) on a rat intestinal epithelial cells IEC-6 to elucidate the mechanisms underlying its effect. First, the results of wound-healing and cell viability assays demonstrated that SPCP promoted migration and proliferation in a dose-dependent manner. Subsequently, when the mechanisms of migration and proliferation promotion by SPCP were confirmed, we found that the epidermal growth factor receptor (EGFR) and mitogen-activated protein (MAPK) signaling pathways were activated by phosphorylation. Cell cycle progression from G0/G1 to S phase was also promoted by SPCP through upregulation of the expression levels of cyclins and cyclin-dependent kinases (Cdks), which regulate cell cycle progression to the S phase. Meanwhile, the expression of cyclin-dependent kinase inhibitors (CKIs), such as p21 and p27, decreased with SPCP. In conclusion, our results indicate that activation of EGFR and its downstream signaling pathway by SPCP treatment regulates cell cycle progression. Therefore, these results contribute to the research on the molecular mechanism for SPCP promoting the migration and proliferation of rat intestinal epithelial cells.

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Cubic Membranes Formation in Synchronized Human Hepatocellular Carcinoma Cells Reveals a Possible Role as a Structural Antioxidant Defense System in Cell Cycle Progression

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.617406 ◽

2020 ◽

Vol 8 ◽

Author(s):

Deqin Kong ◽

Rui Liu ◽

Jiangzheng Liu ◽

Qingbiao Zhou ◽

Jiaxin Zhang ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Cell Cycle ◽

Cell Cycle Progression ◽

S Phase ◽

Carcinoma Cells ◽

Human Hepatocellular Carcinoma ◽

Cycle Progression ◽

Hepatocellular Carcinoma Cells ◽

G2 Phase ◽

Human Hepatocellular Carcinoma Cells

Cubic membranes (CMs) represent unique biological membrane structures with highly curved three-dimensional periodic minimal surfaces, which have been observed in a wide range of cell types and organelles under various stress conditions (e. g., starvation, virus-infection, and oxidation). However, there are few reports on the biological roles of CMs, especially their roles in cell cycle. Hence, we established a stable cell population of human hepatocellular carcinoma cells (HepG2) of 100% S phase by thymidine treatment, and determined certain parameters in G2 phase released from S phase. Then we found a close relationship between CMs formation and cell cycle, and an increase in reactive oxygen species (ROS) and mitochondrial function. After the synchronization of HepG2 cells were induced, CMs were observed through transmission electron microscope in G2 phase but not in G1, S and M phase. Moreover, the increased ATP production, mitochondrial and intracellular ROS levels were also present in G2 phase, which demonstrated a positive correlation with CMs formation by Pearson correlation analysis. This study suggests that CMs may act as an antioxidant structure in response to mitochondria-derived ROS during G2 phase and thus participate in cell cycle progression.

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Effects of Guanine Nucleotide Depletion on Cell Cycle Progression in Human T Lymphocytes

Blood ◽

10.1182/blood.v91.8.2896.2896_2896_2904 ◽

1998 ◽

Vol 91 (8) ◽

pp. 2896-2904 ◽

Cited By ~ 69

Author(s):

Josée Laliberté ◽

Ann Yee ◽

Yue Xiong ◽

Beverly S. Mitchell

Keyword(s):

Cell Cycle ◽

T Cells ◽

T Lymphocytes ◽

Cell Cycle Progression ◽

S Phase ◽

Guanine Nucleotides ◽

Erythroid Cell ◽

Guanine Nucleotide ◽

Cycle Progression ◽

Human T Lymphocytes

Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2–induced elimination of p27Kip1, a CDK inhibitor, and resulted in the retention of high levels of p27Kip1 in IL-2/PHA-L–treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27Kip1 inhibitory activity.

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Cellular Ras and Cyclin D1 Are Required during Different Cell Cycle Periods in Cycling NIH 3T3 Cells

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4623 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4623-4632 ◽

Cited By ~ 50

Author(s):

Masahiro Hitomi ◽

Dennis W. Stacey

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Cyclin D1 ◽

Cell Cycle Progression ◽

Time Lapse ◽

S Phase ◽

3T3 Cells ◽

Cycle Progression ◽

Nih 3T3 ◽

Nih 3T3 Cells

ABSTRACT Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.

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Phosphorylation-induced unfolding regulates p19INK4dduring the human cell cycle

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1719774115 ◽

2018 ◽

Vol 115 (13) ◽

pp. 3344-3349 ◽

Cited By ~ 9

Author(s):

Amit Kumar ◽

Mohanraj Gopalswamy ◽

Annika Wolf ◽

David J. Brockwell ◽

Mechthild Hatzfeld ◽

...

Keyword(s):

Cell Cycle ◽

Structural Biology ◽

Cell Cycle Progression ◽

S Phase ◽

Cycle Progression ◽

Cyclin Dependent Kinases ◽

Ankyrin Repeat Protein ◽

Dependent Protein ◽

Local Unfolding ◽

Molecular Mode

Cell cycle progression is tightly regulated by cyclin-dependent kinases (CDKs). The ankyrin-repeat protein p19INK4dfunctions as a key regulator of G1/S transition; however, its molecular mode of action is unknown. Here, we combine cell and structural biology methods to unravel the mechanism by which p19INK4dcontrols cell cycle progression. We delineate how the stepwise phosphorylation of p19INK4dSer66 and Ser76 by cell cycle-independent (p38) and -dependent protein kinases (CDK1), respectively, leads to local unfolding of the three N-terminal ankyrin repeats of p19INK4d. This dissociates the CDK6–p19INK4dinhibitory complex and, thereby, activates CDK6. CDK6 triggers entry into S-phase, whereas p19INK4dis ubiquitinated and degraded. Our findings reveal how signaling-dependent p19INK4dunfolding contributes to the irreversibility of G1/S transition.

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Growth Characteristics of Anaerobically Treated Early and Late S-Period of Ehrlich Ascites Tumor Cells after Reaeration

Zeitschrift für Naturforschung C ◽

10.1515/znc-1983-3-426 ◽

1983 ◽

Vol 38 (3-4) ◽

pp. 313-318 ◽

Cited By ~ 6

Author(s):

Rainer Merz ◽

Friedhelm Schneider

Keyword(s):

Cell Cycle ◽

Flow Cytometry ◽

Cell Cycle Progression ◽

S Phase ◽

Ehrlich Ascites Tumor Cells ◽

Ascites Tumor ◽

Cycle Progression ◽

Ehrlich Ascites ◽

Main Fraction ◽

S Period

Utilizing centrifugal elutriation, early and late S-phase cells were separated from 4, 8 and 12 h anaerobically cultured Ehrlich Ascites tumor cells strain Karzel. The cytokinetic properties of these fractions after reaeration were studied by flow cytometry and the BrdU-H 33258-technique of flow cytometry. After a 4 h period of anaerobiosis, growth of early S-phase cells is not changed, 8 h deprivation of oxygen causes a delay of cell cycle progression, while the main fraction of 12 h anaerobically treated early S-populations did not divide after reaeration within 24 h. In comparison to early S-phase cells the cell cycle progression of the main fraction of late S-period is accelerated after a 4 h exclusion of oxygen. A fraction of 8 h anaerobically pretreated late S-cells continues to cycle, but a considerable number reinitiates DNA synthesis without preceeding division. Cells with DNA content up to 8 c are detected by flow cytometry. 12 h anaerobically cultured late S-cells do not divide after reaeration, a large number of these cells starts again to synthesize DNA. A considerable part of tetraploid cells retain viability, divide and enter a new cell cycle, another part of the cells disintegrates

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