Differential expression of survivin in bone marrow cells from patients with acute lymphocytic leukemia and chronic lymphocytic leukemia

Abstract

Survivin, a member of the inhibitor of apoptosis protein (IAP) gene family, has been detected widely in fetal tissue and in a variety of human malignancies. In the current study, we investigated the expression of IAP family proteins in bone marrow samples from acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and control cases by quantitative real-time RT-PCR method and an immunohistochemical approach. Overexpression of survivin and cIAP2 mRNA was significant in CLL bone marrow cells (P<0.05, respectively) compared with control samples. By immunohistochemistry, survivin was detected in a few scattered myeloid cells in all cases of control bone marrow. Concerning the ALL bone marrow, more than half the cases demonstrated positive expression of survivin (8 out of 13), while the majority of CLL cases (20 out of 21) exhibited intense expression of survivin. The differential subcellular localization of survivin was distinct between ALL and CLL cases. ALL cells essentially revealed nuclear localization of survivin as well as cytoplasmic signals in some cases, while CLL cells from the majority of cases predominantly showed cytoplasmic expression. Next, RT-PCR was performed for the expression of survivin and its splicing variant, survivin-2B and survivin-ΔEx3 in ALL and CLL cells, as the distribution of these variants would be regulated by nuclear/cytoplasmic transport system. In both ALL and CLL bone marrow samples, the expression of wild-type survivin was more predominant than that of survivin-2B or survivin-ΔEx3, although the expression of survivin-ΔEx3 was prominent in samples from survivin-expressing ALL cases. Thus, the splicing of survivin mRNA may be differently regulated in ALL and CLL cells, causing distinct manners of nuclear/cytoplasmic transport of survivin protein. In conclusion, our observations indicate a differential regulatory mechanism for the expression of IAP family proteins in ALL and CLL cells, although the functions of IAP families and the mechanisms of nuclear/cytoplasmic transport of survivin should be clarified in future studies.

Introduction

The regulation of apoptotic cell death may have a profound effect on the pathogenesis and progression of hematological malignancies. Chronic lymphocytic leukemia (CLL) is characterized by clonal expansion of relatively mature B cells with a high percentage of cells arrested in the non-proliferative G0/G1 cell cycle phase [1], [2]. The progressive rise of lymphocytes, despite the very low proportion of proliferating cells, has led to the notion that the pathogenesis of CLL is primarily related to defective apoptosis. In contrast, acute lymphocytic leukemia (ALL) cells exhibit highly proliferative character with a very low percentage of apoptotic cells [1], [3], [4]. Thus, ALL and CLL cells may be regulated by different types of cell-proliferation/cell-death signaling pathway. To begin to clarify the antiapoptotic pathways in lymphocytic leukemias, the expression and modulation of the family of inhibitor of apoptosis proteins (IAPs), especially survivin, were investigated and compared in control, ALL and CLL bone marrow samples.

Survivin is expressed widely in fetal tissues, but becomes restricted during development, and appears to be negligibly expressed in the majority of terminally differentiated adult tissues [5], [6]. However, analysis of the differences in gene expression between normal and tumor cells has revealed that survivin is one of the genes most consistently overexpressed in tumor cells relative to normal tissue [7]. In fact, survivin is prominently expressed in transformed cell lines and in many of the human cancers including hematopoietic cell tumors [8].

As with other IAP family proteins, survivin blocks apoptosis induced by a variety of apoptotic triggers [9], [10]. Although the exact biochemical mechanism by which survivin suppresses apoptosis has been debated, survivin is known to bind directly to and inhibit caspase-3 and -7, which act as terminal effectors in apoptotic protease cascades [10], [11]. Survivin is usually detected in the cytoplasm of tumor cells, and is therefore widely regarded as a cytoplasmic protein [5], [12], [13]. However, several studies have shown nuclear accumulation of survivin in gastric cancer cells [14] and lung cancer cells [15]. Thus, the mechanisms that control its nuclear-cytoplasmic localizations in tumor cells are still controversial.

Many cellular proteins either reside in the nucleus or shuttle between the nucleus and the cytoplasm across the nuclear envelope. In a recent study, survivin was shown to be a nuclear shuttling protein that was actively exported from the nucleus via the chromosome region maintenance 1 (CRM1)-dependent pathway [15]. CRM1 was shown to be a receptor for the nuclear export signal that bound to the nuclear export sequences of the proteins. Thus, the molecular export sequences are very important in determining the subcellular localization of proteins. Differences in the amino acid sequence of the carboxy-terminal domain of survivin determine the dramatically different localization of survivin and its splice variant, survivin-ΔEx3. Survivin-ΔEx3 lacks exon 3 but has additional sequences that could mediate its strong nuclear accumulation. Therefore, wild-type survivin localizes to the cytoplasm, while survivin-ΔEx3 accumulates in the nucleus.

Here, in the present study, overall survivin expression was significantly up-regulated in the bone marrow cells from ALL and CLL compared with the control bone marrow. However, different localization of survivin was shown by the nuclear expression in ALL and the cytoplasmic expression in CLL. Expression of other IAPs including NAIP, cIAP1, cIAP2 and XIAP, all of which appeared to suppress apoptosis by caspase and procaspase inhibition [16], [17], [18], [19] was also determined in these samples and the significance of IAP family protein expression in lymphocytic leukemias was discussed.