Genetic alterations of the retinoblastoma-related gene RB2/p130 identify different pathogenetic mechanisms in and among Burkitt's lymphoma subtypes

Abstract

Alterations of cell cycle-associated genes probably contribute to the pathogenesis of Burkitt's Lymphoma (BL), in addition to c-myc translocation. Mutations disrupting the nuclear localization signal of the retinoblastoma-related gene RB2/p130 have been documented recently in BL cell lines and primary tumors. Given the importance of the RB2/p130 gene in controlling cell growth, mutations of this gene may result in uncontrolled cell proliferation. We tested the expression and genomic organization of the RB2/p130 gene in relation to the proliferative features of a series of BL samples collected from the endemic and sporadic regions, regardless of whether the samples were acquired immune deficiency syndrome (AIDS)-related. The expression of the Rb2/p130, p107, and cell proliferation-related proteins (cyclin A and B) was determined by immunohistochemistry. The structures of exons 19 through 22 of the RB2/p130 gene, encoding for the B domain and C terminus, were analyzed by polymerase chain reaction (PCR) analysis and single-strand conformation polymorphism (SSCP) technique. The direct PCR products were sequenced to identify the actual mutations. Our results suggest that BL is composed of a mixture of molecular types with distinct genetic and phenotypic patterns, probably resulting from different pathogenetic mechanisms. In endemic BL, the RB2/p130 gene is mutated in most of the cases, and the protein is restricted to the cytoplasm. In AIDS-related BL, high levels of nuclear expression of the wild-type pRb2/p130, p107, and cell proliferation-related proteins were detected. This finding is in line with the molecular mechanisms observed in virus-linked oncogenesis. Sporadic BLs were mainly characterized by the low nuclear values of the wild-type pRb2/p130 and, conversely, the high values of p107. The increased cell proliferation due to different alterations of cell growth control by Rb-related proteins may be the first step in lymphomagenesis, during which additional genetic changes, including missense mutations of c-myc, may subsequently occur.

Figure 1.

Insertions and point mutation found in RB2/p130 gene sequences of BLs, which affect a bipartite NLS. The nucleotide localization is derived from the numbering scheme used by Mayol et al.

Figure 2.

Comparison of percentages of nuclear staining of pRB2/p130 and p107 for each case. Top and right side of the plot report the probability density functions (p.d.f.) of the variables for which the cut-off values were determined.

Figure 3.

Immunostaining of pRb2/p130 and p107 in BL. a and b: Cases with cytoplasmic staining of pRb2/p130 (a) and corresponding values of p107 <45% (b), pRb2^-/p107^-/RB2mutated; c and d: cases with nuclear values of pRb2/p130 <45% (c) but values of p107>45% (d), pRb2^-/p107⁺/RB2 wild type; e and f: cases with nuclear values of both pRb2/p130 (e) and p107 (f) > 45%, pRb2⁺/p107⁺/RB2 wild type. ENVISION⁺/HRP method (see text); original magnifications, ×970 (a and b); ×850 (b–f).