High FLT3 mRNA Expression in Acute Myeloid Leukemia May Be Functionally an Alternative to Mutational Activation of the Receptor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3374-3374 ◽  
Author(s):  
Florian C. Kuchenbauer ◽  
Wolfgang Kern ◽  
Claudia Schoch ◽  
Alexander Kohlmann ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
De Novo ◽  
Expression Level ◽  
Alternative Mechanism ◽  
Expression Levels ◽  
Flt3 Mutation ◽  
Flt3 Mutations ◽  
Flt3 Expression ◽  
Mutational Activation

Abstract Mutations of the FLT3 gene are detectable in approximately 30% of all adult AML. These mutations lead to an autoactivation of the receptor inducing increased proliferation of the leukemic clone. An alternative mechanism of FLT3 activation might be mRNA overexpression. In the presented study the FLT3 expression level in 208 adult AML patients and 8 healthy donors was assessed by real time PCR and correlated to several parameters. In all patients cytomorphology, cytogenetics, and FLT3 mutation status was assessed. Significant differences of FLT3-expression levels were found in certain AML subgroups. The highest expression levels were found in FAB subtypes M5 and the lowest in M3. In total, increasing levels were shown in the following order: M3 <M3v <M6 <M2 <M4eo <M4 <M0 <M1 <M5a <M5b. Independent analysis of FLT3 expression in different cytogenetic AML subgroups showed the lowest median in the t(15;17) group, followed by t(8;21), inv(16), normal, complex karyotype and the highest median in the t(11q23) group. No difference was observed between the group of secondary AML following MDS, therapy related AML and the de novo AML (p=0.868, p=0.562, and p=0.570, respectively). Compared to clinical parameters, FLT3 expression correlated with high percentage of bone marrow blasts (p=0.0005) and high leukocyte count (p<0.001). In contrast to previous studies no difference in FLT3 expression levels was detected between AML with (n=74) and without (n=130) any FLT3 mutation. Assessment of FLT3 RNA by microarray analysis and FLT3 receptor surface expression (CD135) detected by flow cytometry correlated significantly with FLT3-expression as assessed by real time PCR (p<0.001, each). To analyze whether high FLT3 expression is a prognostic parameter 118 AML cases with normal karyotype were devided into two groups. Group 1 (n=75) was defined to have less and group 2 (n=43) more than the median of the FLT3 expression level found in the total group. No impact on OS and EFS could be shown (608 vs. 311 days, p=0.1283 and 398 vs. 208 day, p=0.3056). In conclusion, these data support the hypothesis that FLT3 activation through mRNA overexpression is an alternative mechanism to FLT3 mutations. Especially as it was found extremely high in 11q23 AML, that rarely reveal FLT3 mutations.

scholarly journals Changes in the expression of galanin and galanin receptors in the wall of the colon in pigs experimentally infected with Brachyspira hyodysenteriae

2014 ◽  
Vol 58 (1) ◽  
pp. 23-28 ◽  
Author(s):  
Krzysztof Wąsowicz ◽  
Piotr Podlasz ◽  
Małgorzata Chmielewska ◽  
Katarzyna Łosiewicz ◽  
Jerzy Kaleczyc ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Experimental Colitis ◽  
Expression Level ◽  
Colonic Wall ◽  
Brachyspira Hyodysenteriae ◽  
Expression Levels ◽  
Gapdh Expression ◽  
Galanin Receptors ◽  
Different Levels

Abstract The expression of galanin (GAL) and its three receptors (GalR1, GalR2, and GalR3) were studied with real-time PCR in the colonic wall of pigs suffering from experimental colitis caused by the infection with Brachyspira hyodysenteriae. The expression was studied in the muscular membrane, mucosa/submucosa layer, and in lymphocytes isolated from mucosa/submucosa. The expression levels were normalized to glyceraldehyde-6-phosphate dehydrogenase (GAPDH) expression and compared to expression levels in control animals. GAL expression was found in all three studied compartments of the colonic wall. A significant decrease in GAL expression level was found in the mucosa/submucosa and in isolated lymphocytes, whereas the decrease was much less profound in the muscular membrane. In the case of galanin receptors their expression was found in all studied compartments of the colonic wall, however at different levels, as compared to GAPDH expression. The decrease of galanin receptors expression was found in all studied compartments of the colonic wall of the sick animals.

In Acute Promyelocytic Leukemia (APL) with Multiple Relapses Additional Prognostic Information Is Provided by FLT3 and Telomerase (hTERT) Quantitative Real-Time PCR.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4408-4408
Author(s):  
Paolo Bernasconi ◽  
Silvia Calatroni ◽  
Barbara Rocca ◽  
Paola Maria Cavigliano ◽  
Carlo Castagnola ◽  
...  
Keyword(s):  
Real Time ◽  
Induction Chemotherapy ◽  
Real Time Pcr ◽  
Clinical Relapse ◽  
Htert Expression ◽  
Quantitative Real Time Pcr ◽  
Molecular Remission ◽  
Expression Levels ◽  
Flt3 Expression ◽  
Multiple Relapses

Abstract The present study, carried out in two APL with multiple relapses, was aimed at determining 1) which correlation does exist between FLT3/hTERT expression levels and PML-RARA results; 2) whether high FLT3 and hTERT expression levels might be predictive of relapse; 3) whether FLT3 expression is better than FLT3 Internal Tandem Duplication (ITD) for evaluating disease outcome. Relative quantifications of FLT3/hTERT transcripts were performed by real-time PCR using SybrGreen I. For FLT3 calibration total RNA from a normal subject was used, for hTERT total RNA from K562 cells. In both cases the ΔΔCt method was used for quantification. On clinical diagnosis one patient with a WBC of 124.0x109/L and a PML-RARA fusion at PML BCR1 presented the FLT3/hTERT genes highly expressed. On qualitative PCR the patient also showed the ITD of FLT3. He was treated with the AIDA protocol and succeeded in achieving a haematological but not molecular remission. During CR FLT3/hTERT expression remained high and the ITD was never detected. Fourteen months later when on first clinical relapse FLT3 expression abruptly increased, the ITD reappeared, hTERT levels were still high. A re-induction chemotherapy induced a second haematological but not molecular remission lasting five months. FLT3 as well as hTERT expression levels became similar to those of the control, and FLT3 ITD disappeared. A progressive increase of FLT3 expression and an abrupt increase of hTERT expression preceded the second relapse which was accompanied by the reappearance of the ITD. After re-induction chemotherapy FLT3/hTERT expression dropped down to values of the control. A third CR was obtained but the patient remained PML/RARA and Flt3 ITD positive and soon after died of a CNS relapse. The other patient was treated in another Centre and came to our observation in haematological CR. At that time he was PML-RARA negative with high FLT3/hTERT expression. Eight months later he was still in clinical but not molecular CR having a PML-RARA fusion at BCR3, high FLT3/hTERT expression levels and presenting FLT3 ITD. One month later when clinical relapse occurred FLT3 expression levels were unchanged, hTERT expression dropped down to normal values and FLT3 ITD was still present. A re-induction chemotherapy induced a second CR with alternatively positive and negative PML-RARA results, high FLT3 and low hTERT expression levels. The patient underwent an allogeneic bone marrow transplant from an unrelated donor but five months later he relapsed for the second time with an abrupt rise of hTERT expression that preceded a quick increase of FLT3 expression. A third clinical but not molecular CR was achieved after chemotherapy, but the patient remained PML-RARA positive with a normal FLT3/hTERT expression. Two months later a rapid increase of hTERT expression preceded that of FLT3 and the occurrence of the third relapse. In conclusion i) increased FLT3 and hTERT levels during CR are associated with alternative positive/negative PML-RARA results on nested RT PCR and are always predictive of pending relapse; ii) on disease recurrence a marked elevation of hTERT expression often preceded that of FLT3; iii) quantitative real-time PCR of the FLT3 gene was more effective in predicting disease outcome than the ITD, this last being discovered only when FLT3 expression was already high.

scholarly journals The Effects of miR-136-5p-Mediated Regulation of A20 in Astrocytes from Cultured Spinal Cord Cultured Cells In Vitro

2017 ◽  
Vol 41 (4) ◽  
pp. 1596-1604 ◽  
Author(s):  
Xiaoming Peng ◽  
Xiongzhi Shi ◽  
Jinmin Zhao ◽  
Jichen He ◽  
Keke Li ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Inflammatory Cytokine ◽  
Cultured Cells ◽  
Expression Level ◽  
Control Group ◽  
Interleukin 17 ◽  
Expression Levels ◽  
Inhibition Group

Background/Aims: This study focused on investigating the regulatory mechanism of miR-136-5p in mouse astrocytes stimulated with interleukin-17(IL-17). Methods: C57BL/6 mouse astrocytes were stimulated with IL-17 (100ng/ml) for various periods of time (0-48 hours) and at various doses (0-200 ng), and the expression levels of inflammatory cytokine and chemokine genes (IL-6, TNF-α, MCP-1, MCP-5 and MIP-2) were then detected by real-time PCR. The expression of the A20 gene was measured with real-time PCR in cells that were stimulated with IL-17 (50 ng/ml) for various periods of time (0-48 hours). C57BL/6 mouse astrocytes were transfected with Ctrl-anti-miR-136-5p or LNA -anti-miR-136-5p for 48 h. Thereafter, the cells were stimulated with or without IL-17 (50ng/ml) for 6 h. The level of A20 protein (TNFα-induced protein 3, TNFAIP3) was detected by Western blot analysis. Results: (1) Compared with the DMEM control group, within six hours, IL-17 stimulation significantly increased the expression levels of inflammatory cytokine and chemokine genes and clearly decreased the expression level of the A20 protein. (2) Without IL-17 stimulation, the expression level of the miR-136-5p gene was significantly decreased, whereas in the miR-136-5p-inhibition group, the A20 protein expression was elevated. IL-17 stimulation slightly decreased the expression of the A20 protein in the miR-136-5p-inhibition group, but it was still slightly higher than in the control group. Conclusion: This study demonstrated that miR-136-5p affected the expression of A20 in IL-17-stimulated astrocytes.

Detection of ERG and MN1 Gene Expression in Adult Patients with De Novo Acute myeloid Leukemia(AML) by Real-Time Quantitative Polymerase Chain Reaction and Its Clinical Significance.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4706-4706
Author(s):  
Bing Xu ◽  
Xutao Guo ◽  
Guoshu Chen ◽  
Xiaoyan Song ◽  
Pengcheng Shi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Clinical Significance ◽  
De Novo ◽  
Risk Group ◽  
Expression Level ◽  
Chain Reaction ◽  
Expression Levels ◽  
Polymerase Chain

Abstract Abstract 4706 Introdution Current study found that certain genetic abnormalities are related to the prognosis of AML, such as ERG and MN1 gene expression. However, little is known about the clinical significance of ERG and MN1 expression level in Chinese patients with AML and and their correlation. Patients and methods A real time quantitative reverse transcriptase polymerase chain reaction method was established for detecting ERG and MN1 gene expression levels in 87 de novo AML patients. Results The median expression levels of ERG and MN1 in AML patient were statistically higher than that in normal control group (P<0.001). ERG gene expression had no correlation with MN1 gene expression (P=0.194) in AML patients. ERG and MN1 gene expressions were equally distributed among the FAB subtypes (P=0.850 and 0.870). Spearman's rank correlation showed that leukocyte counts and lactate dehydrogenase were significantly related to high ERG expression (P=0.005 and 0.032), but not significant correlation was found between hemoglobin and platelet counts. The ERG expression level of cases with middle and low risk group was lower than that of cases with high risk group (P<0.036). There was no significant difference of CR rates in high and low ERG and MN1 expression groups after chemical theropy (P=0.968 and 0.695). During the follow-up of one year, the cumulative relapse rates of high ERG expression groups was significantly higher than that of low ERG expression groups(P=0.039), and high ERG expression cases have a significantly worse OS than low ERG expression cases(P=0.005). Conclusions There isn't a linear correlation between ERG gene expression and MN1 gene expression in AML. Overexpression of ERG gene is a independent prognositic factor for AML. Quantification of the two gene expression together is not more effective to the judgement of prognosis in AML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The FMS like Tyrosine Kinase 3 (FLT3) Is Overexpressed in a Subgroup of Multiple Myeloma Patients with Inferior Prognosis

Cancers ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 2341
Author(s):  
Normann Steiner ◽  
Karin Jöhrer ◽  
Selina Plewan ◽  
Andrea Brunner-Véber ◽  
Georg Göbel ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Tyrosine Kinase ◽  
Real Time ◽  
Cell Lines ◽  
Real Time Pcr ◽  
Tyrosine Kinase Receptor ◽  
Bone Marrow Biopsies ◽  
Flt3 Inhibitors ◽  
Flt3 Expression

Therapy resistance remains a major challenge in the management of multiple myeloma (MM). We evaluated the expression of FLT3 tyrosine kinase receptor (FLT3, CD135) in myeloma cells as a possible clonal driver. FLT3 expression was analyzed in bone marrow biopsies of patients with monoclonal gammopathy of undetermined significance or smoldering myeloma (MGUS, SMM), newly diagnosed MM (NDMM), and relapsed/refractory MM (RRMM) by immunohistochemistry (IHC). FLT3 gene expression was analyzed by RNA sequencing (RNAseq) and real-time PCR (rt-PCR). Anti-myeloma activity of FLT3 inhibitors (midostaurin, gilteritinib) was tested in vitro on MM cell lines and primary MM cells by 3H-tymidine incorporation assays or flow cytometry. Semi-quantitative expression analysis applying a staining score (FLT3 expression IHC-score, FES, range 1–6) revealed that a high FES (>3) was associated with a significantly shorter progression-free survival (PFS) in NDMM and RRMM patients (p = 0.04). RNAseq and real-time PCR confirmed the expression of FLT3 in CD138-purified MM samples. The functional relevance of FLT3 expression was corroborated by demonstrating the in vitro anti-myeloma activity of FLT3 inhibitors on FLT3-positive MM cell lines and primary MM cells. FLT3 inhibitors might offer a new targeted therapy approach in a subgroup of MM patients displaying aberrant FLT3 signaling.

scholarly journals A De Novo Transcriptome and Valid Reference Genes for Quantitative Real-Time PCR in Colaphellus bowringi

PLoS ONE ◽  
2015 ◽  
Vol 10 (2) ◽  
pp. e0118693 ◽  
Author(s):  
Qian-Qian Tan ◽  
Li Zhu ◽  
Yi Li ◽  
Wen Liu ◽  
Wei-Hua Ma ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
De Novo ◽  
Quantitative Real Time Pcr ◽  
De Novo Transcriptome

46 EXPRESSION OF TRANSCRIPTION FACTORS SPECIFIC TO THE TROPHOBLAST LINEAGE IN MOUSE SOMATIC NUCLEAR TRANSFER EMBRYOS

2008 ◽  
Vol 20 (1) ◽  
pp. 103
Author(s):  
T. Mitani ◽  
M. Nishiwaki ◽  
M. Anzai ◽  
H. Kato ◽  
Y. Hosoi ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Transfer ◽  
Ectopic Expression ◽  
Expression Level ◽  
Pcr Analysis ◽  
Cell Stage ◽  
Cdx2 Expression ◽  
Morula Stage

Somatic cell nuclear transfer (SCNT) embryos can develop at relatively high rates during the preimplantation period; however, most of these fail after implantation. Development of extraembryonic tissue is indispensable for normal embryonic development. Hence, an abnormality of trophoblast development might be a significant factor in post-implantation lethality of SCNT embryos. A transcription factor, caudal-related homeobox 2 (Cdx2), appears to be involved in the segregation of ICM and trophectoderm (TE) in preimplantation embryos (Niwa et al. 2005 Cell 123, 917–929). Both Cdx2 and Oct3/4 are expressed in all cells at the morula stage, and then Cdx2 expression becomes restricted to the TE and Oct3/4 to the ICM as the blastocyst develops. Mouse embryos deficient in Cdx2 are able to develop to normal blastocysts but die soon after implantation, probably because of defects in the TE lineage. Moreover, dysplasia of the spongiotrophoblast layer might attribute to an abnormality of Tpbpa expression in mouse SCNT embryos (Wakisaka-Saito et al. 2006 Biochem. Biophys. Res. Commun. 349, 106–114). In this study, we examined the expression profiles of transcription factors implicated in trophoblast development in mouse SCNT embryos and intracytoplasmic sperm injection (ICSI) embryos by immunohistochemistry and real-time PCR analysis. SCNT embryos were produced according to the method reported previously (Wakayama et al. 1998 Nature 394, 369–374). In brief, B6D2F1 and B6C3F1 female mice were used for the collection of recipient oocytes and donor cells, respectively. After nuclear transfer, the oocytes were activated and cultured in KSOM to the morula and blastocyst stages. Immunohistochemical analysis demonstrated that in ICSI embryos Cdx2 was only partially expressed at the 8-cell stage but completely in early morulae. In contrast, in SCNT embryos, it was absent at the 8-cell stage and appeared partially at the early morula stage. Thereafter, Cdx2 expression became restricted to the TE cells in both the ICSI and the SCNT blastocysts. However, ectopic expression of Oct3/4 was observed in the TE cells of SCNT, but not in ICSI blastocysts. Real-time PCR analysis showed that at the 8-cell stage, Cdx2 was expressed in ICSI but not in SCNT embryos. In addition, the expression level of Cdx2 in SCNT embryos at the blastocyst stage was only half that in ICSI embryos (P < 0.05). However, there was no significant difference in expression level of Oct3/4 between ICSI and SCNT embryos. Eomesodermin (Eomes) is also implicated in trophoblast development and its expression depends on Cdx2, BMP4, and FGF4. In SCNT embryos, the expression level of Eomes was also only half that in ICSI embryos. These results indicate that the delayed expression of Cdx2 in SCNT embryos may lead to the ectopic expression of Oct3/4 in blastocysts and, along with the limited expression of Cdx2 and Eomes, may contribute to disorders in the function of the trophoblast lineage for normal placental development. This work was supported by a Grant-in-Aid for the 21st Century Center of Excellence Program of the MEXT, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science.

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