Evaluation of Focal Adhesion Kinase Gene Expression in Leukemia and Cancer Cell Lines Using Combined Molecular Cytogenetic Investigations and Quantitative Real Time RT-PCR.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4282-4282
Author(s):  
Lydia Campos ◽  
Andrei Tchirkov ◽  
Jerome Cornillon ◽  
Mirella Mihaescu ◽  
Michel Giollant ◽  
...  
Keyword(s):  
Western Blot ◽  
Focal Adhesion Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Focal Adhesion ◽  
Genomic Dna ◽  
Chromosome 8 ◽  
Rt Pcr ◽  
Molecular Cytogenetic ◽  
Chromosomal Origin

Abstract FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase protein implicated in integrin-mediated signal pathways. The human gene fak is located on chromosome 8q near c-myc. Cells that overexpress FAK show increased migration and increased cell survival with apoptosis inhibition. The aim of the present study was to investigate the genomic or post-genomic origin of this overexpression in seven tumor cell lines with different levels of FAK expression: K562, an erythroblastic cell line; U937, a monoblastic cell line; KG1a, a poorly differenciated myeloblastic cell line; HL 60, a promyelocytic cell line; Jurkat, a lymphoblastic cell line; A549, a bronchial adenocarcinoma cell line; M4Beu, a lymph node metastasis melanoma cell line. Total or partial chromosome 8 qualitative or quantitative abnormalities and the presence of double minute chromosomes bearing coding genomic DNA fragments were investigated using both conventional cytogenetic (standard karyotype after R- and G- banding) and molecular cytogenetic such as multicolor karyotype (M-FISH), FISH and comparative genomic hybridization (CGH). The mRNA expression was investigated using quantitative real time RT-PCR and the protein level using immunocytochemistry and western blot (immunoblot) analysis. Except for the Jurkat cell line, karyotypes revealed complex modifications. FISH and M-FISH allowed to precisely identify the chromosomal origin of the genomic material on each chromosome and CGH allowed to characterize relative loss and gain of coding genomic DNA and to identify their chromosomal origin. Summarized results are shown in the following table: No detectable RT-PCR product was observed for HL60 and U937 cell lines. The preliminary results show that there was usually a good correlation between PCR data and western blot analysis. However, FAK overexpression for KG1a cell line most probably has a genomic origin, while in K562 and A549 cell lines the origin is most probably at the transcription or post-transcription level without any abnormality of chromosome 8. Dysregulation of fak gene is observed in hematopoietic as well as solid tulor cell lines, and may contribute to leukemogenesis.</CENTER> Cell line FISH 8q24-qter CGH Chr. 8 RT-PCR RT-Q-PCR Western blot RT-Q-PCR results in copy number ratio K562 Normal Normal + 130.1 ++++ KG1a Amplified enh(8)(q11qter) + 37.5 ++++ Jurkat Normal Normal + 8.7 + (low) U937 Normal enh(8)(pterqter) 0 0 0 HL60 Amplified amp(8)(q23-24) 0 0 0 M4Beu Normal Normal + 43.5 0 A549 Normal Normal + 46 ++++

Newly established Askin tumor cell line and overexpression of focal adhesion kinase in Ewing sarcoma family of tumors cell lines

2003 ◽  
Vol 146 (2) ◽  
pp. 102-109 ◽  
Author(s):  
Hiroshi Moritake ◽  
Tohru Sugimoto ◽  
Hiroshi Kuroda ◽  
Fumio Hidaka ◽  
Yukiko Takahashi ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Focal Adhesion Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
Focal Adhesion ◽  
Ewing Sarcoma ◽  
Tumor Cell Line ◽  
Askin Tumor

MEK 1/2 Inhibitor U0126 Reversed Imatinib Resistance in IM-Resistant K562R

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4914-4914
Author(s):  
Yuping Gong ◽  
Juan Lin ◽  
Ting Niu
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Signaling Pathway ◽  
Cell Lines ◽  
Mtt Assay ◽  
K562 Cell ◽  
Single Agent ◽  
Rt Pcr ◽  
Imatinib Resistance ◽  
Ic50 Values

Abstract Abstract 4914 Objective To study the anti-leukemic activity of MEK inhibitor U0126 alone or in combination with imatinib and explored the reversing mechanism to imatinib resistance in imatinib resistant K562R cell line. Methods Cytotoxicity of drug was detected by the MTT assay in the IM-sensitive cell line K562 and IM-resistant cell line K562R. Western Blot assay were employed to examine the expression of p-cAbl, p-Lyn, p-STAT5, ERK signaling pathway (p-cRaf, p-MEK, p-ERK), PI3K/AKT/mTOR signaling pathway(p-AKT, p-mTOR, p-4EBP1) and the apoptosis related proteins (Bax, Bcl2). The apoptosis rates were analyzed by Annexin V/PI double staining flow cytometry assay. The levels of lyn and erk1/2 gene were assayed by RT-PCR. Results 1. BCR-ABL-independent activation of Lyn and ERK1/2 may be the IM resistant mechanism in K562R cell line. The IC50 value of K562R cell line(1. 505±0. 459 μmol/L) inhibited by imatinib for 72 hours was higher than the values of K562(0. 159±0. 032μmol/L) and the Resistant Fold(RF)was 9. 465. Western Blot assays showed that p-Lyn, p-MEK and p-ERK of ERK pathway, p-mTOR and p-4EPB1 were over-expressed in K562R cell line relative to K562 cell line. However, the levels of p-cAbl, p-STAT5, p-Raf, p-AKT of PI3K/AKT/mTOR were similar in the K562R and K562 cell lines. The treatment of IM could reduce the expression of p-cAbl and p-MEK, but not that of p-Lyn, p-ERK, p-mTOR, p-4EBP1, and even up-regulate the p-Lyn, p-ERK, p-mTOR. The expression of Bax, Bcl2 and the apoptosis rates were the similar in both cell lines. 2. MEK1/2 inhibitor U0126 could reverse the IM resistance in K562R cell line. MTT assay showed single-agent U0126 is more sensitive to K562R than K562. The IC50 values of the two cell lines were 34. 235±5. 658 μmol/L and 85. 824±4. 474 μmol/L respectively. The combination of imatinib and U0126 markedly enhanced inhibitory effect as measured by MTT assay in K562R cell line, combination with 10μmol/L U0126, the IC50 values of IM was 0. 134±0. 059μmol/L, which reduced to 8. 9% of single IM treatment. 3. The reversing mechanism of U0126 to imatinib resistance in K562R cell line. Western Blot showed single IM up-regulated the p-Lyn and p-ERK, while U0126 reduced the expression of them, the combination of the IM and U0126 could synergisticly reduce the p-Lyn expression and neutralize the up-regulation of p-ERK caused by IM single agent. Single IM also up-regulated the p-mTOR in K562R while U0126 reduced it, single IM or U0126 had no influence on p-4EBP1 in K562R, the combination of the two drugs could synergisticly reduce the p-4EBP1 and neutralize the up-regulation of p-mTOR caused by IM single agent. IM but not U0126could reduce p-cAbl and the combination of the two was more effective than IM treatment. IM alone or combination with U0126 could not regulate p-STAT5 expression in K562R. RT-PCR showed that neither IM treatment nor its combination with U0126 could change the level of lyn and erk1, 2 gene in the cell lines. Conclusions 1. BCR-ABL-independent activation of Lyn and ERK1/2 involved in IM resistance mechanism in IM-resistant K562R cell line. Imatinib alone could up-regulated the expression of the p-Lyn, p-ERK, p-mTOR in K562R cell line. MEK1/2 inhibitor U0126 could reverse the IM resistance by reducing the expression of the p-Lyn, p-ERK, p-mTOR, p-4EBP1 of IM-resistant K562R cell line, and the combination of U0216 and Imatinib could synergisticly depress up-regulation of the p-Lyn, p-ERK, p-mTOR and p-4EBP1 caused by IM. Grant support: National Natural Science Foundation of China (No. 30770912), Foundation of the Science & Technology Department of Sichuan Province (No. 2008SZ0017). Disclosures: No relevant conflicts of interest to declare.

Synthesis and Characterization of Quinoline-3-Carboxamide Derivatives as Inhibitors of the ATM Kinase

2020 ◽  
Vol 20 (23) ◽  
pp. 2070-2079
Author(s):  
Srimadhavi Ravi ◽  
Sugata Barui ◽  
Sivapriya Kirubakaran ◽  
Parul Duhan ◽  
Kaushik Bhowmik
Keyword(s):  
Western Blot ◽  
Cancer Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cancer Cell Lines ◽  
Atm Kinase ◽  
Cytotoxicity Studies

Background: The importance of inhibiting the kinases of the DDR pathway for radiosensitizing cancer cells is well established. Cancer cells exploit these kinases for their survival, which leads to the development of resistance towards DNA damaging therapeutics. Objective: In this article, the focus is on targeting the key mediator of the DDR pathway, the ATM kinase. A new set of quinoline-3-carboxamides, as potential inhibitors of ATM, is reported. Methods: Quinoline-3-carboxamide derivatives were synthesized and cytotoxicity assay was performed to analyze the effect of molecules on different cancer cell lines like HCT116, MDA-MB-468, and MDA-MB-231. Results: Three of the synthesized compounds showed promising cytotoxicity towards a selected set of cancer cell lines. Western Blot analysis was also performed by pre-treating the cells with quercetin, a known ATM upregulator, by causing DNA double-strand breaks. SAR studies suggested the importance of the electron-donating nature of the R group for the molecule to be toxic. Finally, Western-Blot analysis confirmed the down-regulation of ATM in the cells. Additionally, the PTEN negative cell line, MDA-MB-468, was more sensitive towards the compounds in comparison with the PTEN positive cell line, MDA-MB-231. Cytotoxicity studies against 293T cells showed that the compounds were at least three times less toxic when compared with HCT116. Conclusion: In conclusion, these experiments will lay the groundwork for the evolution of potent and selective ATM inhibitors for the radio- and chemo-sensitization of cancer cells.

scholarly journals Chinese hamster ovary cell line DXB-11: chromosomal instability and karyotype heterogeneity

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Victoria I. Turilova ◽  
Tatyana S. Goryachaya ◽  
Tatiana K. Yakovleva
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Chromosome 8 ◽  
Ovary Cell ◽  
Differential Staining ◽  
Nucleolar Organizer Regions ◽  
Karyotype Heterogeneity

Abstract Background Chinese hamster ovary cell lines, also known as CHO cells, represent a large family of related, yet quite different, cell lines which are metabolic mutants derived from the original cell line, CHO-ori. Dihydrofolate reductase-deficient DXB-11 cell line, one of the first CHO derivatives, serves as the host cell line for the production of therapeutic proteins. It is generally assumed that DXB-11 is identical to DUKX or CHO-DUK cell lines, but, to our knowledge, DXB-11 karyotype has not been described yet. Results Using differential staining approaches (G-, C-banding and Ag-staining), we presented DXB-11 karyotype and revealed that karyotypes of DXB-11 and CHO-DUK cells have a number of differences. Although the number of chromosomes is equal—20 in each cell line—DXB-11 has normal chromosomes of the 1st and 5th pairs as well as an intact chromosome 8. Besides, in DXB-11 line, chromosome der(Z9) includes the material of chromosomes X and 6, whereas in CHO-DUK it results from the translocation of chromosomes 1 and 6. Ag-positive nucleolar organizer regions were revealed in the long arms of chromosome del(4)(q11q12) and both chromosome 5 homologues, as well as in the short arms of chromosomes 8 and add(8)(q11). Only 19 from 112 (16.96%) DXB-11 cells display identical chromosome complement accepted as the main structural variant of karyotype. The karyotype heterogeneity of all the rest of cells (93, 83.04%) occurs due to clonal and nonclonal additional structural rearrangements of chromosomes. Estimation of the frequency of chromosome involvement in these rearrangements allowed us to reveal that chromosomes 9, der(X)t(X;3;4), del(2)(p21p23), del(2)(q11q22) /Z2, der(4) /Z7, add(6)(p11) /Z8 are the most stable, whereas mar2, probably der(10), is the most unstable chromosome. A comparative analysis of our own and literary data on CHO karyotypes allowed to designate conservative chromosomes, both normal and rearranged, that remain unchanged in different CHO cell lines, as well as variable chromosomes that determine the individuality of karyotypes of CHO derivatives. Conclusion DXB-11and CHO-DUK cell lines differ in karyotypes. The revealed differential instability of DXB-11 chromosomes is likely not incidental and results in karyotype heterogeneity of cell population.

scholarly journals Sirtuin 2 in Endometrial Cancer: A Potential Regulator for Cell Proliferation, Apoptosis and RAS/ERK Pathway

2020 ◽  
Vol 19 ◽  
pp. 153303382098078
Author(s):  
Yanjuan Guo ◽  
Nannan Zhao ◽  
Jianli Zhou ◽  
Jianxin Dong ◽  
Xing Wang
Keyword(s):  
Cell Proliferation ◽  
Endometrial Cancer ◽  
Western Blot ◽  
Cell Line ◽  
Cell Lines ◽  
Erk Pathway ◽  
Uterine Epithelial Cell ◽  
Knock Down ◽  
Ishikawa Cells ◽  
And Control

Objective: The present study aimed to explore the function of sirtuin 2 (SIRT2) on cell proliferation, apoptosis, rat sarcoma virus (RAS)/ extracellular signal-regulated kinase (ERK) pathway in endometrial cancer (EC). Methods: SIRT2 expression in human EC cell lines and human endometrial (uterine) epithelial cell (HEEC) line was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot. SIRT2 knock-down and control knock-down plasmids were transfected into HEC1A cells, respectively; SIRT2 overexpression and control overexpression plasmids were transfected into Ishikawa cells, respectively. After transfection, SIRT2, HRas proto-oncogene, GTPase (HRAS) expressions were evaluated by RT-qPCR and western blot. ERK and phosphorylated ERK (pERK) expressions were evaluated by western blot. Meanwhile, cell proliferation and cell apoptosis were measured. Results: Compared to normal HEEC cell line, SIRT2 mRNA and protein expressions were increased in most human EC cell lines (including HEC1A, RL952 and AN3CA), while were similar in Ishikawa cell line. In HEC1A cells, SIRT2 knock-down decreased cell proliferation but increased apoptosis. In Ishikawa cells, SIRT2 overexpression induced cell proliferation but inhibited apoptosis. For RAS/ERK pathway, SIRT2 knock-down reduced HRAS and inactivated pERK in HEC1A cells, whereas SIRT2 overexpression increased HRAS and activated pERK in Ishikawa cells, suggesting that SIRT2 was implicated in the regulation of RAS/ERK pathway in EC cells. Conclusion: SIRT2 contributes to the EC tumorigenesis, which appears as a potential therapeutic target.

scholarly journals Candida albicans Expresses a Focal Adhesion Kinase-Like Protein That Undergoes Increased Tyrosine Phosphorylation upon Yeast Cell Adhesion to Vitronectin and the EA.hy 926 Human Endothelial Cell Line

2002 ◽  
Vol 70 (7) ◽  
pp. 3804-3815 ◽  
Author(s):  
Giorgio Santoni ◽  
Roberta Lucciarini ◽  
Consuelo Amantini ◽  
Jordan Jacobelli ◽  
Elisabetta Spreghini ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Candida Albicans ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Yeast Cell ◽  
Focal Adhesion Kinase ◽  
Cell Line ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Human Endothelial Cell Line

ABSTRACT The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of αvβ3 and αvβ5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.

scholarly journals Elevated Expression of the Testis-specific Gene WBP2NL in Breast Cancer

2015 ◽  
Vol 7 ◽  
pp. BIC.S19079 ◽  
Author(s):  
Seyedmehdi Nourashrafeddin ◽  
Mehdi Dianatpour ◽  
Mahmoud Aarabi ◽  
Maryam Beigom Mobasheri ◽  
Golnesa Kazemi-oula ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Line ◽  
Cell Lines ◽  
Specific Gene ◽  
Rt Pcr ◽  
Noncancerous Tissue ◽  
Ww Domain ◽  
Group I ◽  
Elevated Expression ◽  
Cancer Tissues

Breast cancer is one of the most common causes of cancer death in women; therefore, the study of molecular aspects of breast cancer for finding new biomarkers is important. Recent studies have shown that WW domain-binding protein 2 (WBP2) is important for the oncogenic property of breast cancer. WWP2 N-terminal-like ( WBP2NL) is a testis-specific signaling protein that induces meiotic resumption and oocyte activation events. Our previous study revealed that WBP2NL gene expression is elevated in actively dividing cells and it might be associated with cellular proliferation and tumorigenic process. However, the clinical relevance and importance of WBP2NL gene in cancer has not been understood yet. Therefore, we were interested in analyzing the expression of WBP2NL gene in human breast cancer tissues and breast cancer cell lines, for the first time. We used reverse transcription-polymerase chain reaction (RT-PCR) and semi-nested RT-PCR to evaluate the expression of WBP2NL in malignant breast cancer and adjacent noncancerous tissue (ANCT) samples, as well as MCF-7 and MDA-MB-231 cell lines. The WBP2NL gene was expressed in 45 out of 50 (90%) breast cancer tissues and overexpressed in the MDA-MB-231 cell line. We suggest that WBP2NL may play roles in breast cancer activation maybe through binding to a group I WW domain protein. The elevated expression of WBP2NL gene in breast cancer and MDA-MB-231 cell line leads us to suggest that WBP2NL might be considered as a novel prognostic factor for early diagnosis of breast cancer.

scholarly journals Knockdown of NOLC1 Inhibits PI3K-AKT Pathway to Improve the Poor Prognosis of Esophageal Carcinoma

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Fanguo Kong ◽  
Yansheng Shang ◽  
Xingyuan Diao ◽  
Jiaguo Huang ◽  
Hui Liu
Keyword(s):  
Overall Survival ◽  
Western Blot ◽  
Esophageal Carcinoma ◽  
Cell Line ◽  
Cell Lines ◽  
Poor Prognosis ◽  
Caspase 3 ◽  
Cyclin B1 ◽  
Akt Pathway ◽  
Pi3k Inhibitor Ly294002

Objective. Esophageal carcinoma (ESCA) is a common malignant gastrointestinal tumor. The abnormal expression of NOLC1 is involved in the tumorigenesis of various human tumors, whereas the function and mechanism of NOLC1 in ESCA remain unclear. In this study, we explored the relationship between NOLC1 and poor prognosis of ESCA, and its role and mechanism in the occurrence of ESCA. Methods. The NOLC1 expression in ESCA tissues and cell lines was determined by qRT-PCR, immunohistochemistry, or western blot. The Kaplan–Meier method was conducted to estimate the overall survival. Cox regression analysis was carried out to examine the association between patient characteristics and prognosis. A recombined lentiviral vector containing NOLC1 was applied for transfecting ESCA cells (Eca109 and TE-13) and established a stable cell line with low NOLC1 expression or high NOLC1 expression, in the absence or presence of PI3K inhibitor (LY294002) treatment. Cell proliferation, apoptosis rate, invasion ability, migration ability, and PI3K/AKT pathway were detected by CCK8 assay, flow cytometry, Transwell assay, wound-healing assay, and western blot. Results. NOLC1 overexpression was observed in ESCA tissues and ESCA cell lines (EC9706, Eca109, TE-13, Kyse170, T.TN) compared with adjacent normal tissues and normal esophageal cell line HEEC. NOLC1 overexpression was markedly associated with bigger tumor size, lymph node metastasis, and advanced TNM stage. Patients with NOLC1 overexpression have shorter overall survival than that of those with low NOLC1 expression. NOLC1 overexpression was considered to be an independent poor prognostic factor affecting overall survival. NOLC1 knockdown inhibited proliferation, migration, invasion, and cyclin B1 expression and promoted the apoptosis and cleaved-caspase-3 expression of Eca109 and TE-13 cells. NOLC1 overexpression accelerated proliferation, migration, invasion, and cyclin B1 expression and inhibited the apoptosis and cleaved-caspase-3 expression of ESCA cells via activating PI3K/AKT pathway. Rescue experiments showed that PI3K inhibitor (LY294002) could reverse the phenomenon caused by NOLC1 overexpression. Conclusion. NOLC1 may be a marker for poor prognosis. It can participate in the occurrence and development of ESCA via the PI3K/AKT pathway.

scholarly journals A Novel, Myeloid Transcription Factor, C/EBPε, Is Upregulated During Granulocytic, But Not Monocytic, Differentiation

Blood ◽  
1997 ◽  
Vol 90 (7) ◽  
pp. 2591-2600 ◽  
Author(s):  
Roberta Morosetti ◽  
Dorothy J. Park ◽  
Alexey M. Chumakov ◽  
Isabelle Grillier ◽  
Masaaki Shiohara ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transcription Factor ◽  
Hematopoietic Stem Cells ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Nb4 Cells

Human C/EBPε is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBPε, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBPε mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBPε was the only C/EBP family member that was easily detected by RT-PCR. No C/EBPε mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBPε. Northern blot and RT-PCR analyses showed that C/EBPε mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBPε protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBPε protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38−), purified from humans had very weak expression of C/EBPε mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBPε mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBPε, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBPε is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

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