Elsevier

Leukemia Research

Volume 29, Issue 12, December 2005, Pages 1435-1441
Leukemia Research

Decreased expression of CCAAT/enhancer binding protein ζ (C/EBPζ) in patients with different myeloid diseases

https://doi.org/10.1016/j.leukres.2005.05.020Get rights and content

Abstract

CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors that have been implicated in diverse cellular functions such as cellular differentiation and proliferation, and inflammatory processes. C/EBPζ, also known as GADD153, CHOP10, and DDIT3 has been found associated with the development of myxoid liposarcoma and the progression of melanoma. To investigate the correlation of C/EBPζ transcript levels with the development of leukemia, samples from 187 patients with myelodysplastic syndrome (MDS), acute myeloid leukemia (AML), and chronic myeloid leukemia (CML) were examined for C/EBPζ mRNA using real-time quantitative PCR (RQ-PCR). RQ-PCR analysis demonstrated the median levels of C/EBPζ were significantly decreased in MDS, AML, and CML patients compared with normal controls (1.40, 0.96, 2.60 versus 14.69, P < 0.0001). Significant differences were also observed between patients with CML and with AML or MDS. These results suggest that the insufficient dosage of C/EBPζ might be involved in the development of leukemia.

Introduction

The CCAAT/enhancer binding proteins (C/EBPs) are a family of transcription factors with homologous structures as well as functions, and comprised of six members: C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPɛ, and C/EBPζ [1], [2]. These six proteins contain a conserved bZIP domain at the C-terminus and play important roles for diverse cellular responses, including the regulation of cellular differentiation and proliferation, the control of apoptosis, immune and inflammatory processes [3], [4]. In vitro experiments and mouse knockout models revealed the central roles of C/EBPs in normal granulopoiesis. Profound abnormalities of the haematopoietic system are seen in C/EBPα- and C/EBPɛ-null mice. C/EBPα-deficient mice develop an early block in the maturation of granulocytes, with only myeloblasts of the granulocytic lineages in peripheral blood and bone marrow [5]. C/EBPɛ-deficient mice fail to generate functional neutrophils and eosinophils and showed an increased rate of myeloid proliferation and apoptosis [6], [7]. A recent study disclosed that the expression level of C/EBPα transcript was down regulated by AML1-ETO fusion protein and its alteration might be involved in the block in granulocytic differentiation [8]. Furthermore, mutations in C/EBPα including substitution, deletion, insertion, or duplication have been identified in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [9], [10], [11]. Although no mutations in C/EBPɛ were identified in patients with MDS and AML [12], deletion or insertion has been observed in patients with a rare congenital disorder, neutrophil-specific granule deficiency (SGD) [13], [14]. More recently, a study found an important role of C/EBPδ in the maintenance of genomic integrity [15].

C/EBPζ, also named growth arrest and DNA-damage-inducible gene 153 (GADD153), DNA-damage-inducible transcript 3 (DDIT3), CHOP10 [16], [17], is the latest cloned C/EBP family member. The feature of ubiquitous expression indicates its important role in physiological and pathophysiological conditions. The expression of C/EBPζ can be induced by a wide variety of treatments such as DNA alkylation, nutritional deprivation, and radiation, resulting in growth arrest and increased apoptosis [18], [19], [20]. Given the part that C/EBPζ play in the control of cellular growth and cellular differentiation, we assumed that the decreased expression of C/EBPζ cannot induce cell cycle arrest to allow time for the damaged DNA to be repaired before the entrance of the cell cycle from G1 into S phase, resulting in the increased susceptibility of offspring cells to genomic instability and cancer development. In the gene expression profiling of the bone marrow mononuclear cells (BMNCs) from MDS patients, we found that C/EBPζ transcript was markedly down regulated in MDS patients compared with normal controls (Leukemia, submitted). To further explore the potential role of C/EBPζ in the development of leukemia, we examined the expression level of C/EBPζ mRNA in patients with MDS, AML, and chronic myeloid leukemia (CML) by using real-time quantitative PCR (RQ-PCR).

Section snippets

Patients and samples

The BMNCs from 187 individuals with different types of myeloid disorders were studied. The diagnosis of these patients was made according to the French-American-British (FAB) classification [21], [22], [23] combined to karyotyping analysis, including 61 MDS, 54 AML, and 72 CML. The MDS group consisted of 34 cases with refractory anaemia (RA), 20 RA with excess blasts (RAEB), and 7 RAEB in transformation (RAEBt). The AML group consisted of 7 cases with subtype M1, 15 M2, 15 M3, 4 M4, 7 M5, and 6

Sensitivity, reproducibility and efficiency of RQ-PCR

Six runs of amplifications were performed on the limited dilution series of respective plasmids on different days. The maximal sensitivity reached 10 copies/μL for each plasmid, however, the reproducible sensitivity was 100 copies/μL. Coefficient of variation (CV) calculated for each concentration indicated a good reproducibility, varying from 0.46% to 2.39%. The amplified plots and standard curves in one run of the third day are shown in Fig. 1. PCR efficiencies calculated from the slope of

Discussion

We found that the expression levels of C/EBPζ were decreased in primary leukemic cells and cell lines of all types of myeloid malignancies. A prior study also showed that C/EBPζ mRNA was down regulated not only in peripheral blood mononuclear cells but also in granulocytes of CML patients using microarray analysis [27]. Previous publications have revealed that C/EBPζ gene plays an important role in endoplasmic reticulum (ER) stress [1], [2], [4], [20]. Several animal models have further

Acknowledgements

This study was funded a grant (ZS0201) from the Scientific and Technological Bureau of Suzhou.

Contributions. Jun Qian conducted the experimental design and sample detection and wrote the manuscript. Zixing Chen contributed to the experimental design and the revision of manuscript. Jiang Li contributed to the clone of C/EBPζ product. Wei Wang and Jiannong Cen contributed to the sample collection.

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