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1.
Cell Cycle ; 19(4): 464-478, 2020 02.
Article in English | MEDLINE | ID: mdl-31959066

ABSTRACT

Microtubule-targeting agents (MTAs) are the most effective chemotherapeutics used in cancer therapy to date, but their clinical use is often hampered by the acquisition of resistance. Thereby, elucidation of the molecular signaling pathways activated by novel FDA-approved MTAs such as eribulin is important for future therapeutic applications. In contrast to several reports, we show here that regardless of the presence of caspase-3, clinically relevant concentrations of eribulin and the classical MTA paclitaxel predominantly induce caspase-independent cell death in MCF-7 breast carcinoma cells. On the molecular level, several key proteins involved in apoptosis such as p53, Plk1, caspase-2, and Bim as well as the two MAPKs ERK and JNK were activated by both compounds to a similar extent. However, none of them proved to be important for eribulin- and paclitaxel-induced cytotoxicity, as their siRNA-mediated knockdown or inactivation by small molecule inhibitors did not alter cell death rates. In contrast, knockdown of the anti-apoptotic Bcl-2 protein, which becomes heavily phosphorylated at Ser70 during MTA treatment, resulted surprisingly in a reduction of MTA-mediated cell death. This phenomenon can be most likely explained by our observation that the absence of Bcl-2 slowed down cell cycle progression resulting in fewer cells entering mitosis, thereby delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle differently, they exhibit no functional differences in downstream molecular cell death signaling in MCF-7 breast cancer cells.


Subject(s)
Caspase 3/metabolism , Furans/pharmacology , Ketones/pharmacology , Microtubules/metabolism , Paclitaxel/pharmacology , Signal Transduction , Anthracenes/pharmacology , Butadienes/pharmacology , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Humans , Microtubules/drug effects , Mitogen-Activated Protein Kinases/metabolism , Nitriles/pharmacology , Phosphorylation/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Signal Transduction/drug effects
2.
Cell Death Differ ; 27(4): 1274-1285, 2020 04.
Article in English | MEDLINE | ID: mdl-31511650

ABSTRACT

In recent years it has become more and more apparent that the regulation of gene expression by RNA-binding proteins (RBPs) is of utmost importance for most cellular signaling pathways. RBPs control several aspects of RNA biogenesis including splicing, localization, stability, and translation efficiency. One of these RBPs is RBM47 that recently has been suggested to function as a tumor suppressor as it was shown to suppress breast and colon cancer progression. Here we demonstrate that RBM47 is an important regulator of basal and DNA damage-induced p53 and p21WAF1/CIP1 protein expression. Knockdown of RBM47 by siRNAs results in a strong reduction in p53 mRNA and protein levels due to an impaired p53 promoter activity. Accordingly, overexpression of Flag-RBM47 enhances p53 promoter activity demonstrating that RBM47 regulates p53 at the transcriptional level. By controlling p53, knockdown of RBM47 concomitantly decreases also p21 expression at the transcriptional level, driving irradiated carcinoma cell lines from different entities into cell death rather than into senescence. Thus, RBM47 represents a novel molecular switch of cell fate decisions that functions as a regulator of the p53/p21-signaling axis.


Subject(s)
Cell Lineage/genetics , Cyclin-Dependent Kinase Inhibitor p21/genetics , RNA-Binding Proteins/metabolism , Signal Transduction , Transcription, Genetic , Tumor Suppressor Protein p53/genetics , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Gene Knockdown Techniques , Humans , Promoter Regions, Genetic , Tumor Suppressor Protein p53/metabolism
3.
J Biol Chem ; 292(20): 8331-8341, 2017 05 19.
Article in English | MEDLINE | ID: mdl-28348086

ABSTRACT

The cyclin-dependent kinase inhibitor p21 is an important player in stress pathways exhibiting both tumor-suppressive and oncogenic functions. Thus, expression of p21 has to be tightly controlled, which is achieved by numerous mechanisms at the transcriptional, translational, and posttranslational level. Performing immunoprecipitation of bromouridine-labeled p21 mRNAs that had been incubated before with cytoplasmic extracts of untreated HCT116 colon carcinoma cells, we identified the DEAD-box RNA helicase DDX41 as a novel regulator of p21 expression. DDX41 specifically precipitates with the 3'UTR, but not with the 5'UTR, of p21 mRNA. Knockdown of DDX41 increases basal and γ irradiation-induced p21 protein levels without affecting p21 mRNA expression. Conversely, overexpression of DDX41 strongly inhibits expression of a FLAG-p21 and a luciferase construct, but only in the presence of the p21 3'UTR. Together, these data suggest that this helicase regulates p21 expression at the translational level independent of the transcriptional activity of p53. However, knockdown of DDX41 completely fails to increase p21 protein levels in p53-deficient HCT116 cells. Moreover, posttranslational up-regulation of p21 achieved in both p53+/+ and p53-/- HCT116 cells in response to pharmaceutical inhibition of the proteasome (by MG-132) or p90 ribosomal S6 kinases (by BI-D1870) is further increased by knockdown of DDX41 only in p53-proficient but not in p53-deficient cells. Although our data demonstrate that DDX41 suppresses p21 translation without disturbing the function of p53 to directly induce p21 mRNA expression, this process indirectly requires p53, perhaps in the form of another p53 target gene or as a still undefined posttranscriptional function of p53.


Subject(s)
3' Untranslated Regions/physiology , Cyclin-Dependent Kinase Inhibitor p21/biosynthesis , DEAD-box RNA Helicases/metabolism , Protein Biosynthesis/physiology , Cell Line, Tumor , Cyclin-Dependent Kinase Inhibitor p21/genetics , DEAD-box RNA Helicases/genetics , Gene Knockdown Techniques , Humans , Protein Biosynthesis/drug effects , Pteridines/pharmacology , Ribosomal Protein S6 Kinases, 90-kDa/antagonists & inhibitors , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
4.
BMC Genomics ; 15: 906, 2014 Oct 17.
Article in English | MEDLINE | ID: mdl-25326207

ABSTRACT

BACKGROUND: The human pathogen Trichomonas vaginalis is a parabasalian flagellate that is estimated to infect 3% of the world's population annually. With a 160 megabase genome and up to 60,000 genes residing in six chromosomes, the parasite has the largest genome among sequenced protists. Although it is thought that the genome size and unusual large coding capacity is owed to genome duplication events, the exact reason and its consequences are less well studied. RESULTS: Among transcriptome data we found thousands of instances, in which reads mapped onto genomic loci not annotated as genes, some reaching up to several kilobases in length. At first sight these appear to represent long non-coding RNAs (lncRNAs), however, about half of these lncRNAs have significant sequence similarities to genomic loci annotated as protein-coding genes. This provides evidence for the transcription of hundreds of pseudogenes in the parasite. Conventional lncRNAs and pseudogenes are expressed in Trichomonas through their own transcription start sites and independently from flanking genes in Trichomonas. Expression of several representative lncRNAs was verified through reverse-transcriptase PCR in different T. vaginalis strains and case studies exclude the use of alternative start codons or stop codon suppression for the genes analysed. CONCLUSION: Our results demonstrate that T. vaginalis expresses thousands of intergenic loci, including numerous transcribed pseudogenes. In contrast to yeast these are expressed independently from neighbouring genes. Our results furthermore illustrate the effect genome duplication events can have on the transcriptome of a protist. The parasite's genome is in a steady state of changing and we hypothesize that the numerous lncRNAs could offer a large pool for potential innovation from which novel proteins or regulatory RNA units could evolve.


Subject(s)
Pseudogenes , RNA, Long Noncoding/genetics , RNA, Protozoan/genetics , Trichomonas vaginalis/genetics , Gene Duplication , Gene Expression Profiling , Sequence Analysis, RNA
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