Growth, differentiation, and malignant transformation of pre-B cells mediated by inducible activation of v-Abl oncogene

The nonreceptor tyrosine kinase, encoded by the v-Abl oncogene of Abelson murine leukemia virus induces transformation of progenitor B cells. The v-Abl oncogene promotes cell cycle progression and inhibits pre-B cell differentiation. The temperature-sensitive form of Abelson murine leukemia virus offers a reversible model to study the role of v-Abl in regulating growth and differentiation. Inactivation of v-Abl elevates p27 and Foxo3a levels and activates NF-kappaB/Rel, which leads to G1 arrest and induction of Ig L chain gene rearrangement, respectively. In turn, v-Abl reactivation reduces p27 and Foxo3a levels, thus permitting G1-arrested cells to reenter the cell cycle. However, the cell lines derived from SCID mice that are defective in the catalytic subunit of DNA-dependent protein kinase retain elevated levels of p27 and Foxo3a proteins despite reactivation of v-Abl. Consequently, these cells are locked in the G1 phase for an extended period of time. The few cells that manage to bypass the G1 arrest become tumorigenic and fail to undergo pre-B cell differentiation induced by v-Abl inactivation. Deregulation of p27, Foxo3a, c-myc, and NF-kappaB/Rel was found to be associated with the malignant transformation of SCID temperature-sensitive form of Abelson murine leukemia virus pre-B cells.     

Negative regulation of HDM2 to attenuate p53 degradation by ribosomal protein L26

HDM2 is a p53-specific E3 ubiquitin ligase. Its overexpression leads to excessive inactivation of tumor protein p53, diminishing its tumor suppressor function. HDM2 also affects the cell cycle, apoptosis and tumorigenesis through interacting with other molecules, including several ribosomal proteins. To identify novel HDM2 regulators, we performed a yeast two-hybrid screening using HDM2 as bait. Among the candidates, ribosomal protein L26 (RPL26) was characterized as a novel HDM2-interactor. The interaction between HDM2 and RPL26 was further validated by in vivo and in vitro assays. RPL26 modulates the HDM2–p53 interaction by forming a ternary complex among RPL26, HDM2 and p53, which stabilize p53 through inhibiting the ubiquitin ligase activity of HDM2. The ribosomal stress caused by a low dose of Act D enhances RPL26–HDM2 interaction and activates p53. Overexpression of RPL26 results in activating of p53, inhibits cell proliferation and induces a p53-dependent cell cycle arrest. These results provide a novel regulatory mechanism of RPL26 to activate p53 by inhibiting HDM2.     

SUMO-Specific Protease 2 Is Essential for Modulating p53-Mdm2 in Development of Trophoblast Stem Cell Niches and Lineages

SUMO-specific protease 2 (SENP2) modifies proteins by removing SUMO from its substrates. Although SUMO-specific proteases are known to reverse sumoylation in many defined systems, their importance in mammalian development and pathogenesis remains largely elusive. Here we report that SENP2 is highly expressed in trophoblast cells that are required for placentation. Targeted disruption of SENP2 in mice reveals its essential role in development of all three trophoblast layers. The mutation causes a deficiency in cell cycle progression. SENP2 has a specific role in the G–S transition, which is required for mitotic and endoreduplication cell cycles in trophoblast proliferation and differentiation, respectively. SENP2 ablation disturbs the p53–Mdm2 pathway, affecting the expansion of trophoblast progenitors and their maturation. Reintroducing SENP2 into the mutants can reduce the sumoylation of Mdm2, diminish the p53 level and promote trophoblast development. Furthermore, downregulation of p53 alleviates the SENP2-null phenotypes and stimulation of p53 causes abnormalities in trophoblast proliferation and differentiation, resembling those of the SENP2 mutants. Our data reveal a key genetic pathway, SENP2–Mdm2–p53, underlying trophoblast lineage development, suggesting its pivotal role in cell cycle progression of mitosis and endoreduplication.     

High abundance of CDC45 inhibits cell proliferation through elevation of HSPA6

ObjectivesCDC45 is the core component of CMG (CDC45‐MCMs‐GINS) complex that plays important role in the initial step of DNA replication in eukaryotic cells. The expression level of cdc45 is under the critical control for the accurate cell cycle progression. Loss‐of‐function of cdc45 has been demonstrated to inhibit cell proliferation and leads to cell death due to the inhibition of DNA replication and G1‐phase arrest. An increasing of CDC45 inhibits cell proliferation as well. Nevertheless, a systematic analysis of the effect of high dose of CDC45 on cell physiology and behaviors is unclear. In the present study, we aimed to investigate the effects and mechanisms of high dose of CDC45 on cell behaviors.Materials and MethodsWe overexpressed cdc45 in cultured cell lines, Ciona and Drosophila embryos, respectively. The cell cycle progression was examined by the BrdU incorporation experiment, flow cytometry and PH3 (phospho‐Histone 3) staining. RNA‐sequencing analysis and qRT‐PCR were carried out to screen the affected genes in HeLa cells overexpressing cdc45. siRNA‐mediated knockdown was performed to investigate gene functions in HeLa cells overexpressing cdc45.ResultsWe found that high level of cdc45 from different species (human, mammal, ascidian, and Drosophila) inhibited cell cycle in vitro and in vivo. High dose of CDC45 blocks cells entering into S phase. However, we failed to detect DNA damage and cell apoptosis. We identified hspa6 was the most upregulated gene in HeLa cells overexpressing cdc45 via RNA‐seq analysis and qRT‐PCR validation. Overexpression of Hs‐hspa6 inhibited proliferation rate and DNA replication in HeLa cells, mimicking the phenotype of cdc45 overexpression. RNAi against hspa6 partially rescued the cell proliferation defect caused by high dose of CDC45.ConclusionsOur study suggests that high abundance of CDC45 stops cell cycle. Instead of inducing apoptosis, excessive CDC45 prevents cell entering S phase probably due to promoting hspa6 expression.

CDC45 is essential for DNA replication. Surprisingly high dose of CDC45 inhibits cell proliferation and blocks cell entering S phase without inducing apoptosis nor aneuploidy as expected. The overexpressed CDC45 induces the elevation of HSPA6, which in turn inhibits cell proliferation.     

MicroRNA-149 Inhibits Proliferation and Cell Cycle Progression through the Targeting of ZBTB2 in Human Gastric Cancer

An increasing body of evidence indicates that miR-149 can both suppress and promote tumor growth depending on the tumor type. However, the role of miR-149 in the progression of gastric cancer (GC) remains unknown. Here we report that miR-149 is a tumor suppressor in human gastric cancer. miR-149 expression is decreased in GC cell lines and clinical specimens in comparison to normal gastric epithelial cell and tissues, respectively. The expression levels of miR-149 also correlate with the differentiation degree of GC cells and tissues. Moreover, ectopic expression of miR-149 in gastric cancer cells inhibits proliferation and cell cycle progression by down-regulating ZBTB2, a potent repressor of the ARF-HDM2-p53-p21 pathway, with a potential binding site for miR-149 in its mRNA''s 3′UTR. It is also found that ZBTB2 expression increases in GC cells and tissues compared to normal gastric epithelial cell and tissues, respectively. Silencing of ZBTB2 leads to suppression of cell growth and cell cycle arrest in G0/G1 phase, indicating that ZBTB2 may act as an oncogene in GC. Furthermore, transfection of miR-149 mimics into gastric cancer cells induces down-regulation of ZBTB2 and HDM2, and up-regulation of ARF, p53, and p21 compared to the controls. In summary, our data suggest that miR-149 functions as a tumor suppressor in human gastric cancer by, at least partially through, targeting ZBTB2.     

Activation of GPER Induces Differentiation and Inhibition of Coronary Artery Smooth Muscle Cell Proliferation

Background

Vascular pathology and dysfunction are direct life-threatening outcomes resulting from atherosclerosis or vascular injury, which are primarily attributed to contractile smooth muscle cells (SMCs) dedifferentiation and proliferation by re-entering cell cycle. Increasing evidence suggests potent protective effects of G-protein coupled estrogen receptor 1 (GPER) activation against cardiovascular diseases. However, the mechanism underlying GPER function remains poorly understood, especially if it plays a potential role in modulating coronary artery smooth muscle cells (CASMCs).

Methodology/Principal Findings

The objective of our study was to understand the functional role of GPER in CASMC proliferation and differentiation in coronary arteries using from humans and swine models. We found that the GPER agonist, G-1, inhibited both human and porcine CASMC proliferation in a concentration- (10⁻⁸ to 10⁻⁵ M) and time-dependent manner. Flow cytometry revealed that treatment with G-1 significantly decreased the proportion of S-phase and G2/M cells in the growing cell population, suggesting that G-1 inhibits cell proliferation by slowing progression of the cell cycle. Further, G-1-induced cell cycle retardation was associated with decreased expression of cyclin B, up-regulation of cyclin D1, and concomitant induction of p21, and partially mediated by suppressed ERK1/2 and Akt pathways. In addition, G-1 induces SMC differentiation evidenced by increased α-smooth muscle actin (α-actin) and smooth muscle protein 22α (SM22α) protein expressions and inhibits CASMC migration induced by growth medium.

Conclusion

GPER activation inhibits CASMC proliferation by suppressing cell cycle progression via inhibition of ERK1/2 and Akt phosphorylation. GPER may constitute a novel mechanism to suppress intimal migration and/or synthetic phenotype of VSMC.     

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal

Differentiation of most cell types requires both establishment of G₁ arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21^–/– cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21^–/– cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat–p16^Ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.     

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.     

MiR-24 Tumor Suppressor Activity Is Regulated Independent of p53 and through a Target Site Polymorphism

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3′ UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.     

Eupatorin-Induced Cell Death in Human Leukemia Cells Is Dependent on Caspases and Activates the Mitogen-Activated Protein Kinase Pathway

Eupatorin is a naturally occurring flavone that inhibits cell proliferation in human tumor cells. Here we demonstrate that eupatorin arrests cells at the G₂-M phase of the cell cycle and induces apoptotic cell death involving activation of multiple caspases, mitochondrial release of cytochrome c and poly(ADP-ribose) polymerase cleavage in human leukemia cells. This flavonoid induced the phosphorylation of members of the mitogen-activated protein kinases and cell death was attenuated by inhibition of c-jun N-terminal kinases/stress activated protein kinases. Eupatorin-induced cell death is mediated by both the extrinsic and the intrinsic apoptotic pathways and through a mechanism dependent on reactive oxygen species generation.     

The cyclin-dependent kinase inhibitor p21 (WAF1) is required for survival of differentiating neuroblastoma cells.

We are employing recent advances in the understanding of the cell cycle to study the inverse relationship between proliferation and neuronal differentiation. Nerve growth factor and aphidicolin, an inhibitor of DNA polymerases, synergistically induce neuronal differentiation of SH-SY5Y neuroblastoma cells and the expression of p21WAF1, an inhibitor of cyclin-dependent kinases. The differentiated cells continue to express p21WAF1, even after removal of aphidicolin from the culture medium. The p21WAF1 protein coimmunoprecipitates with cyclin E and inhibits cyclin E-associated protein kinase activity. Each of three antisense oligonucleotides complementary to p21WAF1 mRNA partially blocks expression of p21WAF1 and promotes programmed cell death. These data indicate that p21WAF1 expression is required for survival of these differentiating neuroblastoma cells. Thus, the problem of neuronal differentiation can now be understood in the context of negative regulators of the cell cycle.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

Inhibition of the NAD-Dependent Protein Deacetylase SIRT2 Induces Granulocytic Differentiation in Human Leukemia Cells

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.     

Cell cycle arrest and reversion of Ras-induced transformation by a conditionally activated form of mitogen-activated protein kinase kinase kinase 3

Signal-induced proliferation, differentiation, or stress responses of cells depend on mitogen-activated protein kinase (MAPK) cascades, the core modules of which consist of members of three successively acting kinase families (MAPK kinase kinase [MAP3K], MAPK kinase, and MAPK). It is demonstrated here that the MEKK3 kinase inhibits cell proliferation, a biologic response not commonly associated with members of the MAP3K family of kinases. A conditionally activated form of MEKK3 stably expressed in fibroblasts arrests these cells in early G1. MEKK3 critically blocks mitogen-driven expression of cyclin D1, a cyclin which is essential for progression of fibroblasts through G1. The MEKK3-induced block of cyclin D1 expression and of cell cycle progression may be mediated via p38 MAPK, a downstream effector of MEKK3. The MEKK3-mediated block of proliferation also reverses Ras-induced cellular transformation, suggesting possible tumor-suppressing functions for this kinase. Together, these results suggest an involvement of the MEKK3 kinase in negative regulation of cell cycle progression, and they provide the first insights into biologic activities of this kinase.     

Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Autologous c-kit⁺ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G₁ phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G₂/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.