Redox regulation of cell proliferation: Bioinformatics and redox proteomics approaches to identify redox-sensitive cell cycle regulators

Abstract Abstract 1238 PF4 (CXCL4), a platelet specific chemokine released in large amounts from activated platelet α -granules, is a negative regulator of megakaryopoiesis. In mouse studies, we have shown that PF4 levels regulate steady-state platelet count and impact chemotherapy and radiation-induced thrombocytopenia. In a clinical study in leukemia patients, we found that PF4 levels were inversely related to steady-state platelet count and to recovery after chemotherapy. The molecular basis for the effect of PF4 in megakaryopoiesis is largely unknown. Our studies in cell models suggested that PF4 might act through the cell surface receptor low-density lipoprotein related protein-1 (LRP1). Using an early megakaryoblastic cell line, which expresses LRP1, Meg-like cell line (Meg01), we show that PF4 exerts an anti-proliferative effect on the cells through inactivation of cell cycle regulators CDC2 (CDK1) and CDK2. PF4 treatment (200 μg/ml for 48 hrs) of Meg01 cells induced a decrease in cells in G1 (from 68% of cells to 51%, p=0.001) with a concurrent increase in the percentage of cells in S (12% of cells to 21%, p = 0.02 for no PF4 vs. PF4 treatment) and G2 (from 20% to 28% of cells) phase, without significant bromodeoxyuridine (BrdU) incorporation by the cells in the S phase, suggesting that PF4 causes a cell cycle arrest resulting in decreased cell proliferation. The cell cycle arrest and lack of BrDU incorporation was confirmed in primary murine Megs. No apoptosis was detected in PF4 treated Meg01 or primary cells. To determine the molecular mechanisms by which PF4 causes cell cycle arrest, we used Western blots interrogating cell cycle proteins. We detected a transient increase in the inhibitory phosphorylation (at Tyr15) of CDC2 after PF4 treatment, as well as a decrease in phosphorylation of the activating site (Thr160) on CDK2. In addition, we found PF4 treatment resulted in the degradation of Cdc25c, the upstream phosphatase of Tyr15 of CDC2. In primary murine Megs, we detected a significant decrease of total CDC2, biologically equivalent to the CDC2 inactivation seen in Meg01 cells. The CDK inhibitor Roscovitine inhibited Meg01 cell proliferation and had minimum additive effect with PF4. Overexpression of the constitutively active CDC2 mutant CDC2AF with the inhibitory phosphorylation sites Thr14 and Tyr15 replaced by Ala and Phe, respectively, desensitized the cells to PF4 treatment. These results suggested that PF4 inhibits megakaryopoiesis by decreasing the proliferation of megakaryocytes in their early developmental stage by inactivating cell cycle regulators CDC2 and CDK2. Unraveling the mechanisms by which PF4 inhibits megakaryopoiesis may lead to the development of novel therapeutics to regulate platelet counts. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Cyclin G1 inhibits the proliferation of mouse endometrial stromal cell in decidualization

Archives of Biological Sciences ◽

10.2298/abs160124080x ◽

2017 ◽

Vol 69 (1) ◽

pp. 71-81

Author(s):

Qian Xu ◽

Dong-zhi Yuan ◽

Sheng Zhang ◽

Ting Qu ◽

Shi-mao Zhang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Stromal Cells ◽

Stromal Cell ◽

Embryo Implantation ◽

Negative Regulator ◽

Cell Cycle Regulators ◽

Cyclin G1 ◽

Decidual Cells

Uterine stromal cell decidualization is a dynamic physiological process in which cell proliferation, differentiation and apoptosis are orchestrated and occur in a temporal and cell-specific manner. This process is important for successful embryo implantation. Many cell-cycle regulators are involved in decidualization. The protein cyclin G1 is a unique regulator of the cell cycle with dual functions in cell proliferation. It was reported that cyclin G1 is expressed in mouse uterine stromal cells during the period of peri-implantation. To prove the function of cyclin G1 in mouse uterine stromal cells during this period, immunohistochemistry was used to stain mouse uterine tissues on days 4-8 of pregnancy. The results showed obvious spatial and temporal expression of cyclin G1 in uterine stromal cells, and that it is expressed in the cells of the primary decidual zone (PDZ) on day 5 and secondary decidual zone (SDZ) on days 6 and 7, when the stromal cells experienced active proliferation and differentiation was initiated. Applying the decidualization model of cultured primary stromal cells in vitro, we further revealed that the expression of cyclin G1 is associated with decidualization of stromal cells induced by medroxyprogesterone acetate (MPA) and estradiol-17? (E2). RNA interference was used for the knockdown of cyclin G1 in the induced decidual cells. Flow cytometry analysis indicated that the proportion of cells in the S stage was increased, and decreased in the G2/M phase. Our study indicates that cyclin G1, as a negative regulator of the cell cycle, plays an important role in the process of decidualization in mouse uterine stromal cells by inhibiting cell-cycle progression.

Download Full-text

Molecular and Functional Effects of the Novel MEK Inhibitor PD0325901 in Preclinical Models of Human Leukemias.

Blood ◽

10.1182/blood.v108.11.254.254 ◽

2006 ◽

Vol 108 (11) ◽

pp. 254-254

Author(s):

Michele Milella ◽

Maria Rosaria Ricciardi ◽

Chiara Gregorj ◽

Fabiana De Cave ◽

Steven L. Abrams ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Cell Line ◽

Cell Lines ◽

Therapeutic Target ◽

Hematological Malignancies ◽

Mek Inhibitor ◽

Sensitive Cell ◽

Fold Reduction

Abstract The Raf/MEK/ERK signaling module plays a pivotal role in the regulation of cell proliferation, survival, and differentiation. Our group, among others, has recently demonstrated that this pathway is frequently dysregulated in hematological malignancies and may constitute an attractive therapeutic target, particularly in AML. Here we investigated the effects of PD0325901, a novel MEK inhibitor, on phospho-protein expression, gene expression profiles, cell proliferation, and apoptosis in cell line models of AML, ALL, multiple myeloma (MM), ex vivo-cultured primary AML blasts, and oncogene-transformed hematopoietic cells. AML cell lines (OCI-AML2, OCI-AML3, HL-60) were strikingly sensitive to PD0325901 (IC50: 5–19 nM), NB4 (APL) and U266 (MM) showed intermediate sensitivity (IC50: 822 and 724 nM), while all the lymphoid cell lines tested and the myeloid cell lines U937 and KG1 were resistant (IC50 > 1000 nM). Cell growth inhibition was due to inhibition of cell cycle progression and induction of apoptosis. A statistically significant reduction in the proportion of S-phase cells (p=0.01) and increase in the percentage of apoptotic cells (p=0.019) was also observed in 18 primary AML samples in response to 100 nM PD0325901. Analysis of the correlation between sensitivity/resistance to PD0325901 and Ras/Raf mutation status is currently ongoing. PD0325901 effects were also examined in a panel of IL-3-dependent murine myeloid FDC-P1 cell lines transformed to grow in response to 11 different oncogenes in the absence of IL-3. Fms-, Ras-, Raf-1-, B-Raf-, MEK1-, IGF-1R-, and STAT5a-transformed FDC-P1 cells were very sensitive to PD0325901 (IC50: ~ 1 nM), while A-Raf-, BCR-ABL-, EGFR- or Src-transformed cells were 10 to 100 fold less sensitive (IC50: 10 to 100 nM); the parental, IL-3 dependent FDC-P1 cell line had an IC50 > 1000 nM. Analysis of the phosphorylation levels of 18 different target proteins after treatment with 10 nM PD0325901 showed a 5- to 8-fold reduction in ERK-1/2, observed only in sensitive cell lines, and a 2-fold reduction in JNK and STAT3 phosphorylation. PD0325901 (10 nM) treatment also profoundly altered the gene expression profile of the sensitive cell line OCI-AML3: 96 genes were modulated after 24 h (37 up- and 59 down-regulated), most of which involved in cell cycle regulation. Changes in cyclin D1 and D3, cyclin E, and cdc 25A were also validated at the protein level. Overall, PD0325901 shows potent growth-inhibitory and pro-apoptotic activity, indicating that MEK may be an appropriate therapeutic target in an array of different hematological malignancies. Further preclinical/clinical development of this compound is warranted, particularly in myeloid leukemias.

Download Full-text

Obatoclax, a BH3 Mimetic, Enhances Cisplatin-Induced Apoptosis and Decreases the Clonogenicity of Muscle Invasive Bladder Cancer Cells via Mechanisms That Involve the Inhibition of Pro-Survival Molecules as Well as Cell Cycle Regulators

International Journal of Molecular Sciences ◽

10.3390/ijms20061285 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1285 ◽

Cited By ~ 2

Author(s):

Thomas Steele ◽

George Talbott ◽

Anhao Sam ◽

Clifford Tepper ◽

Paramita Ghosh ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Bladder Cancer ◽

Invasive Bladder Cancer ◽

Dependent Manner ◽

Cell Cycle Regulators ◽

Muscle Invasive Bladder Cancer ◽

Expression Levels ◽

Muscle Invasive ◽

Bh3 Mimetic

Several studies by our group and others have determined that expression levels of Bcl-2 and/or Bcl-xL, pro-survival molecules which are associated with chemoresistance, are elevated in patients with muscle invasive bladder cancer (MI-BC). The goal of this study was to determine whether combining Obatoclax, a BH3 mimetic which inhibits pro-survival Bcl-2 family members, can improve responses to cisplatin chemotherapy, the standard of care treatment for MI-BC. Three MI-BC cell lines (T24, TCCSuP, 5637) were treated with Obatoclax alone or in combination with cisplatin and/or pre-miR-34a, a molecule which we have previously shown to inhibit MI-BC cell proliferation via decreasing Cdk6 expression. Proliferation, clonogenic, and apoptosis assays confirmed that Obatoclax can decrease cell proliferation and promote apoptosis in a dose-dependent manner. Combination treatment experiments identified Obatoclax + cisplatin as the most effective treatment. Immunoprecipitation and Western analyses indicate that, in addition to being able to inhibit Bcl-2 and Bcl-xL, Obatoclax can also decrease cyclin D1 and Cdk4/6 expression levels. This has not previously been reported. The combined data demonstrate that Obatoclax can inhibit cell proliferation, promote apoptosis, and significantly enhance the effectiveness of cisplatin in MI-BC cells via mechanisms that likely involve the inhibition of both pro-survival molecules and cell cycle regulators.

Download Full-text

Cell cycle regulatory control for uterine stromal cell decidualization in implantation

Reproduction ◽

10.1530/rep-08-0539 ◽

2009 ◽

Vol 137 (6) ◽

pp. 889-899 ◽

Cited By ~ 67

Author(s):

Sanjoy K Das

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Stromal Cell ◽

Molecular Mechanisms ◽

Embryo Implantation ◽

Regulatory Control ◽

Cell Cycle Regulators ◽

Cell Proliferation And Differentiation ◽

Decidual Cells ◽

Uterine Stromal Cell

Uterine stromal cell decidualization is integral to successful embryo implantation, which is a gateway to pregnancy establishment. This process is characterized by stromal cell proliferation and differentiation into decidual cells with polyploidy. The molecular mechanisms that are involved in these events remain poorly understood. The current concept is that locally induced factors with the onset of implantation influence uterine stromal cell proliferation and/or differentiation through modulation of core cell cycle regulators. This review will aim to address the currently available knowledge on interaction between growth factor/homeobox and cell cycle regulatory signaling in the progression of various aspects of decidualization.

Download Full-text

Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

Cell Cycle ◽

10.1080/15384101.2015.1120925 ◽

2016 ◽

Vol 15 (2) ◽

pp. 196-212 ◽

Cited By ~ 198

Author(s):

Suzan Ruijtenberg ◽

Sander van den Heuvel

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cell Proliferation ◽

Specific Gene ◽

Cell Cycle Regulators ◽

Cell Type ◽

Specific Gene Expression ◽

Cell Proliferation And Differentiation ◽

Proliferation And Differentiation ◽

Cell Type Specific

Download Full-text

The miRNA bantam regulates growth and tumorigenesis by repressing the cell cycle regulator tribbles

Life Science Alliance ◽

10.26508/lsa.201900381 ◽

2019 ◽

Vol 2 (4) ◽

pp. e201900381 ◽

Cited By ~ 5

Author(s):

Stephan U Gerlach ◽

Moritz Sander ◽

Shilin Song ◽

Héctor Herranz

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Target Genes ◽

Hippo Pathway ◽

Growth Control ◽

Tissue Growth ◽

Tumor Formation ◽

Hippo Signaling ◽

Cell Cycle Regulators ◽

The Hippo Pathway

One of the fundamental issues in biology is understanding how organ size is controlled. Tissue growth has to be carefully regulated to generate well-functioning organs, and defects in growth control can result in tumor formation. The Hippo signaling pathway is a universal growth regulator and has been implicated in cancer. In Drosophila, the Hippo pathway acts through the miRNA bantam to regulate cell proliferation and apoptosis. Even though the bantam targets regulating apoptosis have been determined, the target genes controlling proliferation have not been identified thus far. In this study, we identify the gene tribbles as a direct bantam target gene. Tribbles limits cell proliferation by suppressing G2/M transition. We show that tribbles regulation by bantam is central in controlling tissue growth and tumorigenesis. We expand our study to other cell cycle regulators and show that deregulated G2/M transition can collaborate with oncogene activation driving tumor formation.

Download Full-text

Effect of selinexor (KPT-330), a novel oral selective inhibitor of nuclear export (SINE), on tumor suppressors and cell cycle proteins in prostate cancer cells and regression of castration-resistant patient-derived xenograft tumor growth.

Journal of Clinical Oncology ◽

10.1200/jco.2015.33.7_suppl.277 ◽

2015 ◽

Vol 33 (7_suppl) ◽

pp. 277-277

Author(s):

Sankar N. Maity ◽

Guanglin Wu ◽

Jing-Fang Lu ◽

Anh Hoang ◽

Yosef Landesman ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Cycle ◽

Cell Proliferation ◽

Tumor Suppressors ◽

Nuclear Export ◽

Small Cell ◽

Nuclear Accumulation ◽

Cell Cycle Regulators ◽

Castration Resistant ◽

Cargo Proteins

277 Background: Androgen deprivation, anti-androgen and androgen biosynthesis inhibitor treatment can initially control the metastatic prostate cancer (PCa), but treatment-refractory progression frequently follows, with the loss of tumor suppressors (TSPs) and increased expression of cell cycle proteins. Inhibition of the nuclear export protein, Exportin 1 (XPO1) leads to nuclear accumulation of cargo proteins such as TSPs & cell-cycle regulators implicated in castration-resistant PCa (CRPC) progression. XPO1 and specific cargo genes are overexpressed in metastatic CRPC relative to benign & primary prostate tumors, implicating XPO1 activity as playing a role in disease progression. Selinexor (KPT-330), a novel, oral SINE currently in Phase 1/2 for both hematological and solid tumors, has potent activity against CRPC. We hypothesized this activity is due selinexor induced nuclear expression of TSPs. Methods: To test this hypothesis, we treated selected PCa cell lines and patient-derived xenografts (PDXs, two adenocarcinomas and one small cell carcinonoma) with selinexor to determine the effect on survival and cargo protein localization. Results: Treatment with selinexor markedly inhibited PCa cell proliferation in vitro, activated the tumor suppressor TP53 & inhibited cell-cycle regulators. Also, treatment of the PDXs with selinexor for at least 3 weeks significantly inhibited tumor growth & reduced the prostate-specific antigen level in the adenocarcinomas. Selinexor increased cell death in all three PDX tumors and reduced cell proliferation in the adenocarcinomas, but not in the small-cell tumor. Expression analyses demonstrated that selinexor induced nuclear accumulation of different cargo proteins unique to the PCa model, accounting for PDX-specific regression. Conclusions: These results point to an anti-tumorigenic effect of selinexor treatment across a spectrum of hormone-refractory PCa that may provide insight into the drivers of PCa treatment resistance and heterogeneity.

Download Full-text