Expression and phosphorylation status of retinoblastoma protein in adult T-cell leukemia/lymphoma

Abstract

The deletion or hyperphosphorylation of the retinoblastoma protein (pRB), is reported to progress various tumors. But its relevance to adult T-cell leukemia/lymphoma (ATL) remains to be elucidated. To better understand the role of pRB in ATL, we examined the expression and phosphorylation status of pRB in three ATL cell lines and 43 clinical samples, eight peripheral blood samples and 35 lymph node samples, from patients with ATL by Western blotting. In addition, 30 lymph node sections were also evaluated immunohistochemically. As a result, Western blotting analysis revealed that the pRB in the ATL cell lines was in the hyperphosphorylated, but that in 39 of 43 clinical samples, pRB was exclusively in the hypophosphorylated form. Four peripheral blood samples were negative for pRB. Immunohistochemistry revealed that the lymph nodes of all of 30 patients tested were positive for pRB at various staining levels, weak, mild, and strong. But weak expression may be essentially negative for pRB function. Patients with weak pRB expression in their lymph nodes lived significantly shorter lives than those with mild expression. Surprisingly, patients with strong expression also showed a significantly worse prognosis than those with mild expression. Although only the absence of pRB expression was considered previously to be indicative of RB functional loss, it has been reported recently that overexpression of pRB is correlated with progression of disease in patients with advanced bladder carcinoma or follicular lymphoma. These findings indicate that pRB controls tumor proliferation not only as a cell cycle regulator but also by other mechanisms, possibly through the inhibition of apoptosis, as suggested by recent findings in an osteosarcoma cell line, Saos-2. In conclusion, pRB may play an essential role in its hypophosphorylated form for progression of ATL, as well as a cell cycle promoter in hyperphosphorylated or negative/excessive reduced form.

Introduction

Adult T-cell leukemia/lymphoma (ATL) is a hematological malignancy with poor prognosis [1], but its relationship to oncogenes or tumor suppressor genes remains to be elucidated. Human retinoblastoma susceptibility gene (Rb1) is one of the best characterized tumor suppressor genes; it is localized on chromosome 13q14 and encoded by 27 exons dispersed over 180 kilobases (kb) in the genomic DNA [2], [3]. A 4.7-kb mRNA transcript from this gene is expressed in all normal tissues [4], [5]. The gene encodes a nuclear phosphoprotein termed retinoblastoma protein (pRB), which consists of 928 amino acids [6]. Abnormalities of the Rb1 gene have been reported in a variety of solid cancers, including small cell lung carcinoma [7] and bladder carcinoma [8] as well as retinoblastoma tumors. Deletions and mutations of the RB have been reported in various hematological malignancies, such as acute myeloid leukemia (AML) [9], chronic lymphocytic leukemia [10], chronic myeloid leukemia [11] and lymphomas [12]. Hangaishi et al. reported extensive studies of pRB and its gene expression in various primary lymphoid malignancies, including ATL. They found that five of ten ATL patients lacked pRB, although none of them showed deletion of the RB gene [13]. Hatta et al. showed that homozygous deletion of exon 1 of the RB gene was observed in two of 40 ATL cases [14]. The phosphorylation of pRB, occurring in mid/late G1, reverses its growth-inhibitory effect and enables cells to proceed from G1 to S phase. Hyperphoshorylation in hematological malignancy, such as AML [9], was reported, while this form of pRB is absent in CLL [10]. But the expression and phosphorylation status of pRB in ATL remains to be analyzed.

In this study, we performed Western blotting in three ATL cell lines and 43 clinical samples of peripheral blood or lymph nodes from patients with ATL to examine the phosphorylation status as well as the expression of pRB. Simultaneously, we immunostained 30 lymph node samples of patients with ATL to detect expression and localization of pRB. Both methods cooperated well to analyze the pRB in ATL. Finally, we analyzed the correlation between the survival time and the immunostaining levels of pRB in 30 lymph node samples, because previous reports did not describe such an analysis [13], [14].