Expression of the Transcription Factor PAX5/BSAP in Childhood Acute Leukemia Cells and Haematological Tumor Cell Lines.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4295-4295
Author(s):  
Bei Zhang ◽  
Li-Jun Tie ◽  
Qidong Ye ◽  
Yanxia Zhao ◽  
Long-Jun Gu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Tumor Cell ◽  
B Cell ◽  
Mrna Expression ◽  
Real Time ◽  
Cell Lines ◽  
Clinical Samples ◽  
Tumor Cell Lines ◽  
B Lineage ◽  
Pax5 Gene

Abstract PAX5 gene is a paired-box PAX gene family member,and encodes the transcription factor BSAP(B-cell specific activator protein) which is a key regulator of B-cell development and differentiation.Dysregulation of PAX5 gene function may contribute to tumorigenesis in lymphoid malignancies.But up to now,a detailed examination of PAX5/BSAP expression in acute leukemia(mainly acute B-lineage lymphoblastic leukemia) has not been reported.In this study,a real-time RT-PCR assay for the relative quantitation of PAX5 and CD19 mRNA expression was developed.It was applied on 6 haematological tumor cell lines and bone marrow cells of 6 normal children,58 previously untreated and 4 relapse acute leukemic children,including 39 cases of B-ALL,10 cases of T-ALL,and 13 cases of AML.PAX5 and CD19 mRNA expression were detected in B-cell lines tested,but almost not in other T- and myeloid cell lines.Among clinical samples,expression of PAX5 mRNA in B-ALL was significantly higher than that in T-ALL and AML(P=0.029 and P=0.013,respectively).PAX5 expression was significantly lower in T-ALL and AML than normal controls.The mRNA levels of PAX5 between T-ALL and AML had not any difference.Individual difference of PAX5 mRNA expression levels in children with B-ALL was great.Because binding sites for BSAP have been identified in the promoters of CD19,the study found that in B-ALL,there was clear correlation between the level of PAX5 expression and that of CD19,which was also studied by real-time RT-PCR.BSAP expression by Western Blotting analysis was also performed in haematological tumor cells,including 6 haematological tumor cell lines and 4 clinical samples(2 cases of B-ALL,1 case of T-ALL,and 1 case of AML).The results of Western Blotting analysis showed a 52-KD BSAP band in B lineage cells,but not in T- and myeloid lineage cells.The intensity of BSAP bands was in accordance with PAX5 mRNA expression level detected by real-time RT-PCR.It was concluded that PAX5 transcripts are readily detectable and quantified in clinical materials with B-ALL by real-time RT-PCR.The strong PAX5 mRNA expression in some B-ALL can be considered to be particularly interesting for further analysis.

Activation of the heat shock transcription factor by hypoxia in normal and tumor cell lines in vivo and in vitro

1992 ◽  
Vol 23 (4) ◽  
pp. 891-897 ◽  
Author(s):  
Amato J. Giaccia ◽  
Elizabeth A. Auger ◽  
Albert Koong ◽  
David J. Terris ◽  
Andrew I. Minchinton ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Tumor Cell ◽  
Cell Lines ◽  
Heat Shock Transcription Factor ◽  
Tumor Cell Lines

scholarly journals Real-Time RT-PCR Quantification of Human Telomerase Reverse Transcriptase Splice Variants in Tumor Cell Lines and Non–Small Cell Lung Cancer

2007 ◽  
Vol 53 (1) ◽  
pp. 53-61 ◽  
Author(s):  
Eleni Mavrogiannou ◽  
Areti Strati ◽  
Aliki Stathopoulou ◽  
Emily G Tsaroucha ◽  
Loukas Kaklamanis ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Real Time ◽  
Cell Lines ◽  
Splice Variant ◽  
Splice Variants ◽  
Telomerase Activity ◽  
Tumor Cell Lines ◽  
Rt Pcr ◽  
Human Telomerase Reverse Transcriptase ◽  
Tissue Samples

Abstract Background: We developed and validated a real-time reverse transcription (RT)–PCR for the quantification of 4 individual human telomerase reverse transcriptase (TERT) splice variants (α+β+, α−β+, α+β−, α−β−) in tumor cell lines and non–small cell lung cancer (NSCLC). Methods: We used in silico designed primers and a common TaqMan probe for highly specific amplification of each TERT splice variant, PCR transcript–specific DNA external standards as calibrators, and the MCF-7 cell line for the development and validation of the method. We then quantified TERT splice variants in 6 tumor cell lines and telomerase activity and TERT splice variant expression in cancerous and paired noncancerous tissue samples from 28 NSCLC patients. Results: In most tumor cell lines, we observed little variation in the proportion of TERT splice variants. The α+β− splice variant showed the highest expression and α−β+ and α−β− the lowest. Quantification of the 4 TERT splice variants in NSCLC and surrounding nonneoplastic tissues showed the highest expression percentage for the α+β− variant in both NSCLC and adjacent nonneoplastic tissue samples, followed by α+β+, with the α−β+ and α−β− splice variants having the lowest expression. In the NSCLC tumors, the α+β+ variant had higher expression than other splice variants, and its expression correlated with telomerase activity, overall survival, and disease-free survival. Conclusions: Real-time RT-PCR quantification is a specific, sensitive, and rapid method that can elucidate the biological role of TERT splice variants in tumor development and progression. Our results suggest that the expression of the TERT α+β+ splice variant may be an independent negative prognostic factor for NSCLC patients.

The NF-κB transcription factor and cancer: high expression of NF-κB- and IκB-related proteins in tumor cell lines

1994 ◽  
Vol 47 (1) ◽  
pp. 145-149 ◽  
Author(s):  
Vincent Bours ◽  
Emmanuel Dejardin ◽  
Francine Goujon-Letawe ◽  
Marie-Paule Merville ◽  
Vincent Castronovo
Keyword(s):  
Transcription Factor ◽  
Tumor Cell ◽  
Cell Lines ◽  
High Expression ◽  
Tumor Cell Lines ◽  
Related Proteins

Patulin Acts Synergistically with Doxorubicin and Bortezomib in Cytotoxicity against Hematological Malignancies

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2664-2664
Author(s):  
Alex Hessel ◽  
Malefa Tselanyane ◽  
Fengrong Wang ◽  
Ebenezer David ◽  
Sagar Lonial ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Cell ◽  
B Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Mechanism Of Action ◽  
Primary Tumor ◽  
Dose Effect ◽  
Tumor Cell Lines ◽  
Synergism And Antagonism

Abstract Patulin (4-Hydroxy-4H-furo 3,2-C-pyran-2(6H)-one) is a first-in-class mycotoxin under development as a novel chemotherapeutic agent. The mechanism of action of Patulin has been reported to include activation of mitogen activated protein kinases (MAPKs) and generation of reactive oxygen species (ROS). We have previously shown Patulin to have activity against myeloma, leukemia, and lymphoma cell lines, as well as primary tumor cells in clinical samples from patients with these diagnoses (Wang BLOOD 2007). Moreover, we have shown that Patulin specifically and potently targets tumor cells over normal cells and effectively killed primary tumor cells of patients with refractory illness. The aim of this study was to determine whether Patulin acts synergistically with the topoisomerase inhibitor doxorubicin or the proteasome inhibitor bortezomib. We hypothesize that drugs with non-cross-reactive modes of action could be complementary. Human tumor cell lines from B-cell lymphoma (DAUDI), T-Cell leukemia and lymphoma (Jurkat and H9, respectively), and myeloid leukemia (HL60) malignancies were tested for their sensitivity to single agents Patulin, bortezomib, and doxorubicin as well as Patulin in combination with the latter two agents (Patulin and bortezomib, P + B; Patulin and doxorubicin, P + D). Cells were treated with a range of concentrations of each single agent and the drugs in combination over 24 hours. Following treatment, cell metabolic activity was assessed using a microculture tetrazolium (MTT) assay and cell viability was assessed by flow cytometry using Annexin V and propidium iodide (PI) staining. Dose-effect curves, median effect plots, and combination index (CI) values were generated in the Compusyn software program for each target cell population. Median-effect doses (IC50s) of individual drugs were interpolated using the y-intercept of median-effect plots. Three dose-effect data points were used to create a range of CI values at different fractions of affect (fa). The lower and upper values of the CI range were used to characterize drug combinations as synergistic, antagonistic, or additive based on Chou’s Symbols for Synergism and Antagonism using CI analysis (Table 1). The role for ROS in the mechanism of action of Patulin was confirmed by flow cytometry showing increased levels of ROS in cell lines following Patulin exposure. Preincubation of cell lines with N-acetyl cysteine (NAC) or concurrent exposure to Patulin and NAC abrogated the cytotoxic activity of the mycotoxin. H9 cells were most sensitive to the effects of Patulin, with an IC50 of 1.2 μM. Combinations of P + B acted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cells; however, P + B also demonstrated moderate antagonism against the Jurkat and H9 cell lines (Table 2). Likewise, combinations of P + D interacted synergistically against Jurkat, H9, DAUDI, and HL60 tumor cell lines while simultaneously demonstrating strong antagonism against the H9 cell line. Patulin kills leukemia and lymphoma cells via generation of intracellular ROS. Synergy of Patulin with either bortezomib or doxorubicin in leukemia and lymphoma cell lines indicates a distinct mechanism of action for the mycotoxin and compared to other chemotherapeutics and supports the rationale for continued development of Patulin as a novel chemotherapeutic mycotoxin. TABLE 1. Chou’s Symbols for Synergism and Antagonism using CI Analysis CI Description < 0.1 Very Strong Synergism 0.1–0.3 Strong Synergism 0.3–0.7 Synergism 0.7–0.85 Moderate Synergism 0.85–0.90 Slight Synergism 0.90–1.10 Nearly Additive 1.10–1.20 Slight Antagonism 1.20–1.45 Moderate Antagonism 1.45–3.3 Antagonism 3.3–10 Strong Antagonism > 10 Very Strong Antagonism TABLE 2. IC50 of single agents patulin, bortezomib, and doxorubicin and CI in hematological cancer cell lines Tumor cell line IC50 of patulin (μM) IC50 of bortezomib (nM) IC50 of doxorubicin (μM) CI: P + B CI: P + D T-Cell Jurkat 1.16 5600 5.2 0.6 -- 1.2 0.1 -- 1.2 H9 1.2 14 0.33 0.4 -- > 1 0.3 -- > 1 B-Cell DAUDI 0.98 1.1 0.19 < 0.1 -- 0.1 < 0.1 -- 0.3 Myeloid HL60 1.7 0.42 0.07 < 0.1 -- 0.1 < 0.1

scholarly journals Phosphorylation by c-Jun NH2-terminal Kinase 1 Regulates the Stability of Transcription Factor Sp1 during Mitosis

2008 ◽  
Vol 19 (3) ◽  
pp. 1139-1151 ◽  
Author(s):  
Jian-Ying Chuang ◽  
Yi-Ting Wang ◽  
Shiu-Hwa Yeh ◽  
Yi-Wen Liu ◽  
Wen-Chang Chang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Tumor Cell ◽  
Cell Lines ◽  
Hela Cells ◽  
Kinase Inhibitors ◽  
Degradation Pathway ◽  
Glioma Cell Line ◽  
Tumor Cell Lines ◽  
Epithelial Tumor ◽  
Transcription Factor Sp1

The transcription factor Sp1 is ubiquitously expressed in different cells and thereby regulates the expression of genes involved in many cellular processes. This study reveals that Sp1 was phosphorylated during the mitotic stage in three epithelial tumor cell lines and one glioma cell line. By using different kinase inhibitors, we found that during mitosis in HeLa cells, the c-Jun NH2-terminal kinase (JNK) 1 was activated that was then required for the phosphorylation of Sp1. In addition, blockade of the Sp1 phosphorylation via inhibition JNK1 activity in mitosis resulted in the ubiquitination and degradation of Sp1. JNK1 phosphorylated Sp1 at Thr278/739. The Sp1 mutated at Thr278/739 was unstable during mitosis, possessing less transcriptional activity for the 12(S)-lipoxygenase expression and exhibiting a decreased cell growth rate compared with wild-type Sp1 in HeLa cells. In N-methyl-N-nitrosourea–induced mammary tumors, JNK1 activation provided a potential relevance with the accumulation of Sp1. Together, our results indicate that JNK1 activation is necessary to phosphorylate Sp1 and to shield Sp1 from the ubiquitin-dependent degradation pathway during mitosis in tumor cell lines.

Low or Undetectable Levels of Thrombopoietin Receptor (Tpo-R; c-mpl) Expression on Tumor Cell Lines and Primary Tumors Compared with Epo-R, ErbB2, and IGF-1R

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 5394-5394 ◽  
Author(s):  
Connie L Erickson-Miller ◽  
Antony Chadderton ◽  
Anna Gibbard ◽  
Jennifer Kirchner ◽  
Lone Ottesen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Tumor Cell ◽  
Mrna Expression ◽  
Cell Lines ◽  
American Association ◽  
Breast Tumors ◽  
Tumor Cell Lines ◽  
Qrt Pcr ◽  
Thrombopoietin Receptor ◽  
Low Levels

Abstract INTRODUCTION: Recent discovery of thrombopoietin receptor (Tpo-R; c-mpl) agonists and thrombopoietin mimetics has warranted a better understanding of their effects on solid and liquid tumors. These agents bind to different components of the Tpo-R and therefore signal differently. Previously published data on Tpo-R agonists have shown antiproliferative effects on leukemia and lymphoma cells in vitro (Kalota A, Gewirtz AM. American Association for Cancer Research. 2008. Abstract 2392; Erickson-Miller CL, et al. American Association for Cancer Research. 2008. Abstract 5691). Although the expression of Tpo-R is well documented on cells of the megakaryocyte lineage, there is little quantitative information available on the expression of Tpo-R on tumors. METHODS & RESULTS: To better define Tpo-R expression, we first performed quantitative reverse-transcriptase PCR (qRT-PCR) on 378 tumor cell lines available from the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ). Tpo-R was consistently expressed at low levels, with a mean normalized abundance of 1,447 and a mode of 621. Only 3 cell lines expressed Tpo-R mRNA below the limits of reliable quantitation (SAOS-2, SF-539, and WIDR; bone, brain, and colon tumor cell lines, respectively). In comparison, the erythropoietin receptor (Epo-R) was expressed in low-to-moderate levels (mean, 12,587; mode, 7,811) and ErbB2 was expressed at higher levels (mean, 280,190; mode, 40,828), with expression, as expected, much higher among the breast tumor cell lines. IGF-1R was also expressed at higher levels (mean, 78,977; mode, 56,624). Three cell lines had greater than 9,500 normalized abundance: HEL 92.1.7, KG-1 (2 erythroleukemia cell lines), and NCI-H510 (lung cancer cell line). To determine if these trends also occurred in patient tumor samples, microarray data were examined from 118 breast cancer, 29 non-small cell lung cancer (NSCLC), and 151 renal cell carcinoma (RCC) samples collected prior to treatment in GlaxoSmithKline clinical trials. Robust multiarray average (RMA) analysis was used to determine relative mRNA expression levels. Tpo-R mRNA levels were too low for accurate measurement in all breast cancer and RCC samples, but were detectable at low levels in 14 (48%) NSCLC samples (Table 1). In contrast, Epo-R was expressed in 75% breast cancer samples, in all NSCLC samples, and in 87% RCC samples. ErbB2 was expressed in all breast samples, 97% of the NSCLC, and in 81% RCC samples. IGF-1R was expressed in 86% breast cancer samples, all NSCLC samples, and 54% RCC samples. For breast tumors, the levels of Tpo-R message expression rank as follows: Tpo-R<Epo-R<IGF-1R<ErbB2. To determine the relationship between Tpo-R message expression and Tpo-R protein expression, Western blot analyses were performed on several of the tumor cell lines, including 2 with the highest Tpo-R mRNA expression as determined by qRT-PCR (HEL 92.1.7 and NCI-H510) and 2 with undetectable Tpo-R mRNA expression (ML-2 and NCI-H360). Western blots demonstrated that Tpo-R protein was detectable in the lysates of HEL 92.1.7 cells and normal human platelets, which were used as a positive control. However, Tpo-R protein was not detected in NCI-H510, ML-2, or NCI-H360 cells. Thus, even the high levels of Tpo-R mRNA in NCI-H510 cells did not correlate to detectable Tpo-R protein expression. CONCLUSIONS: In summary, low or undetectable levels of Tpo-R mRNA expression were observed in tumor cell lines and in limited samples of patient tumors, compared with Epo-R, ErbB2, and IGF-1R. In the tumor cells tested, Tpo-R protein was not always detectable, even when Tpo-R mRNA was expressed. Table 1. Number (%) of primary tumor samples with accurately detectable Tpo-R mRNA expression from RMA analysis of microarray data. Breast tumors N = 118 NSCLC N = 29 RCC N = 151 Tpo-R (c-mpl), n (%) 0 14 (48) 0 Epo-R, n (%) 89 (75) 29 (100) 132 (87) ErbB2, n (%) 118 (100) 28 (97) 122 (81) IGF-1R, n (%) 102 (86) 29 (100) 81 (54)

ABT-737 Sensitizes B Cell Tumors for Killing by CD19-Retargeted T Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4032-4032
Author(s):  
Hannah S C Karlsson ◽  
Camilla Lindqvist ◽  
Gabriella Paul-Wetterberg ◽  
Helena Jernberg Wiklund ◽  
Kenneth Nilsson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
B Cell ◽  
Cell Lines ◽  
Single Agent ◽  
Tumor Cell Lines ◽  
Tumor Cell Death ◽  
T Cell Therapy ◽  
Apoptosis Pathways

Abstract Abstract 4032 Introduction: T cells expressing tumor-targeting chimeric antigen receptors are showing promise in clinical trials for patients with B cell leukemia and lymphoma. However, increased levels of anti-apoptotic proteins, a common trait among B-cell tumors, may hamper treatment efficacy. ABT-737 is a small molecule inhibitor of anti-apoptotic proteins such as BCL-2, BCL-xL, BCL-w, and MCL-1, which induces apoptosis via the intrinsic apoptosis pathway in contrast to T-cells that utilize the extrinsic pathway controlled by death receptors and their ligands. ABT-737 has been shown to efficiently promote apoptosis in B-cell tumors as exemplified in models of pre-B-ALL. Recently, ABT-737 was shown to synergize with TRAIL to induce apoptosis. This prompted us to investigate if ABT-737 could be combined with T-cell therapy to enhance tumor cell death. Methods: PBMCs from healthy donors and patients with pre-B-ALL was genetically engineered with a second generation chimeric antigen receptor (CAR) targeting CD19 on B-cells. The T-cells and ABT-737 were tested both individually, and in combination, for their cytotoxic capacity in in vitro assays such as flow cytometry and the Caspase-Glo® 3/7 assay. The effects were studied in a panel of B-cell tumor cell lines (Daudi, U698, Karpas422, DG75, Nall-1) since they may exhibit different apoptosis resistance profiles. The expression of anti-apoptosis molecules in these cell lines was investigated by PCR. Results: PCR confirmed expression of BCL family proteins in the cell lines tested. CD19-targeting T-cells specifically induced apoptosis in CD19+ tumor cells. Similarly, but less efficiently, ABT-737 as single agent increased apoptosis in the various tumor cell lines. When combining T-cell and ABT-737 therapy, the tumor cell death was significantly increased to that of single agent treatment. The effect varied from additive to synergistic effects. The tumor cell lines did not change the level of antigen presenting molecules (MHC I and II), death receptors (Fas) or adhesion or costimulatory molecules (ICAM-I, CD80, CD86) upon ABT-737 treatment. Hence, the effect did not likely represent increased killing by enhanced physical interaction between T-cells and tumors but rather simultaneous engagement of both intrinsic and extrinsic apoptosis pathways. Conclusion: The apoptosis inducer ABT-737 is potently enhancing CD19-targeting T-cell therapy. By triggering both intrinsic and extrinsic apoptosis pathways also resistant tumors may succumb to treatment. Disclosures: Simonsson: Novartis, BMS, Merck, Pfizer: Consultancy, Honoraria.

Phosphodiesterase 10, a novel target in colon cancer.

2014 ◽  
Vol 32 (3_suppl) ◽  
pp. 503-503
Author(s):  
Suzanne Russo ◽  
Nan Li ◽  
Kevin Lee ◽  
Yaguang Xi ◽  
Bing Zhu ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Mouse Model ◽  
Tumor Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Human Tumor ◽  
Clinical Samples ◽  
Mrna Levels ◽  
Tumor Cell Lines ◽  
Sirna Knockdown

503 Background: Elevation of intracellular cGMP is known to inhibit tumor proliferation and induce apoptosis, although the phosphodiesterase (PDE) isozymes that regulate cGMP levels in tumor cells have not been well studied. We report first evidence that PDE10 is elevated in colon tumors compared with normal colon and suggest that PDE10 inhibitors can be used for the treatment or prevention of colon cancer. Methods: PDE10 protein and mRNA levels were measured in human colon tumor cells (HT29, HCT116, SW480, Caco2), normal colonocytes (NCM460), human clinical samples, and ApcMin/+ mouse model. Two chemically distinct PDE10 selective inhibitors, PQ-10 and Pf-2545920, were tested against the cell lines. The NCI-60 panel of human tumor cell lines was also screened against Pf-2545920 to identify potential differences in sensitivity among histologically diverse tumor types. We also performed siRNA knockdown studies in colonocytes and tumor cell lines. To determine the effect of the PDE10 siRNA knockdown on cyclic nucleotide hydrolysis, whole cell lysates from transfected cells were assayed for PDE activity using cGMP or cAMP as substrates. Results: PDE10 levels were low in normal colonocytes (NCM460) and elevated in tumor cell lines. Similarly, PDE10 was elevated human clinical specimens and the ApcMin+/ mouse model compared with normal mucosa. PDE10 inhibitors and siRNA selectively inhibited colonic tumor growth while stable knockdown inhibited colony formation and increased doubling time. Pf-2545920 also supressed growth of all cell lines within the NCI-60 panel. In comparison with lysates from vector control cells, transfection with PDE10 siRNA reduced cGMP hydrolysis by ~35% in both HCT116 and HT29 cell lines, but did not affect cGMP hydrolysis in colonocytes; siRNA did not significantly affect cAMP degradation in all 3 cell lines. Conclusions: PDE10 plays a role in colon tumorgenesis whereby inhibitors can selectively suppress tumor cell growth. The mechanism by which PDE10 inhibition affects growth appears to involve activation of cGMP/PKG signalling. PDE10 represents a novel anticancer target for the treament and prevention of colon cancer.

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