scholarly journals Distribution and Abundance of Messenger Ribonucleic Acid for Growth Hormone Receptor Isoforms in Human Tissues1

2000 ◽  
Vol 85 (8) ◽  
pp. 2865-2871
Author(s):  
Mercedes Ballesteros ◽  
Kin-Chuen Leung ◽  
Richard J. M. Ross ◽  
Tiina P. Iismaa ◽  
Ken K. Y. Ho
Keyword(s):  
Cell Lines ◽  
Ribonucleic Acid ◽  
Full Length ◽  
Liver Fat ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Messenger Ribonucleic Acid ◽  
Receptor Isoforms ◽  
Alternatively Spliced ◽  
Exon 9

Two alternatively spliced exon 9 variants of human GH receptor (GHR) messenger ribonucleic acid (mRNA), GHR-(1–279) and GHR-(1–277), were recently identified in liver. They encode receptor proteins lacking most of the intracellular domain and inhibit GH action in a dominant negative manner. Little is known about tissue distribution and abundance of these GHR isoforms. We have developed quantitative RT-PCR assays specific for the full-length and truncated GHRs and investigated their expression in various human tissues and cell lines. The mRNA of full-length GHR and GHR-(1–279) were readily detectable in all tissues investigated, with liver, fat, muscle, and kidney showing high levels of expression. These two receptor isoforms were also detected in a range of human cell lines, with strongest expression in IM9, a lymphoblastoid cell line. In contrast, GHR-(1–277) message was expressed at low levels in liver, fat, muscle, kidney, and prostate and in trace amount in IM9 cells. Full-length GHR was the most abundant isoform, accounting for over 90% of total receptor transcripts in liver, fat, and muscle for quantitative RT-PCR. However, liver had 2- to 4-fold more full-length receptor mRNA and 16- to 40-fold more GHR-(1–277) mRNA than fat and muscle, whereas the mRNA levels of GHR-(1–279) were similar in the three tissues. GHR-(1–279) constituted less than 4% in liver and 7–10% in fat and muscle. GHR-(1–277) accounted for 0.5% of total GHR transcripts in liver and less than 0.1% in the other two tissues. These data suggest that the absolute and relative abundance of mRNA of the three GHR isoforms may be tissue specific. The regulation of expression of exon 9 alternatively spliced GHR variants may provide a potential mechanism for modulation of GH sensitivity at the tissue level.

scholarly journals Identification of a Truncated Dual Oxidase 2 (DUOX2) Messenger Ribonucleic Acid (mRNA) in Two Rat Thyroid Cell Lines. Insulin and Forskolin Regulation of DUOX2 mRNA Levels in FRTL-5 Cells and Porcine Thyrocytes

Endocrinology ◽  
2003 ◽  
Vol 144 (2) ◽  
pp. 567-574 ◽  
Author(s):  
Stanislas Morand ◽  
Orquidea Filipe Dos Santos ◽  
Renée Ohayon ◽  
Jacques Kaniewski ◽  
Marie-Sophie Noel-Hudson ◽  
...  
Keyword(s):  
Cell Lines ◽  
Ribonucleic Acid ◽  
Thyroid Cell ◽  
Mrna Levels ◽  
Messenger Ribonucleic Acid ◽  
Dual Oxidase ◽  
Rat Thyroid

scholarly journals c-kit expression in human megakaryoblastic leukemia cell lines

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2133-2144 ◽  
Author(s):  
ZB Hu ◽  
W Ma ◽  
CC Uphoff ◽  
H Quentmeier ◽  
HG Drexler
Keyword(s):  
Growth Factors ◽  
Cell Lines ◽  
Leukemia Cell ◽  
Northern Blotting ◽  
Lymphoma Cell ◽  
Mrna Levels ◽  
Bryostatin 1 ◽  
Rt Pcr ◽  
Megakaryoblastic Leukemia ◽  
Leukemia Cell Lines

Abstract A panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all- trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage- colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregulation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.

A Novel Mechanism of ADAMTS13 Deficiency in Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3668-3668
Author(s):  
Wenhua Zhou ◽  
Lingli Dong ◽  
Eric E. Bouhassira ◽  
Han-Mou Tsai
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Mammalian Cell ◽  
Activity Level ◽  
Full Length ◽  
Mouse Strains ◽  
Activity Levels ◽  
Adamts13 Activity ◽  
Rt Pcr ◽  
Mammalian Cell Lines

Abstract Background. ADAMTS13, a circulating metalloprotease that cleaves conformationally altered von Willebrand factor (VWF), is critical for preventing microvascular thrombosis. Deficiency of the protease, due to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura. In the course of investigating the regulation of ADAMTS13, we noted that mice differed widely in their plasma ADAMTS13 activity levels. In order to understand the factors affecting plasma ADAMTS13 levels, we examined the molecular basis of ADAMTS13 variation in different strains of mice. Methods. Plasma ADAMTS13 activity level was determined by previously described SDS PAGE and immunoblotting. ADAMTS13 transcripts were analyzed by RT PCR, RACE, nucleotide sequencing, and real-time RT PCR. Plasmids containing the cDNA of mouse ADAMTS13 were constructed for transfection of mammalian cell lines. Results. The mouse strains FVB/NJ and 129X1/SvJ differed from C57BL/6J and DBA/2J by more than 10 folds in their plasma ADAMTS13 activity levels (3.09 +/− 0.45 vs 0.24 +/− 0.11 U/mL for FVB/NJ and C57BL/6J respectively, P < 0.0001). To determine the causes of the difference, we analyzed the ADAMTS13 transcripts by using RT PCR and RACE, which showed that the FVB/NJ mice contained the predicted full-length ADAMTS13 transcript with a domain structure similar to human ADAMTS13, while the C57BL/6J mice contained at least 4 isoforms: the full-length transcript, one internal splicing isoform, and two truncated forms that ended with an extraneous sequence homologous to the long-terminal repeat (LTR) of an retrotransposone of the IAP type. Comparison of the genomic sequences showed that the ADAMTS13 gene of C57BL/6J mice contained in its intron #23 an IAP-type retrotransposone sequence whose LTR sequence with a stop codon was included in the mouse transcripts. The IAP retrotransposone sequence, which contained one base substitution at the 5′-end 4-base repeat (tgtt>g) and was flanked at both ends by a 6-base repeat (cactag), was also present in the DBA/2J but not the 129X1/SvJ strains of mice. Real-time RT PCR showed that the FVB/NJ and C57BL/6J mice had similar levels of ADAMTS13 transcripts in the liver. Nevertheless in the C57BL/6J mice the IAP-truncated forms accounted for >90% of the ADAMTS13 transcripts. Expression of mouse ADAMTS13 cDNA in mammalian cell lines revealed that the both the full-length and the IAP-truncated forms of the ADAMTS13 protease were similar in VWF-cleaving activity. Conclusion. This study shows the presence of intragenic retrotransposone in the ADAMTS13 gene of some mouse strains. The presence of an IAP-retrotransposone within the ADAMTS13 gene of C57BL/6J mice affects the splicing of the ADAMTS13 transcripts, creating truncated forms of the protease that lack the last two TSP-1 and the CUB domains but remain proteolytically active in cleaving VWF. The lower plasma ADAMTS13 activity level of C57BL/6J may result from abnormal intravascular clearance of the protease or other post-secretory events.

Alternation of Cellular Morphology and Proliferation Induced by microRNA in Human Leukemia Cell Line, UT-7/EPO.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4203-4203
Author(s):  
Nobuyoshi Kosaka ◽  
Yusuke Yamamoto ◽  
Nami Nogawa ◽  
Keiichi Sugiura ◽  
Hiroshi Miyazaki ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Macrocytic Anemia ◽  
Leukemia Cell ◽  
Leukemia Cell Line ◽  
Human Leukemia ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Morphological Studies

Abstract Mature microRNA (miRNA) originated from primary miRNA (pri-miRNA) is a new group of potential regulator for cell differentiation, apoptosis, proliferation and oncogenesis. Some miRNAs were recently identified in hematopoietic cells, while the roles of miRNAs in erythrocytic and megakaryocytic cells had not been well examined. As a first step to explore for miRNAs specific for hematopoietic lineage, the expressions of several known primary microRNAs in erythrocytic and megakaryocytic cell lines, such as TF-1, HL-60, HEK293 and UT-7 leukemia cells, were examined by RT-PCR. We consequently focused on the pri-miR-10a, a primary transcript of miR-10a located within Hox gene clusters, and found the significant expression in TF-1 cells and UT-7/EPO cells. The UT-7/EPO cells were a subline established from the original UT-7 cells, as well as UT-7/GM and UT-7/TPO cells; therefore it was suitable for the further comparative analysis. Interestingly, in UT-7/EPO cells, the expression of pri-miR-10a increased under stimulation of erythropoietin (EPO; 1U/mL and 10U/mL). Based on these observations, it was postulated that pri-miR-10a might involve in modulating erythrocyte differentiation or proliferation. To clarify the role of pri-miR-10a in UT-7/EPO, we have established clonal cell lines by transfecting UT-7/EPO cells with either the control vector or the pri-miR-10a expression vector pCMV-pri-miR10a. Overexpression of pri-miR-10a in the UT-7/EPO cell line (miR10a-UT-7/EPO) was confirmed by RT-PCR. MiR10a-UT-7/EPO showed higher proliferation rate even at low concentration of EPO (0.1 mU/mL). Overexpression of pri-miR-10a did not appear to affect HOXB4 and HOXA1 expression, as similar mRNA levels were seen in both cell lines. It was notable that the cellular size of miR10a-UT-7/EPO became larger than its parental cells. Morphological studies of miR10a-UT-7/EPO were performed in detail. It is possible that miR-10a was capable to modulate morphological features particularly in cellular size relating to cell cycle regulation. For instance, loss of the E2F family members result in marked macrocytic anemia with megaloblastic features in adult mice (Mol Cell. 2000 Aug;6(2):281–91., Mol Cell Biol. 2003 May;23(10):3607–22., Blood. 2006 Aug 1;108(3):886–95.). Data presented here hypothesized that the roles of miR-10a in erythroid cells are tightly associated with cell cycle.

The Brd-Inhibitor OTX015 Shows Pre-Clinical Activity in Anaplastic Large T-Cell Lymphoma (ALCL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4872-4872 ◽  
Author(s):  
Michela Boi ◽  
Paola Bonetti ◽  
Maurilio Ponzoni ◽  
Maria Grazia Tibiletti ◽  
Anastasios Stathis ◽  
...  
Keyword(s):  
Cell Cycle ◽  
T Cell ◽  
Cell Lines ◽  
Proliferative Activity ◽  
Research Funding ◽  
Cell Lymphoma ◽  
G1 Arrest ◽  
Apparent Difference ◽  
Mrna Levels ◽  
Rt Pcr

Abstract Abstract 4872 Background: ALCL, is clinically/biologically heterogeneous disease, including ALK+ and ALK- systemic forms. Despite the progresses in understanding the molecular pathogenesis of ALCL, the therapy is still based on chemotherapy, thus the identification of new treatment modalities is needed. Bromodomain-containing proteins are components of transcription factors complexes and determinants of epigenetic memory. Inhibitors of BRD2/3/4, members of the Bromodomain and Extraterminal (BET) family, have recently shown antitumor activity in different hematological malignancies models. Here, we report anti-proliferative activity of OTX015, a novel selective orally bioavailable BRD2/3/4 inhibitor, in a panel of ALCL cell lines. Material and Methods: Eight established human cell lines derived from ALK+ and ALK- anaplastic large cell lymphoma (ALCL) were treated with increasing doses of OTX015 (OncoEthix SA) and MTT assays were performed after 72h exposure. For cell cycle analysis, cells were treated and stained with Click-iT Edu Flow Cytometry Assay Kits (Invitrogen) and 7-AAD and analyzed for DNA content using a FACScan flow cytometer. Results were analyzed with FlowJo 7.6.3 software. RNA was extracted using the Qiagen RNAEasy kit and reverse-transcribed using the Superscript First-Strand Synthesis System for RT-PCR kit according to the manufacturer's instructions. RT-PCR was performed on using Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For senescence detection, cells were stained using a b-Galactosidase Staining Kit (Calbiochem). Results: We assessed OTX-015 anti-proliferative activity in eight ALCL cell lines. The majority (5/8) of the cell lines were sensitive, with IC50 between 36 and 546 nM. There was no apparent difference between ALK+(6) and ALK- (2) cell lines. Cell cycle analyses revealed G1 arrest and a concomitant decrease of the S phase after 24h OTX015 exposure in 4/4 ALCL cell lines, without an increase in cell death, suggesting a cytostatic effect of OTX015. An increase in the percentage of senescent cells after treatment with the BRD-inhibitor was observed in the most sensitive ALK+ALCL cell line. To understand the mechanism of action of OTX015, we assessed MYC mRNA levels before and after treatment. We observed that OTX015 suppressed the transcription of MYCgene and some of its downstream target genes (such as NCL and CAD) in 4/4 ALCL cell lines, with less efficacy in the most resistant one. Conclusion: OTX015 is a new potent BRD-inhibitor with evident anti-proliferative activity in several ALCL cell lines. The down-regulation of MYC gene, followed by cell cycle G1 arrest and increase of cellular senescence, was observed after OTX015 treatment, appearing one of the possible mechanisms of action of the compound. The compound appears worth of further investigation as a new promising therapeutic agent in ALCL and in other mature T-cell tumors. Disclosures: Bonetti: OncoEthix SA: Research Funding. Cvitkovic:OncoEthix SA: Membership on an entity's Board of Directors or advisory committees. Inghirami:OncoEthix SA: Research Funding. Bertoni:OncoEthix SA: Research Funding.

An Alternatively Spliced Form of CD79b Gene May Account for Altered B-Cell Receptor Expression in B-Chronic Lymphocytic Leukemia

Blood ◽  
1999 ◽  
Vol 93 (7) ◽  
pp. 2327-2335 ◽  
Author(s):  
A. Alfarano ◽  
S. Indraccolo ◽  
P. Circosta ◽  
S. Minuzzo ◽  
A. Vallario ◽  
...  
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Alternatively Spliced

Several functional anomalies of B-chronic lymphocytic leukemia (B-CLL) cells may be explained by abnormalities of the B-cell receptor (BCR), a multimeric complex formed by the sIg homodimer and the noncovalently bound heterodimer Ig/Igβ (CD79a/CD79b). Because the expression of the extracellular Ig-like domain of CD79b has been reported to be absent in the cells of most CLL cases, we have investigated the molecular mechanisms that may account for this defect. Peripheral blood lymphocytes (PBL) from 50 patients and two cell lines (MEC1, MEC2) obtained from the PBL of one of them were studied. MEC1, MEC2, and 75% of CLL cases did not express detectable levels of the extracellular Ig-like domain of CD79b, which was nevertheless present in greater than 80% CD19+ cells from normal donors. In healthy subjects the expression of CD79b was equally distributed in CD5+ and CD5− B-cell subsets. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of CD79b RNA from all patients and from MEC1 and MEC2 cell lines consistently yielded two fragments of different size (709 bp and 397 bp). The 709-bp band corresponds to CD79b entire transcript; the 397-bp band corresponds to an alternatively spliced form lacking exon 3 that encodes the extracellular Ig-like domain. Both fragments were also visible in normal PBL. The expression of the 397-bp fragment was increased in normal activated B cells, while no difference was seen between CD5+ and CD5− B cells. To obtain a more accurate estimate of the relative proportions of the two spliced forms, a radioactive PCR was performed in 13 normal and 22 B-CLL samples and the results analyzed using a digital imager. The mean value of the CD79b to the CD79b internally deleted ratio was 0.64 ± 0.20 SD in normal donors and 0.44 ± 0.27 SD in B-CLL (P = .01). Direct sequencing of 397-bp RT-PCR products and of genomic DNA corresponding to exon 3 from MEC1, MEC2, their parental cells, and five fresh B-CLL samples did not show any causal mutation. Single-strand conformation polymorphism analysis of exon 3 performed in 18 additional B-CLL cases showed a single abnormal shift corresponding to a TGT → TGC polymorphic change at amino acid 122. We propose a role for the alternative splicing of CD79b gene in causing the reduced expression of BCR on the surface of B-CLL cells. As normal B cells also present this variant, the mechanism of CD79b posttranscriptional regulation might reflect the activation stage of the normal B cell from which B-CLL derives.

scholarly journals Suppression of Leptin Receptor Messenger Ribonucleic Acid and Leptin Responsiveness in the Ventromedial Nucleus of the Hypothalamus during Pregnancy in the Rat

Endocrinology ◽  
2005 ◽  
Vol 146 (9) ◽  
pp. 3868-3874 ◽  
Author(s):  
S. R. Ladyman ◽  
D. R. Grattan
Keyword(s):  
Choroid Plexus ◽  
Arcuate Nucleus ◽  
Leptin Receptor ◽  
Leptin Resistance ◽  
Ventromedial Nucleus ◽  
Mrna Levels ◽  
Pregnant Rats ◽  
Messenger Ribonucleic Acid ◽  
Leptin Receptors ◽  
Receptor Isoforms

Abstract Pregnancy in the rat is a state of leptin resistance associated with impaired leptin signal transduction in the hypothalamus. The aim of this study was to determine whether this leptin-resistant state is mediated by a change in the level of leptin receptors in the hypothalamus. Real-time RT-PCR was used to determine levels of mRNA for the various leptin receptor isoforms in a number of microdissected hypothalamic nuclei and the choroid plexus. To investigate the functional activation of the leptin receptor, immunohistochemistry for phosphorylated signal transducer and activator of transcription 3 (pSTAT3) was examined in the arcuate nucleus and the ventromedial nucleus of the hypothalamus (VMH) of fasted diestrous and d-14 pregnant rats after an intracerebroventricular (i.c.v.) injection of either leptin (4 μg) or vehicle. A significant reduction of Ob-Rb mRNA levels was observed in the VMH during pregnancy compared with the nonpregnant controls. Furthermore, in pregnant rats the number of cells positive for leptin-induced pSTAT3 in the VMH was greatly reduced during pregnancy compared with nonpregnant rats. There were no differences in the level of Ob-Rb mRNA or in the number of leptin-induced pSTAT3-positive cells in the arcuate nucleus of nonpregnant and pregnant rats. These data implicate the VMH as a key hypothalamic site involved in pregnancy-induced leptin resistance. There were also reduced levels of mRNA for Ob-Ra, a proposed leptin transporter molecule, in the choroid plexus on d 7 and 21 of pregnancy. Hence, diminished transport of leptin into the brain may also contribute to pregnancy-induced leptin resistance.

scholarly journals A Novel Mechanism in Regulating the Alpha-Subunit of the Epithelial Sodium Channel (α ENaC) by the Alternatively Spliced Form α ENaC-b

2009 ◽  
Vol 2 ◽  
pp. BCI.S880 ◽  
Author(s):  
Marlene F. Shehata
Keyword(s):  
Sodium Channel ◽  
Epithelial Sodium Channel ◽  
Dominant Negative Effect ◽  
Kidney Cortex ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Alternatively Spliced ◽  
Dose Dependent

Introduction In Dahl rats’ kidney cortex, the alternatively spliced form of the epithelial sodium channel α subunit (α ENaC-b) is the most abundant mRNA transcript (32+/-3 fold > α ENaC-wt) as was investigated by quantitative RT-PCR analysis. α ENaC-b mRNA levels were significantly higher in Dahl R versus S rats, and were further augmented by high salt diet. Objectives In the present study, we described the molecular cloning and searched for a possible role of α ENaC-b by testing its potential expression in COS7 cells as well as its impact on α ENaC-wt expression levels when co-expressed in COS7 cells in a dose-dependent manner. Methods Using RT-PCR strategy, the full-length wildtype α ENaC transcript and the alternatively spliced form α ENaC-b were amplified, sequenced, cloned, subcloned into PCMV-sport6 expression vector, expressed and co-expressed into COS7 cells in a dose-dependent manner. A combination of denaturing and native western blotting techniques was employed to examine the expression of α ENaC-b in vitro, and to determine if an interaction between α ENaC-b and α ENaC-wt occurs in vitro, and finally to demonstrate if degradation of α ENaC-wt protein does occur. Results α ENaC-b is translated in COS7 cells. Co-expression of α ENaC-b together with α ENaC-wt reduced α ENaC-wt levels in a dose-dependent manner. α ENaC-wt and α ENaC-b appear to form a complex that enhances the degradation of α ENaC-wt. Conclusions Western blots suggest a novel mechanism in α ENaC regulation whereby α ENaC-b exerts a dominant negative effect on α ENaC-wt expression. This is potentially by sequestering α ENaC-wt, enhancing its proteolytic degradation, and possibly explaining the mechanism of salt-resistance in Dahl R rats.

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