Proliferation-dependent expression and phosphorylation of pRB in B cell non-Hodgkin's lymphomas: dependence on RB1 copy number

Abstract

Some studies have suggested that a significant fraction of nonHodgkin's lymphomas (NHL) do not express pRB protein, possibly due to deletions of RB1. We examined RB1/centromere 17 copy number by fluorescent in situ hybridisation, and pRB expression/phosphorylation by immunohistochemistry (IHC) and immunoblotting (IB) in 66 cases of B cell NHL. Thirteen cases had lost one RB1 copy relative to centromere 17 copy number and total DNA content. Case 458/88 had no RB1 copies. pRB levels were heterogeneous as assessed by IB (0.04-1.12 relative units), but all tumours, except for case 458/88, expressed pRB localised to the nucleus in Ͼ75% of the tumour cells by IHC. The fraction of phosphorylated pRB was correlated with pRB expression (r 2 = 0.56, P Ͻ 0.001). The 14 cases with loss of RB1 had lower pRB expression (median 0.25) than those without (median 0.48, P Ͻ 0.001), but a correlation with S phase fraction (r 2 = 0.43, P Ͻ 0.001; previously published data for tumour-specific S phase and apoptotic fractions) indicated that the variation in pRB expression was due to differences in proliferative activity. Furthermore, the regression lines for pRB expression vs S phase fraction were not different for the cases with or without loss of one RB1 copy (P = 0.5). Cases 154/88 (one RB1 copy) and 258/88 (two RB1 copies), in addition to case 458/88, had low expression of (hypophosphorylated) pRB (0.04, 0.08 and 0.04), despite their high S phase fractions (21%, 17% and 21%). There was no association between pRB expression/RB1 copy number and apoptotic fraction. Neither pRB expression nor loss of RB1 had prognostic value, but cases 154/88, 258/88, and 458/88 had short survival times (5, 3 and 46 months, respectively) compared to the others (median survival: 44 months, P = 0.03). It is suggested that pRB expression and function are normal in 63 of 66 NHL cases, including 12 of 13 lymphomas with loss of one RB1 allele.

Retinoblastoma Protein Is Frequently Absent or Phosphorylated in Anaplastic Large-Cell Lymphoma

“…18,37,38 Complete loss of Rb may be because of chromosomal alterations involving the rb gene at 13q14. Homozygous deletions of 13q14 have been detected in a subset of non-Hodgkin's lymphomas, 18,19 but these studies failed to show a correlation between 13q14 chromosomal alterations and Rb expression. In our study we assessed a subset of 25 ALCL tumors for deletions of the rb gene using FISH methods.…”

“…Immunoblots showed a thick band in positive cell lines, consistent with phosphorylated and hyperphosphorylated forms of Rb. 19 Underphosphorylated Rb was detected at low levels in all four ALCL cell lines ( Figure 1A). Serum starvation of SU-DHL1 and Karpas 299 cells resulted in an increased underphosphorylated Rb/total Rb ratio indicating that a subset of these cells underwent cell-cycle arrest through activation of Rb protein ( Figure 1B).…”

“…10,16 In many tumor types, rb gene alterations may result in complete loss or low levels of Rb expression. 4 Homozygous deletions of the 13q14 locus have been detected in a small subset of lymphoid malignancies, [17][18][19] but these studies have failed to show a correlation between 13q14 deletions and Rb expression in these tumors.…”

Response of malignant B lymphocytes to ionizing radiation: Gene expression and genotype

“…The expression was measured with antibodies from Oncogene Research Products (San Diego, CA; clone 1801 anti-TP53, clone EA10 anti-CDKN1A), Zymed (San Francisco, CA; clone 33-170 anti-PLK1), DakoCytomation (Glostrup, Denmark; clone 124 anti-BCL2), Pharmingen (now part of Becton Dickinson, San Jose, CA; polyclonal anti-MCL1, clone PMG3-245 anti-RB1), as described previously. 19,20 Lysates of 500,000 cells were loaded per lane. The amount of actin (ACTB; Santa Cruz, Santa Cruz, CA; clone C-2 anti-actin) was determined on the same blots to control for gel loading, cell concentration and protein degradation.…”

Translocation t(14;18) and gain of chromosome 18/BCL2: effects on BCL2 expression and apoptosis in B-cell non-Hodgkin's lymphomas

“…25,28,33,34 The anti-BCL2 antiserum C-15 (Santa Cruz Biotech, Santa Cruz, CA, USA) and a monoclonal anti-BCL2 antibody kindly provided by DY Mason (Oxford, UK) were used for IHC and IB, respectively.…”