Down-Regulation of PU.1 Gene in Pediatric Acute Lymphoblastic Leukemia Patients from South of Iran.

Background: Acute lymphoblastic leukemia (ALL) is resulted from the infiltration of high amount of non-differentiated cells in bone marrow. Differentiation of the hematopoietic stem cells into specific cell lineage occurs through a highly regulated pathway which is mainly monitored during transcription step. Expression level and pattern of transcription factors e.g. PU.1 determine fate and developmental phases in this pathway. This study was performed to evaluate the expression level of the PU.1 gene in a group of children suffering from ALL. Materials and Methods: The mRNA expression level of the PU.1 gene was compared between 30 children diagnosed as new cases of ALL and 30 sex- and gender-matched healthy children in the present case-control study. The quantitative real time PCR (qRT-PCR) was used to determine the level of PU.1 gene expression. The data were analyzed using Graph Pad Prism statistical software. Results: The mRNA level of the PU.1 gene was significantly lower in the blood samples of the ALL patients compared to the controls (p= 0.002). Conclusion: The results of the study indicated that the PU.1 gene seemed to have key roles in the differentiation pathway of blood cells.


INTRODUCTION
All blood cell types originate from multi-potent hematopoietic stem cells (HSCs) through a highly orchestrated process, called hematopoiesis 1 . Any disturbance in this highly conserved critical process deregulated the cell decision for proliferation or differentiation which may result in human cancers such as leukemia 2 . Among all types of leukemia, acute lymphoblastic leukemia (ALL) is the most common malignancy in young children. It is characterized by a block of B-and T-cell differentiation and the accumulation of immature B and T lymphoblast, which are called Blymphoblastic ALL and T-cell ALL, respectively 3 . Transcriptional regulation seems crucial for maintaining the sequential stages of the  4,5 . The transcription factor, purine-rich box1 (PU.1 or Spi-1), belongs to the E26 transformation-specific (ETS) family and shows to have a critical role in the health of hematopoiesis 6 . PU.1 gene is differentially expressed in various hematopoietic cells and regulates hematopoiesis in dose-dependent manner 6 . The expression level of PU.1 represents an ascending gradient toward the development of lymphoblast to mature B-cells, but shows attenuation toward the more committed T cells [7][8][9] .
Regarding previous reports on the influence of altered expression of PU.1 on erythroleukemia 10 and myeloid leukemia 11 , we aimed to clarify whether the PU.1 gene expression modification affects B-and T-cell lymphopoiesis and involves in acute lymphoblastic leukemia (ALL) malignancy.

MATERIALS AND METHODS Subjects
This case-control study included 30 new cases of pediatric ALL (mean ± SD: 8.03 ± 3.99 years; 2-17 years) hospitalized in Amir Oncology Hospital, Shiraz, Iran. Twenty-one out of 30 patients were diagnosed as having B-lymphoblastic ALL and the 9 remaining patients were diagnosed with T-cell ALL. Immunology and cytogenetic tests and microscopic observation of the cell morphology were used to confirm the diagnosis. Patients with the age more than 20 years, personal history of any bleeding disorders or other malignancies, history of radiotherapy or chemotherapy at the time of sampling or earlier time, and relapsed ALL were excluded from the study. Thirty healthy subjects (7.86 ± 4.15 years) matched by age and gender were also investigated as a control group. According to all parents in an oral interview, none of the children had prior history of malignancies or bleeding disorders. Written informed consent was signed by all parents (cases and controls) and the procedures of the project were definitely explained to those parents who were interested in detailed information. The study was approved by the Institutional Review Board of our department.

RESULTS
Baseline characteristics of the study population are presented in Table 1. The disease was more frequent in males than in females (63.3% vs. 36.7%).

PU.1 gene expression analysis
Comparison of the expression level of the PU.1 gene between children with ALL and healthy children resulted in the significant difference (p=0.002). The reduced expression level of PU.1 gene was observed in ALL group compared with the control group ( Figure 1).

DISCUSSION
To the best of our knowledge, this is the first report of investigating the expression level of PU.1 gene in pediatric ALL patients in Iran. The results are more considerable since all the ALL patients were described as new cases. Down-regulation of PU.1 gene in our patients creates new insight into the importance of transcription regulation in the pathogenesis of ALL and introduces this gene as a probable molecular target in designing new treatment strategies. Normal hematopoiesis is securely controlled by a small number of lineage-specific transcription factors, so that the disturbed expression or function of this group may be involved in the development of leukemia 12 15 . Despite previous studies on the reduced expression of the PU.1 gene in the CML patients, the direct investigation of PU.1 mRNA level in the blood samples of the ALL patients was not performed 16,17 . But the reduced PU.1 expression was evaluated in bone marrow cells of ALL patients and the related mice models, with a presumption of AML1-ETO involvement 18 or Notch signaling 19 . Heterozygous deletion of the PU.1 locus was supposed to be associated with human AML. In two distinct studies, 126 and 120 AML patients were analyzed using direct sequencing 20,21 . Quantitative real-time PCR; however, revealed a decrease in gene expression 21 . Aside from mutations, a SNP in a distal enhancer of PU.1 gene was also reported a reduction in the PU.1 gene expression 22 . Although it was out of our research scope to look for rare PU.1 mutations in human samples, the lower expression of the gene was observed in our study. A similar finding was also observed in acute promyelocytic leukemia (APL) and considered to be in correlation with APL initiation 23 and progression 24 . The most recent attempt for elucidating the mechanism of PU.1 protein provided this idea that PU.1 supports TRAIL-induced cell death by affecting an NF-κB-related pathway in AML 25 , or a trichostatin A-induced apoptosis in collaboration with Bim during lymphoma 26 . We suggest other researchers share their clinical data International Journal of Hematology Oncology and Stem Cell Research ijhoscr.tums.ac.ir along with their genetic analysis for PU.1 and other newly discovered genes. Here, we reported that the transcriptional level of PU.1 gene is significantly lower in Iranian pediatric patients with ALL, and more studies with larger sample sizes are required to confirm this finding by providing novel PU.1 associated mechanisms.