Overexpression of the Anti-Apoptotic Genes mcl-1, bcl-w, bcl-xL and a1 Is Correlated with Chronic Myelogenous Leukemia Progression and Resistance to Gleevec.

Abstract Chronic myeloid leukemia (CML) is a stem cell disease where t(9;22) translocation is considered the primary molecular event leading to the appearance of the bcr-abl fusion gene and consequent cellular transformation. Bcr-Abl tyrosine-kinase inhibitors have been developed and are fairly successful in the treatment of CML. Despite their outstanding clinical activity in CML, they are not a definitive cure: the efficacy of imatinib mesylate (Gleevec®), for instance, in CML-blastic phase is reduced and reports of resistance and intolerance to it have been published. Since Bcr-abl initiates cellular modifications leading to an extreme resistance to apoptosis, we decided to investigate possible secondary targets for CML therapy. We evaluate the expression of known anti and pro-apoptotic genes in terms of CML progression and response to Gleevec. We studied 10 health controls and 71 CML patients in different phases (20 chronic phase, 20 accelerated phase, 10 blastic phase, 15 cytogenetic remission post-Gleevec® and 6 Gleevec® refractory patients). CML group was constituted by 26 men and 35 women, median age of 51.7 years (range 23–73 years), 5 men and 5 women, median age of 49.3 (range 25–72 years), were healthy controls. Peripheral blood mononuclear cells were isolated and expression of bax, bcl-w, mcl-1, bcl-2, a1 and bcl-xL was analyzed by real-time RT-PCR. Protein expression of Bcl-2 and Bcl-xL were analyzed by western-blot. The results of real-time RT-PCR and western-blot are expressed by relative expression, e.g. ratio of investigated genes or protein to the reference GAPDH gene and protein, respectively. We observed an increase of bcl-w (p<0.001), mcl-1 (p< 0.001), a1 (p<0.01) and bcl-xL (p<0.001) gene expression and a remarkable reduction of bcl-2 (p<0.001) in CML-BP patients (table 1). Patients in remission post-Gleevec® presented an anti-apoptotic gene expression profile similar to controls (p>0.05) and refractory patients profile seem to be analogous to blastic crisis (p>0.05). bax levels did not show significant changes in CML patients in different phases (p>0.05). Bcl-2 and Bcl-xL protein data support real-time RT-PCR findings. Taken together these results suggest that mcl-1, bcl-w, bcl-xL and a1 contribute to disease progression and resistance to treatment in CML patients. Further investigations on the state of the apototic machinery in CML patients should provide new approaches for drug design and consequently new efficient treatment for AP, BC and refractory CML patients. Table 1. Ratio of amplicons of the investigated genes to housekeeping (GAPDH). Gene C CP-CML AP-CML BP-CML CCR-CML R-CML C: control; CP: chronic phase; AP: accelerated phase; BP: blastic crisis; CCR: complete cytogenetic remission; R: refractory patients. Results expressed by mean /SD. mcl-1 3.6/0.9 4.4/1.0 8.5/5.0 12.6/2.7 2.4/0.7 13.7/3.6 a1 329.9/153.3 564.1/349.1 1,7000/564.4 972.4/564.0 434.2/98.1 968.8/2.4 bcl-w 4.2/1.1 12.8/3.2 3.4/0.6 17.2/8.6 3.5/0.8 19.8/3.2 bcl-xL 2.9/1.3 5.4/1.1 10.6/3.5 39.4/6.2 3.0/2.1 42.0/3.5 bcl-2 21.1/5.2 13.8/7.0 6.1/3.1 3.1/1.3 21.7/6.4 3.6/2.2 bax 3.2/1.3 4.2/0.8 3.7/1.3 5.2/2.5 4.5/2.7 4.2/2.7

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Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽

10.1182/blood.v126.23.4213.4213 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4213-4213

Author(s):

Priya Khoral ◽

Robert J Guo ◽

Jahangir Abdi ◽

Hong Chang

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Western Blot ◽

Real Time ◽

Cell Lines ◽

Blot Analysis ◽

Drug Combination ◽

Western Blot Analysis ◽

Rt Pcr ◽

Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

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The Polycistronic miRNA Cluster miR-17-92 Is Over-Expressed in Early Phase Chronic Myeloid Leukemia (CML) CD34+ Cells.

Blood ◽

10.1182/blood.v108.11.741.741 ◽

2006 ◽

Vol 108 (11) ◽

pp. 741-741 ◽

Cited By ~ 1

Author(s):

Letizia Venturini ◽

Karin Battmer ◽

Mirco Castoldi ◽

Beate Schultheis ◽

Andreas Hochhaus ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Mirna Expression ◽

Chronic Phase ◽

K562 Cells ◽

Mature Mirnas ◽

Mirna Cluster ◽

Rt Pcr ◽

Micro Rnas ◽

Cd34 Cells

Abstract Micro RNAs (miRNAs) are small non-coding, single-stranded RNAs regulating gene expression by hybridization to complementary sequences in target mRNAs. Aberrant miRNA expression has been found in a variety of human malignancies including B-cell leukemias and lymphomas. The polycistronic miR-17–92 cluster, which includes seven miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92-1), resides in intron 3 of the C13orf25 gene located on human chromosome 13 and has recently been described to harbor oncogenic potential in a murine transplantation model. In addition, expression of the polycistron can be regulated by c-myc, which is also activated by the Bcr-Abl oncogene product. To study miRNA expression in CML, K562 cells were analyzed by microarray analysis (miCHIP) for normalized expression of 244 miRNAs following treatment either with imatinib to inhibit Bcr-Abl kinase activity or with specific anti bcr-abl shRNA to knock-down gene expression. Both of these treatment strategies led to a 2–4-fold reduction of six mature miRNAs (miR-17-5p, miR-17-3p, miR-18a, miR-20a miR-19b-1, miR-92–1) as compared to untreated control cells. The reduction of the miR-17–92 miRNA cluster expression was confirmed for each individual mature miRNA species encoded in the cluster by real-time RT-PCR. Treatment with either imatinib or anti-bcr-abl shRNA also down-regulates expression of mature miRNA miR-19a (>3-fold) in K562 cells as quantified by real-time RT-PCR. We next used PCR based quantification to analyze miR-17–92 expression in purified normal (n=4) and CML CD34+ cells harvested at diagnosis of chronic phase disease (n=16). Individual mature miRNAs of the cluster were up-regulated (2–6-fold) in CML as compared to normal CD34+ cells. Additionally, the pri-miRNA for the miR-17–92 cluster was up-regulated in 70% (7/10) of CML samples. Interestingly, retrovirus-mediated over-expression of the miR-17-19b-1 cluster in K562 cells mediates enhanced colony-formation of transduced cells. Furthermore, K562 cells expressing high levels of the miR-17-19b-1 cluster are more resistant to proliferation inhibition induced by specific anti-myc RNAi, indicating some functional role of the miR-17-92 cluster on proliferation and survival in this model. In summary, the polycistronic miR-17–92 cluster is up-regulated in early chronic phase CML CD34+ cells and may contribute to cellular transformation by the Bcr-Abl oncogene product.

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Global expression analysis of ECL cells in Mastomys natalensis gastric mucosa identifies alterations in the AP-1 pathway induced by gastrin-mediated transformation

Physiological Genomics ◽

10.1152/physiolgenomics.00216.2003 ◽

2004 ◽

Vol 20 (1) ◽

pp. 131-142 ◽

Cited By ~ 20

Author(s):

M. Kidd ◽

T. Hinoue ◽

G. Eick ◽

K. D. Lye ◽

S. M. Mane ◽

...

Keyword(s):

Gene Expression ◽

Gastric Mucosa ◽

Western Blot ◽

Cell Transformation ◽

Mastomys Natalensis ◽

Rt Pcr ◽

Cell Hyperplasia ◽

Ecl Cells ◽

Ecl Cell ◽

Gene Expression Alterations

Enterochromaffin-like (ECL) cell hyperplasia and then irreversible neoplasia can be generated in the African rodent Mastomys natalensis using the H2 receptor blocker, loxtidine, for 8–16 wk. We used a GeneChip approach complemented by standard technologies to identify gene expression alterations in the gastric mucosa during gastrin-mediated ECL cell transformation. Gastric mucosa (mucosal scrapping) and ECL cell-enriched fractions were obtained from untreated Mastomys (controls) and from animals treated with loxtidine for 8 wk (hyperplasia). Tumor ECL cells were obtained by hand-dissection of gastric ECL cell nodules from animals treated with loxtidine for >16 wk and from a spontaneously developed ECL cell tumor. RNA was isolated, examined on rat U34A GeneChips, and comparison analysis was performed to identify altered gene expression. Alterations in gene expressions were examined further by immunohistochemistry, quantitative RT-PCR (Q-RT-PCR), sequencing and Western blot. GeneSpring analysis demonstrated alterations in few genes (<20) in hyperplastic and tumor mucosa. The histamine H1 receptor was consistently increased in proliferating mucosa. This gene change was confirmed by Q-RT-PCR. Other genes showing alterations included neural-(chromogranin A and somatostatin), cell-cycle-, and AP-1-associated genes. Immunostaining confirmed alterations in neural markers. Cluster analysis of ECL cell-enriched samples demonstrated that c- fos and junD were differently regulated. Q-RT-PCR and Western blot in prospectively collected gastric mucosal samples confirmed the differential expression of Fos and Jun. The negative regulators of AP-1, JunD, and Menin were decreased in tumor mucosa. A missense of unknown function was noted in the menin gene. Hypergastrinemia in an animal model of gastric carcinoids differentially altered the histamine type 1 receptor and gene expression and protein composition of AP-1. These results suggest that expression of this receptor and an altered composition of AP-1 with a loss of inhibition play a role in ECL cell transformation.

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Quantitative real time RT-PCR study of pathogen-induced gene expression in rock bream (Oplegnathus fasciatus): Internal controls for data normalization

Marine Genomics ◽

10.1016/j.margen.2014.03.001 ◽

2014 ◽

Vol 15 ◽

pp. 75-84 ◽

Cited By ~ 10

Author(s):

Bao-cun Zhang ◽

Li Sun ◽

Zhi-zhong Xiao ◽

Yong-hua Hu

Keyword(s):

Gene Expression ◽

Real Time ◽

Internal Controls ◽

Data Normalization ◽

Rt Pcr ◽

Oplegnathus Fasciatus ◽

Rock Bream

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Expression pattern of Wif1 and β-catenin during development of anorectum in fetal rats with anorectal malformations

PeerJ ◽

10.7717/peerj.4445 ◽

2018 ◽

Vol 6 ◽

pp. e4445 ◽

Cited By ~ 4

Author(s):

Xiao Bing Tang ◽

Huan Li ◽

Jin Zhang ◽

Wei Lin Wang ◽

Zheng Wei Yuan ◽

...

Keyword(s):

Western Blot ◽

Real Time ◽

Expression Pattern ◽

Immunohistochemical Staining ◽

Anorectal Malformations ◽

Rt Pcr ◽

Rat Embryos ◽

Fetal Rats ◽

Normal Rat ◽

Wnt Inhibitory Factor 1

Purpose This study was performed to investigate the expression pattern of Wnt inhibitory factor 1 (Wif1) and β-catenin during anorectal development in normal and anorectal malformation (ARM) embryos and the possible role of Wif1 and β-catenin in the pathogenesis of ARM. Methods ARM was induced with ethylenethiourea on the 10th gestational day in rat embryos. Cesarean deliveries were performed to harvest the embryos. The expression pattern of Wif1 and β-catenin protein and mRNA was evaluated in normal rat embryos (n = 288) and ARM rat embryos (n = 306) from GD13 to GD16 using immunohistochemical staining, Western blot, and real time RT-PCR. Results Immunohistochemical staining revealed that in normal embryos Wif1 was constantly expressed in the cloaca from GD13 to GD16. On GD13 and GD14, Wif1-immunopositive cells were extensively expressed in the cloaca. On GD15, the expression of Wif1 were mainly detected on the very thin anal membrane. In ARM embryos, the epithelium of the hindgut and urorectal septum demonstrated faint immunostaining for Wif1 from GD14 to GD16. Western blot and real time RT-PCR revealed that Wif1 and β-catenin protein and mRNA expression level was significantly decreased in the ARM groups compared with the normal group on GD14 and GD15 (p < 0.05). Conclusions This study demonstrated that the expression pattern of Wif1 and β-catenin was disrupted in ARM embryos during anorectal morphogenesis, which demonstrated that downregulation of Wif1 and β-catenin at the time of cloacal separation into the primitive rectum and urogenital septum might related to the development of ARM.

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