scholarly journals The lncRNA TEX41 is upregulated in pediatric B-Cells Acute Lymphoblastic Leukemia and it is necessary for leukemic cell growth

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Francesca Maria Orlandella ◽  
Giovanni Smaldone ◽  
Giuliana Salvatore ◽  
Luigi Vitagliano ◽  
Alessandra Cianflone ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Leukemic Cell ◽  
Cycle Progression ◽  
Total Rna ◽  
Expression Levels

Abstract Background Long non-coding RNAs (lncRNAs) represent a diverse class of RNAs involved in the regulation of various physiological and pathological cellular processes, including transcription, intracellular trafficking, and chromosome remodeling. LncRNAs deregulation was linked to the development and progression of various cancer types, such as acute leukemias. In this context, lncRNAs were also evaluated as a novel class of biomarkers for cancer diagnosis and prognosis. Here, we analyzed TEX41 in childhood B cell acute lymphoid leukemia (B-ALL). Methods Total RNA was extracted from pediatric B-ALL patients (at diagnosis and after induction of therapy) and from healthy subjects. Total RNA was also extracted from different leukemia cell line models. The expression level of TEX41 was evaluated by q-RT-PCR. Also, the dataset deposited by St. Jude Children’s Research Hospital was consulted. Furthermore, the silencing of TEX41 in RS4;11 cell line was obtained by 2′-Deoxy, 2′Fluroarabino Nucleic Acids (2′F-ANAs) Oligonucleotides, and the effect on cell proliferation was evaluated. Cell cycle progression and its regulators were analyzed by flow cytometry and immunoblotting. Results We exploited the St Jude Cloud database and found that TEX41 is a lncRNA primarily expressed in the case of B-ALL (n = 79) while its expression levels are low/absent for T-cell ALL (n = 25) and acute myeloid leukemia (n = 38). The association of TEX41 with B-ALL was confirmed by real-time PCR assays. TEX41 disclosed increased expression levels in bone marrow from patients with B-ALL at diagnosis, while its expression levels became low or absent when retested in Bone Marrow cells of the same patient after 1 month of induction therapy. Also, silencing experiments performed on RS4;11 cells showed that TEX41 downregulation impaired in vitro leukemic cell growth determining their arrest in the G2-M phase and the deregulation of cell cycle proteins. Conclusions Our findings highlight that TEX41 is an upregulated lncRNA in the case of B-ALL and this feature makes it a novel potential biomarker for the diagnosis of this leukemia subtype in pediatric patients. Finally, TEX41 expression seems to be critical for leukemic proliferation, indeed, silencing experiments targeting TEX41 mRNA in the RS4;11 cell line hampered in vitro cell growth and cell cycle progression, by inducing G2-M arrest as confirmed propidium iodide staining and by the upregulation of p53 and p21 proteins.

scholarly journals The in vitro effects of compound-X on growth, morphology, the induction of autophagy and apoptosis, as well as cell cycle progression in a cervical adenocarcinoma cell line

2012 ◽  
Vol 31 (1) ◽  
Author(s):  
S. Marais ◽  
T.V. Mqoco ◽  
B.A. Stander ◽  
R. Prudent ◽  
L. Lafanechère ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Hela Cells ◽  
Cell Cycle Progression ◽  
Cervical Adenocarcinoma ◽  
Growth Morphology ◽  
Adenocarcinoma Cell Line ◽  
Cycle Progression ◽  
Adenocarcinoma Cell

It can be concluded that compound-X induced both autophagy and apoptosis as a means of celldeath in HeLa cells.

Distinct and sequential upregulation of genes regulating cell growth and cell cycle progression during hepatic ischemia-reperfusion injury

2005 ◽  
Vol 289 (4) ◽  
pp. C826-C835 ◽  
Author(s):  
Sharon Barone ◽  
Tomohisa Okaya ◽  
Steve Rudich ◽  
Snezana Petrovic ◽  
Kathy Tenrani ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Growth ◽  
Reperfusion Injury ◽  
Cell Cycle Progression ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Catabolic Pathway ◽  
Cycle Progression

Ischemia-reperfusion injury (IRI) in liver and other organs is manifested as an injury phase followed by recovery and resolution. Control of cell growth and proliferation is essential for recovery from the injury. We examined the expression of three related regulators of cell cycle progression in liver IRI: spermidine/spermine N-acetyltransferase (SSAT), p21 (a cyclin-dependent kinase inhibitor), and stathmin. Mice were subjected to hepatic IRI, and liver tissues were harvested at timed intervals. The expression of SSAT, the rate-limiting enzyme in the polyamine catabolic pathway, had increased fivefold 6 h after IRI and correlated with increased putrescine levels in the liver, consistent with increased SSAT enzymatic activity in IRI. The expression of p21, which is transactivated by p53, was undetectable in sham-operated animals but was heavily induced at 12 and 24 h of reperfusion and declined to undetectable baseline levels at 72 h of reperfusion. The interaction of the polyamine pathway with the p53-p21 pathway was shown in vitro, where activation of SSAT with polyamine analog or the addition of putrescine to cultured hepatocytes induced the expression of p53 and p21 and decreased cell viability. The expression of stathmin, which is under negative transcriptional regulation by p21 and controls cell proliferation and progression through mitosis, remained undetectable at 6, 12, and 24 h of reperfusion and was progressively and heavily induced at 48 and 72 h of reperfusion. Double-immunofluorescence labeling with antibodies against stathmin and PCNA, a marker of cell proliferation, demonstrated colocalization of stathmin and PCNA at 48 and 72 h of reperfusion in hepatocytes, indicating the initiation of cell proliferation. The distinct and sequential upregulation of SSAT, p21, and stathmin, along with biochemical activation of the polyamine catabolic pathway in IRI in vivo and the demonstration of p53-p21 upregulation by SSAT and putrescine in vitro, points to the important role of regulators of cell growth and cell cycle progression in the pathophysiology and/or recovery in liver IRI. The data further suggest that SSAT may play a role in the initiation of injury, whereas p21 and stathmin may be involved in the resolution and recovery after liver IRI.

PI3K/Akt/FOXO3a Pathway Contributes to Thrombopoietin-Induced Proliferation of Primary Megakaryocytes In Vitro and In Vivo Via Modulation of p27Kip1.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 202-202
Author(s):  
Takafumi Nakao ◽  
Amy E Geddis ◽  
Norma E. Fox ◽  
Kenneth Kaushansky
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Wild Type ◽  
Cycle Progression ◽  
P27kip1 Expression ◽  
Cell Counts ◽  
Deficient Mice

Abstract Thrombopoietin (TPO), the primary regulator of megakaryocyte (MK) and platelet formation, modulates the activity of multiple signal transduction molecules, including those in the Jak/STAT, p42/p44 MAPK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathways. In the previous study, we reported that PI3K and Akt are necessary for TPO-induced cell cycle progression of primary MK progenitors. The absence of PI3K activity results in a block of transition from G1 to S phase in these cells (Geddis AE et al. JBC2001276:34473–34479). However, the molecular events secondary to the activation of PI3K/Akt responsible for MK proliferation remain unclear. In this study we show that FOXO3a and its downstream target p27Kip1 play an important role in TPO-induced proliferation of MK progenitors. TPO induces phosphorylation of Akt and FOXO3a in both UT-7/TPO, a megakaryocytic cell line, and primary murine MKs in a PI3K dependent fashion. Cell cycle progression of UT-7/TPO cells is blocked in G1 phase by inhibition of PI3K. We found that TPO down-modulates p27Kip1 expression at both the mRNA and protein levels in UT-7/TPO cells and primary MKs in a PI3K dependent fashion. UT-7/TPO stably expressing constitutively active Akt or a dominant-negative form of FOXO3a failed to induce p27Kip1 expression after TPO withdrawal. Induced expression of an active form of FOXO3a resulted in increased p27Kip1 expression in this cell line. In an attempt to assess whether FOXO3a has an effect of MK proliferation in vivo, we compared the number of MKs in Foxo3a-deficient mice and in wild type controls. Although peripheral blood cell counts of erythrocytes, neutrophils, monocytes and platelets were normal in the Foxo3a-deficient mice, total nucleated marrow cell count of Foxo3a-deficient mice were 60% increased compared with wild type controls. In addition, the increase of MKs was more profound than that of total nucleated marrow cells; CD41+ MKs from Foxo3a-deficient mice increased 2.1-fold, and mature MKs with 8N and greater ploidy increased 2.5-fold, compared with wild type controls. Taken together with the previous observation that p27Kip1-deficient mice also display increased numbers of MK progenitors, our findings strongly suggest that the effect of TPO on MK proliferation is mediated by PI3K/Akt-induced FOXO3a inactivation and subsequent p27Kip1 down-regulation in vitro and in vivo.

scholarly journals A Mammalian Bromodomain Protein, Brd4, Interacts with Replication Factor C and Inhibits Progression to S Phase

2002 ◽  
Vol 22 (18) ◽  
pp. 6509-6520 ◽  
Author(s):  
Tetsuo Maruyama ◽  
Andrea Farina ◽  
Anup Dey ◽  
JaeHun Cheong ◽  
Vladimir P. Bermudez ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Cycle Progression ◽  
S Phase ◽  
Replication Factor C ◽  
Cycle Progression ◽  
Vitro Analysis ◽  
Factor C ◽  
In Vitro Analysis

ABSTRACT Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G1 upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G1 to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.

MLL-AF4 Down-Regulates CDKN1B (P27) Independent of Cell Cycle Progression.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2563-2563
Author(s):  
Zhenbiao Xia ◽  
Relja Popovic ◽  
Tara Lorenz ◽  
Donna Santillan ◽  
Frank Erfurth ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Luciferase Reporter ◽  
Cycle Progression ◽  
Downstream Target

Abstract The MLL gene, involved in many chromosomal translocations associated with acute myeloid and lymphoid leukemia, has more than forty known partner genes with which it is able to form in- frame fusions. MLL fusion genes transform hematopoietic cells in vitro, and cause leukemia in mouse models. However, the mechanism is still not clear. Characterizing important downstream target genes may provide rational therapeutic strategies for the treatment of MLL-associated leukemia. We explored potential downstream target genes of the most prevalent MLL fusion protein, MLL-AF4, which is primarily associated with pro-B ALL and is involved in the majority of infant leukemia. To this end, we developed an inducible MLL-AF4 fusion cell line. Overexpression of MLL-AF4 does not lead to increased proliferation in this cell line, but rather, cell growth is slowed compared to similar cell lines inducibly expressing truncated MLL. To try to understand the reason for slower cell growth, we assayed for expression of several CDK inhibitors. We found that in the MLL-AF4 induced cell line, the amount of CDKN1B (cyclin-dependent kinase inhibitor P27) was dramatically decreased both at the RNA and protein levels, in contrast, the levels of CDKN1A (P21) and CDKN2A (P16) were unchanged. Interestingly, we did not observe an increased percentage of cells in S phase of the cell cycle. To explore whether CDKN1B might be a direct target of MLL-AF4, we employed chromatin immunoprecipitation (ChIP) assays and luciferase reporter gene assays. We observed that MLL-AF4 binds to the CDKN1B promoter in vivo and represses CDKN1B promoter activity. Further, we confirmed CDKN1B promoter binding by ChIP assays in the MLL-AF4 leukemia cell line MV4-11. Our results suggest that the CDKN1B may be a downstream target of MLL-AF4, and that MLL-AF4 inhibits CDKN1B expression independent of cell cycle progression.

SU11657, a FLT3-Targeted Tyrosine Kinase, Has Pro-Apoptotic Activity on Leukemia Cells In Vitro.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2797-2797
Author(s):  
Tiziana Grafone ◽  
Laura Ferraretti ◽  
Emanuela Ottaviani ◽  
Manuela Mancini ◽  
Michela Palmisano ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Juxtamembrane Region ◽  
Cell Extracts ◽  
Relevant Effect ◽  
Wide Range

Abstract Fms-related tyrosine kinase3 (Flt3) is the most commonly mutated gene in human acute myeloid leukemia (AML) and has implicated in its pathogenesis. Constitutive activation of the Flt3 receptor tyrosine kinase, have been linked either by internal tandem duplication (ITD) of the juxtamembrane region or by point mutation in the second tyrosine kinase domain (TKD). To investigate the effect in vitro of SU11657, a new compound FLT3 kinase inhibitors, we analyzed human cell lines from AML patients (MV4-11 and HL60) and blast from patients AML using a wide range of concentrations (1nM-10μM) of this novel agent. In HL-60, FLT3-wt cell line, used as negative control does not show relevant effect after treatment with SU11657. Instead, in MV4-11, FLT3-ITD cell line, we observed a decrease dose-dependent in cell viability after treatment with SU11657. The effects of this compound on cell cycle progression show an accumulation of G1/S phase and an induction of apoptosis at 1-10nM concentration after 24h of treatment. First we observed a dephosphorylation of FLT3 on Tyr591 in whole cell extracts from MV4-11 cells after treatment with SU11657 100nM. We also demonstrated a hypophosphorylation of AKT on Ser473 and a consequently dephosphorylation of BAD on Ser136 at nanomolar concentration. We observed a dephosphorylation of STAT-5 to 100nM of SU11657 at 24h. We evaluated the effects of this new compound in AML primary progenitors that showed FLT3-ITD, FLt3-TKD and FLT3-wt. In the patients with mutation ITD and TKD was evident a modification of cell cycle progression with a decrease in G2/M phase and an increase of subdiploid peak. The effect of SU11657 in patients FLT3-wt was not relevant. Our study thus showed a potential therapeutic usefulness of the drug in treatment of AML. Study of signal transductions and gene profile expression will contribute to further understanding of the drug mechanisms.

Potent cytotoxicity and inhibition of pan-cell cycle progression by Alchemix, a novel alkylating anthraquinone

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 12030-12030
Author(s):  
A. A. Epenetos ◽  
K. Pors ◽  
P. J. Smith ◽  
L. H. Patterson
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Cell Cycle Progression ◽  
Topoisomerase Ii ◽  
Human Tumors ◽  
Drug Resistant ◽  
Cycle Progression ◽  
Topo Ii

12030 Introduction: DNA topoisomerase II (topo II) is crucial to the maintenance of cancer cells in a proliferative state. DNA intercalation is a crucial part of topo II inhibition by DNA affinic anthraquinones. Potent cytotoxicity of anthraquinones, is related to their slow rate of dissociation from DNA, the kinetics of which favours long-term trapping of the topo-DNA complexes. Currently available DNA interacting agents at best promote a transient inhibition of topo II, since the topo-drug-DNA ternary complex is reversed by removal of the intracellular drug pool. Results: Alchemix cell cycle events: DNA content and Cyclin B1 expression were measured using flow cytometry and a p53 functional human osteosarcoma cell line (U2-OS) The results indicate: (ii) slow pan-cell cycle progression and mitotic commitment with a limited expression of G2 arrest, (iii) B1 cyclin tracking reveals that escape from Alchemix-induced cell cycle arrest in G2 is forcing some cells to enter polyploidy via an aberrant mitosis in keeping with topoisomerase II inhibition. Alchemix in vitro activity against the NCI human cell line panel including several drug resistant cancer cell lines had a mean IG50 = 49 nM. 11 of the 24 cell lines tested have an IG50 of <10 nM. Alchemix retains potent activity against chemotherapy resistant tumors including drug resistant ones. Conclusions: Alchemix possesses potent activity across a variety of different human tumors and significantly shows potent activity in cisplatin and anthracyline resistant human tumors Alchemix has pan-cell cycle effects. Multilevel targeting by Alchemix reduces the probability of evasion of cell cycle related pharmacodynamic responses. Results help explain the activity of Alchemix in both cisplatin and anthracycline resistant tumors in vitro and in vivo. [Table: see text]

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