ABSTRACT

The Polycomb group protein Bmi1 is a constituent of the Polycomb repressive complex 1, and it is an important molecule for the regulation of the self-renewal of hematopoietic stem cells. In the field of clinical hematology, there are reports that the level of Bmi1 expression in blast cells is related to the prognosis of acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome. We investigated whether the level of Bmi1 expression in leukemic cells is related to the prognosis and the characteristics of childhood acute lymphoblastic leukemia. In all the leukemic blast cells, Bmi1 gene expression was lower value than that in normal B cells. There were no correlations between the level of Bmi1 gene expression in leukemic blast cells and other parameters, including prognosis. Here, we report that the level of Bmi1 expression in blast cells is not related to the prognosis of pediatric acute lymphoblastic leukemia.

1. INTRODUCTION

Mammalian Polycomb group (PcG) protein complexes can be classified into 2 distinct types, Polycomb represssive complex 1 (PRC1) and PRC2. The PcG protein Bmi1 is a constituent of PRC1 [1-5]. Bmi1 is an important molecule for the self-renewal of hematopoietic stem cells (HSCs). The expression of Bmi1 is generally high in HSCs but decreases after these cells differentiate [6,7]. Some reports in the field of clinical hematology have shown that the expression level of Bmi1 in blast cells of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and myelodysplastic syndrome (MDS) can be related to the prognosis of the disease [8-10]. According to these reports, leukemia cells that have a high Bmi1 expression are correlated with poor prognosis. Cancer stem cells in AML were identified for the first time in 1997. In that study, CD38–/CD34+ cells together with leukemic cells were injected into non-obese diabetic mice with severe combined immunodeficiency disease, and the injected mice developed AML [11]. Subsequently, stem cells of breast cancer, brain tumor, prostate cancer, colon cancer, and pancreatic cancer were successively reported [12-16]. Although there are reports on the stem cells of acute lymphoblastic leukemia, these are fewer than those on other cancers [17,18]. A high expression of Bmi1, which has been reported for many cancer stem cells, might be the reason for some cancers becoming chemoresistant [19,20]. We investigated whether Bmi1 expression is related to the prognosis and cell properties of pediatric acute lymphoblastic leukemia.

2. MATERIALS AND METHODS

2.1. Patients

Because childhood leukemia is usually precursor B leukemia, we limited our investigation to pediatric precursor B acute lymphoblastic leukemia. According to the Declaration of Helsinki, informed consent and permission were obtained from all patients. Leukemic blast cells were collected from patients who had been diagnosed with pediatric precursor B acute lymphoblastic leukemia at Hiroshima university hospital from January 2002 to September 2006. These leukemic blast cells were used for real-time polymerase chain reaction (RT-PCR). Cells were obtained from only those patients in whom the percentage of leukemic cells was higher than 90%, and only CD19+ cells were chosen.

2.2. Flow Cytometric Cell Sorting

The bone marrow-derived cells were labeled with different antibodies to sort each hematopoietic lineage. CD34+/CD38–, CD34+/CD38–/CD10–, CD34+/CD38+/ CD10+, CD3+, and CD19+ cells were treated as HSCs, common myeloid progenitors, common lymphoid progenitors, T cells, and B cells, respectively. Dead cells that positively stained with propidium iodide were excluded. The cells were sorted using a FACS Aria system (BD Biosciences, Bedford, MA). Sorted cells with a purity of at least 95% were used for further experimentation.

2.3. Real Time RT-PCR

The TaqMan RT-PCR was used for quantitative gene analysis (TaqMan PCR; Applied Biosystems, Foster City, CA). The sequences of the specific primers were as follows: Bmi1, sense primer: 5’-AAA TGC ATC GAA CAA CGA GAA TC-3’ and antisense primer: 5’-AAT GAA GTA CCC TCC ACA AAG CA-3’; GAPDH, sense primer: 5’-GAA GGT GAA GGT CGG AGT-3’ and antisense primer: 5’-CTT TAG GGT AGT GGT AGA AG-3’. The sequences of the 5’-FAM3’-TAMRA labeled probes were as follows: Bmi1, 5’-CAG GTG GGG ATT TAG CTC AG-3’ and GAPDH, 5’-CCG ACT CTT GCC CTT CGA AC-3’. The relative quantification (ΔΔCT method) was used for quantitative analysis.

2.4. Statistical Analysis

The correlation coefficient was analyzed with Pearson product-moment correlation coefficient. Welch’s t test was used for analyzed between 2 groups.

3. RESULTS

Firstly, we purified the cell populations of each differentiation stage, including the HSCs from the bone marrow of healthy volunteers and analyzed the expression of the Bmi1 gene. The expression of the Bmi1 gene is high in HSCs, and decreases when the cells enter the differentiation stage, as shown Figure 1(a). This result is in agreement with a previous report [21]. The inhibition of T-cell differentiation has been reported in bmi1-deficient mice [22]. Therefore, it is thought that bmi1 is essential for T-cell maturation. The expression of the Bmi1 gene was high in T cells in moderation. The expression of the Bmi1 gene did not increase when B cells underwent differentiation. These results are summarized in the schema (Figure 1(b)). The expression of Bmi1 in HSCs is 1.0. The expression of the Bmi1 gene was lower than HSCs at all hematopoietic stages. In other words, the expression of the Bmi1 gene decreased during the differentiation of the hematopoietic cells. In addition, it seems that undifferentiated cells have a high level of Bmi1 expression.

Table 1 summarizes the basic data of all participants such as the peripheral blood white blood cell count, cell properties, and age. Unfortunately, 4 patients (Nos. 12 - 14)