scholarly journals AZT on telomerase activity and cell proliferation in HS 839.T melanoma cells

2012 ◽  
Vol 27 (12) ◽  
pp. 855-860 ◽  
Author(s):  
Celestino Prospero de Souza Sobrinho ◽  
Alfredo Gragnani ◽  
Ivan Dunshee Abranches Oliveira Santos ◽  
Andrea Fernandes Oliveira ◽  
Monica Vanucci Nunes Lipay ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Enzyme Activity ◽  
Cell Lines ◽  
Telomerase Activity ◽  
Melanoma Cells ◽  
Control Group ◽  
No Inhibition

PURPOSE: To evaluate telomerase activity and proliferation of HS839.T melanoma cells, subjected to the action of AZT. METHODS: Cells were grown in triplicate, AZT at different concentrations: 50, 100 and 200μM, was added and left for 24 and 48 hours, and its effects were compared with the control group. Telomerase activity was detected by PCR and cell proliferation was evaluated by MTT. RESULTS: After 24 hours, there was no inhibition of cell proliferation or telomerase activity when compared to the control group. After 48 hours, there was a momentary decrease, suggesting that the cell lines used in this study are sensitive to AZT, but quickly recover both the enzyme activity and cell proliferation. CONCLUSION: The action of AZT on the melanoma cells studied, at the concentrations and times tested, did not inhibit telomerase activity nor affect cell proliferation.

scholarly journals Treatment with the Recombinant SLIT2 Protein Delays AML Leukemogenesis In Vivo

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2368-2368
Author(s):  
Luise A de Albuquerque Simoes ◽  
Isabel Weinhäuser ◽  
Diego A Pereira-Martins ◽  
César Alexander Ortiz Rojas ◽  
Thiago Mantello Bianco ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Cancer Progression ◽  
Functional Role ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Leukemic Cells ◽  
Control Group

Abstract Accumulating evidence suggest that the axon guidance molecules SLIT and ROBO are not only implicated in physiological process but also in cancer progression. Depending on the type of cancer the SLIT-ROBO axis can either act as a tumor suppressor gene, in which case the SLIT2 promoter site is frequently hypermethylated or as an oncogene, whereby high expression is often associated with poor prognosis. In the context of acute myeloid leukemia (AML), low expression of SLIT2 has been associated with low overall survival (OS) (Golos et al., 2019), while the functional role of SLIT2 remains largely unknown. Recently, we showed that the knockdown of SLIT2 increased cell proliferation of acute promyelocytic leukemia (APL) cells resulting in a more aggressive course of disease progression in vivo using the murine transgenic APL model (Weinhäuser et al., 2020). Here, we aimed to study the functional role of SLIT2 in a more heterogeneous disease, such as AML. Using different publicly available datasets. (GSE58477, normal karyotype blasts: 62, healthy CD34 +: 10; GSE63409, LSC: 14, HSC: 5) we detected increased methylation at the SLIT2 promoter site of AML leukemic cells compared to healthy CD34 + cells suggesting SLIT2 tumor suppressive functions. In addition, we measured decreased levels of SLIT2 in the bone marrow (BM) plasma of AML patients compared to healthy donors. To assess the biological role of SLIT2, we treated AML cell lines (KASUMI1, MV411, and MOLM13) with recombinant SLIT2 (50 ng/mL) in vitro. Administration of SLIT2 reduced AML cell growth, colony formation and induced cell cycle arrest in the G1 phase for all AML cell lines. Conversely, the knockdown of SLIT2 promoted increased THP-1 and OCI-AML3 cell proliferation. Next, we determined whether the treatment with SLIT2 could delay leukemogenesis in vivo using the AML cell line MV4-11. Engraftment was monitored by luciferase bioluminescent signal and NSGS mice were either treated with recombinant SLIT2 using a dose of 25 ng/g of body weight or vehicle (control group). SLIT2 therapy resulted in a lower disease burden, decreased leukemic infiltration in the BM and spleen, reduced spleen size, and increased OS compared to the control group (p<0.05). In conclusion, we showed that SLIT2 methylation is recurrent in AML patients and that the level of SLIT2 in the plasma of AML patients is reduced. Moreover, SLIT2 treatment appears to have a cytostatic effect on different AML cell lines delaying leukemogenesis in vivo. Overall, our study reveals the therapeutic potential of SLIT2 in hematological malignancies, which could be used as an adjuvant in the clinic. Disclosures No relevant conflicts of interest to declare.

scholarly journals Circular RNA hsa_circ_0000282 contributes to osteosarcoma cell proliferation by regulating miR-192/XIAP axis

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Houkun Li ◽  
Limin He ◽  
Yuan Tuo ◽  
Yansheng Huang ◽  
Bing Qian
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
B Cell Lymphoma ◽  
Luciferase Reporter ◽  
Circular Rnas ◽  
Control Group ◽  
Osteosarcoma Cell ◽  
Luciferase Reporter Gene ◽  
Apoptosis Protein ◽  
Gene Assay

Abstract Background Circular RNAs (circRNAs) have emerged as a novel category of non-coding RNA, which exhibit a pivotal effect on regulating gene expression and biological functions, yet how circRNAs function in osteosarcoma (OSA) still demands further investigation. This study aimed at probing into the function of hsa_circ_0000282 in OSA. Methods The expressions of circ_0000282 and miR-192 in OSA tissues and cell lines were examined by quantitative real-time polymerase chain reaction (qRT-PCR), and the correlation between the expression level of circ_0000282 and clinicopathological features of OSA patients was analyzed. The expressions of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma-2 (Bcl-2) and Bcl-2 associated X protein (Bax) in OSA cells were assayed by Western blot. The proliferation and apoptosis of OSA cells were examined by CCK-8, BrdU and flow cytometry, respectively. Bioinformatics analysis, dual-luciferase reporter gene assay and RIP experiments were employed to predict and validate the targeting relationships between circ_0000282 and miR-192, and between miR-192 and XIAP, respectively. Results Circ_0000282 was highly expressed in OSA tissues and cell lines, which represented positive correlation with Enneking stage of OSA patients and negative correlation with tumor differentiation degree. In vitro experiments confirmed that overexpression of circ_0000282 markedly facilitated OSA cell proliferation and repressed cancer cell apoptosis in comparison to control group. Besides, knockdown of circ_0000282 repressed OSA cell proliferation and promoted apoptosis. Additionally, the binding relationships between circ_0000282 and miR-192, and between miR-192 and XIAP were validated. Circ_0000282 indirectly up-regulated XIAP expression by adsorbing miR-192, thereby playing a role in promoting cancer in OSA. Conclusion Circ_0000282 was a novel oncogenic circRNA in OSA. Circ_0000282/miR-192/XIAP axis regulated OSA cell proliferation apoptosis with competitive endogenous RNA mechanism.

scholarly journals Cylindromatosis Is Required for Survival of a Subset of Melanoma Cells

2020 ◽  
Vol 28 (4) ◽  
pp. 385-398
Author(s):  
Ting La ◽  
Lei Jin ◽  
Xiao Ying Liu ◽  
Ze Hua Song ◽  
Margaret Farrelly ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Biological Significance ◽  
Melanoma Cells ◽  
Interacting Protein ◽  
Induction Of Apoptosis ◽  
Melanoma Cell Lines

The deubiquitinase cylindromatosis (CYLD) functions as a tumor suppressor inhibiting cell proliferation in many cancer types including melanoma. Here we present evidence that a proportion of melanoma cells are nonetheless addicted to CYLD for survival. The expression levels of CYLD varied widely in melanoma cell lines and melanomas in vivo, with a subset of melanoma cell lines and melanomas displaying even higher levels of CYLD than melanocyte lines and nevi, respectively. Strikingly, although short hairpin RNA (shRNA) knockdown of CYLD promoted, as anticipated, cell proliferation in some melanoma cell lines, it reduced cell viability in a fraction of melanoma cell lines with relatively high levels of CYLD expression and did not impinge on survival and proliferation in a third type of melanoma cell lines. The decrease in cell viability caused by CYLD knockdown was due to induction of apoptosis, as it was associated with activation of the caspase cascade and was abolished by treatment with a general caspase inhibitor. Mechanistic investigations demonstrated that induction of apoptosis by CYLD knockdown was caused by upregulation of receptor-interacting protein kinase 1 (RIPK1) that was associated with elevated K63-linked polyubiquitination of the protein, indicating that CYLD is critical for controlling RIPK1 expression in these cells. Of note, microRNA (miR) profiling showed that miR-99b-3p that was predicted to target the 3-untranslated region (3-UTR) of the CYLD mRNA was reduced in melanoma cell lines with high levels of CYLD compared with melanocyte lines. Further functional studies confirmed that the reduction in miR-99b-3p expression was responsible for the increased expression of CYLD in a highly cell line-specific manner. Taken together, these results reveal an unexpected role of CYLD in promoting survival of a subset of melanoma cells and uncover the heterogeneity of CYLD expression and its biological significance in melanoma.

HoxA9 Knockdown Inhibits Proliferation and Induces Cell Death in Human MLL-Rearranged Leukemias.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 734-734
Author(s):  
Jorg Faber ◽  
Andrei V. Krivtsov ◽  
Matthew C. Stubbs ◽  
Renee Wright ◽  
Marry van den Heuvel-Eibrink ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mrna Expression ◽  
Cell Lines ◽  
Hox Genes ◽  
Lentiviral Vector ◽  
Vector System ◽  
Control Group ◽  
Bioluminescent Imaging ◽  
Clinical Prognosis

Abstract Homeobox containing (Hox) genes are implicated in the regulation of normal and leukemic hematopoesis. Using gene expression profiling we and others have previously shown that HoxA9 is highly expressed in lymphoid and myeloid leukemias harboring MLL translocations and that high level HoxA9 expression is associated with poor clinical prognosis. Furthermore, HoxA9 plays variable roles in MLL-fusion induced murine leukemias. In this study we aimed to elucidate the role of aberrant HoxA9 expression in human MLL-rearranged and non-rearranged leukemia’s utilizing an shRNA mediated knockdown approach. To establish an efficient knockdown assay three different shRNA constructs targeting human HoxA9 were synthesized and stably introduced into t(9;11) MOLM14 cells utilizing a lentiviral vector system. The shRNA construct which showed highest efficiency as measured by Taqman quantitative PCR (75–80% knockdown MLL-AF9 RNA) and Western Blot analysis was used for further experiments. In MOLM-14 cells, HoxA9 directed shRNA inhibited cell proliferation starting as early as 48h after transduction as determined by MTT assay, and at 72h demonstrated a markedly increased number of apoptotic cells as measured by Annexin V staining. This effect was rescued by introducing a non-targetable exogenous HoxA9 in MOLM-14 cells. To investigate if the HoxA9 knockdown related effects are specific for MLL rearranged cells we next analyzed cell growth and viability in 17 AML/ALL cell lines (7 MLL-rearranged, 10 non rearranged) after shRNA mediated HoxA9 knockdown. Interestingly, impaired cell proliferation and induction of apoptosis was significantly higher in the MLL rearranged cell lines (mean viability: 51.88%) than in the non-rearranged cells (mean viability: 90.98%; p=0.007). Moreover, the effect was also significantly correlated with the baseline HoxA9 mRNA expression before knockdown, with the greatest effect in cell lines expressing the highest HoxA9 levels (R= 0.8, p=0.00017). These findings prompted us to further analyze the effect of HoxA9 knockdown in MLL rearranged and non-rearranged primary human AML cells (6 MLL rearranged, 6 MLL germline). Similar to our findings in cell lines, we found a significantly higher effect on cell proliferation/viability in association with the presence of an MLL translocation (p=0.005) and a significant correlation with the baseline HoxA9 mRNA expression (R= 0.8, p=0.001). Next, we assessed the in vivo effect of HoxA9 knock down by transplanting luciferase-expressing SEMK2 (t4;11) cells and subsequent bioluminescent imaging. SEMK2 cells were transduced with either HoxA9 directed or control shRNA and intravenously injected into SCID-beige mice. Reconstitution was confirmed by in vivo bioluminescent imaging. 34 days after transplantation all mice in the HoxA9 shRNA group (n=4) are still alive with no signs of leukemia whereas all mice in the control group (n=3) have succumbed. Taken together our data implicates an important role for aberrant HoxA9 expression in human MLL rearranged leukemia cells.

scholarly journals Up-Regulating ERIC by CRISPR-dCas9-VPR Inhibits Cell Proliferation and Invasion and Promotes Apoptosis in Human Bladder Cancer

2021 ◽  
Vol 8 ◽  
Author(s):  
Jiangeng Yang ◽  
An Xia ◽  
Huajie Zhang ◽  
Qi Liu ◽  
Hongke You ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Bladder Cancer ◽  
Urothelial Carcinoma ◽  
Cell Lines ◽  
Expression Patterns ◽  
Regulation Of Gene Expression ◽  
Negative Control Group ◽  
Negative Control ◽  
Control Group ◽  
Bladder Urothelial Carcinoma

LncRNAs are defined as non-coding RNAs that are longer than 200 nucleotides in length. The previous studys has shown that lncRNAs played important roles in the regulation of gene expression and were essential in mammalian development and disease processes. Inspired by the observation that lncRNAs are aberrantly expressed in tumors, we extracted RNA from Bladder urothelial carcinoma and matched histologically normal urothelium from each patient and bladder carcinoma cell lines. Then, we reversed transcribed them into cDNA.Last, we investigated the expression patterns of ERIC by the fluorescence quantitative PCR in bladder cancer tissues and cell lines. CRISPR-dCas9-VPR targeting ERIC plasmid was transfected into T24 and 5637 cells, and cells were classified into two groups: negative control (NC) and ERIC overexpression group. MTT assay, transwell assay, and flow cytometry were performed to examine changes in cell proliferation, invasiveness, and apoptosis. We found that the expression of ERIC was down-regulated in bladder urothelial carcinoma compared to matched histologically normal urotheliam. The differences of the expression of this gene were large in the bladder cancer lines. Compared with the negative control group, the ERIC overexpression group showed significantly decreased cell proliferation rate (t = 7.583, p = 0.002; t = 3.283, p = 0.03) and invasiveness (t = 11.538, p < 0.001; t = 8.205, p = 0.01); and increased apoptotic rate (t = −34.083, p < 0.001; t = −14.316, p < 0.001). Our study lays a foundation for further study of its pathogenic mechanism in bladder cancer.

scholarly journals Heterogeneity of Melanoma Cell Responses to Sleep Apnea-Derived Plasma Exosomes and to Intermittent Hypoxia

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4781
Author(s):  
Abdelnaby Khalyfa ◽  
Wojciech Trzepizur ◽  
Alex Gileles-Hillel ◽  
Zhuanhong Qiao ◽  
David Sanz-Rubio ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Intermittent Hypoxia ◽  
Melanoma Cells ◽  
Cell Proliferation And Migration ◽  
Exosomal Mirnas ◽  
And Migration ◽  
Melanoma Cell Lines ◽  
Proliferation And Migration

Obstructive sleep apnea (OSA) is associated with increased cutaneous melanoma incidence and adverse outcomes. Exosomes are secreted by most cells, and play a role in OSA-associated tumor progression and metastasis. We aimed to study the effects of plasma exosomes from OSA patients before and after adherent treatment with continuous positive airway pressure (CPAP) on melanoma cells lines, and also to identify exosomal miRNAs from melanoma cells exposed to intermittent hypoxia (IH) or normoxia. Plasma-derived exosomes were isolated from moderate-to-severe OSA patients before (V1) and after (V2) adherent CPAP treatment for one year. Exosomes were co-incubated with three3 different melanoma cell lines (CRL 1424; CRL 1619; CRL 1675) that are characterized by genotypes involving different mutations in BRAF, STK11, CDKN2A, and PTEN genes to assess the effect of exosomes on cell proliferation and migration, as well as on pAMK activity in the presence or absence of a chemical activator. Subsequently, CRL-1424 and CRL-1675 cells were exposed to intermittent hypoxia (IH) and normoxia, and exosomal miRNAs were identified followed by GO and KEG pathways and gene networks. The exosomes from these IH-exposed melanoma cells were also administered to THP1 macrophages to examine changes in M1 and M2 polarity markers. Plasma exosomes from V1 increased CRL-1424 melanoma cell proliferation and migration compared to V2, but not the other two cell lines. Exposure to CRL-1424 exosomes reduced pAMPK/tAMPK in V1 compared to V2, and treatment with AMPK activator reversed the effects. Unique exosomal miRNAs profiles were identified for CRL-1424 and CRL-1675 in IH compared to normoxia, with six miRNAs being regulated and several KEGG pathways were identified. Two M1 markers (CXCL10 and IL6) were significantly increased in monocytes when treated with exosomes from IH-exposed CRL-1424 and CRL-1625 cells. Our findings suggest that exosomes from untreated OSA patients increase CRL-1424 melanoma malignant properties, an effect that is not observed in two other melanoma cell lines. Exosomal cargo from CRL-1424 cells showed a unique miRNA signature compared to CRL-1675 cells after IH exposures, suggesting that melanoma cells are differentially susceptible to IH, even if they retain similar effects on immune cell polarity. It is postulated that mutations in STK-11 gene encoding for the serine/threonine kinase family that acts as a tumor suppressor may underlie susceptibility to IH-induced metabolic dysfunction, as illustrated by CRL-1424 cells.

scholarly journals YY1 Promotes Telomerase Activity and Laryngeal Squamous Cell Carcinoma Progression Through Impairment of GAS5-Mediated p53 Stability

2021 ◽  
Vol 11 ◽  
Author(s):  
Xudong Wei ◽  
Fenglei Liu ◽  
Xuelian Jiang ◽  
Xiaoyan Xu ◽  
Tianhao Zhou ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Squamous Cell Carcinoma ◽  
Cell Carcinoma ◽  
Telomere Length ◽  
Cell Lines ◽  
Colony Formation ◽  
Laryngeal Squamous Cell Carcinoma ◽  
Telomerase Activity ◽  
P53 Stability

Yin Yang 1 (YY1) is a key transcription factor that exerts functional roles in the cell biological process of various cancers. The current study aimed to elucidate the role and mechanism of YY1 in laryngeal squamous cell carcinoma (LSCC). YY1 mRNA and protein expression in human LSCC cell lines was detected by RT-qPCR and Western blot analysis. An interaction of YY1, GAS5, and p53 protein stability was predicted and confirmed by bioinformatics, ChIP, Co-IP, RIP, and FISH assays. Following loss- and gain-function assays, LSCC cell proliferation, colony formation, cell cycle, telomere length and telomerase activity were evaluated by CCK-8 assay, colony formation assay, flow cytometry, and PCR-ELISA, respectively. Nude mice were xenografted with the tumor in vivo. LSCC cell lines presented with upregulated expression of YY1, downregulated GAS5 expression, and decreased p53 stability. YY1 inhibited the expression of GAS5, which in turn recruited p300 and bound to p53, thus stabilizing it. Moreover, YY1 could directly interact with p300 and suppressp53 stability, leading to enhancement of cell proliferation, telomere length and telomerase activity in vitro along with tumor growth in vivo. Collectively, YY1 can stimulate proliferation and telomerase activity of LSCC cells through suppression of GAS5-dependent p53 stabilization or by decreasing p53 stability via a direct interaction with p300, suggesting that YY1 presents a therapeutic target as a potential oncogene in LSCC development and progression.

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