A novel form of pRb expressed during normal myelopoiesis and in tumour-associated macrophages

Abstract

The retinoblastoma (Rb) tumour suppressor promotes cell cycle exit, terminal differentiation and survival during normal development and is functionally inactivated in most human cancers. We have identified a novel myeloid-specific form of retinoblastoma protein (pRb), termed deltaRb-p70, that exists in vivo as an N-terminally truncated form of full-length pRb. DeltaRb-p70 appears to be the product of alternative translation and is expressed in primary myeloid cells in fetal liver, bone marrow and spleen. It is also expressed in the human myelomonocytic cell line U937 and is down-regulated as U937s are induced to differentiate. We have also detected deltaRb-p70 expression in primary human breast tumours and we have determined that deltaRb-p70 is specifically expressed in tumour-associated macrophages. These data identify a novel mechanism for regulating pRb expression that is unique to the myeloid system.

Figure 1

Identification of myeloid specific forms of pRb. (a) Immunoblot analysis of pRb, p107 and p130 expression in primary mouse fetal liver. (b) Immunoblot analysis of pRb expression in whole cell lysates from E13.5 mouse embryos. (c) Immunoblot analysis of pRb expression in adult tissues. (d) Immunoblot analysis of pRb expression in flow cytometric sorted populations of fetal liver cells, including differentiating erythroblasts (TER119⁺), haematopoietic progenitors (cKit⁺) and myeloid cells (Mac‐1⁺ Gr1⁺ Mac‐1⁺ F4‐80⁺).

Figure 2

Mechanism of synthesis of ΔRb. (a) Primers spanning the indicated exons of mouse Rb give rise to the predicted fragment sizes when used in RT‐PCR on RNA derived from E15.5 fetal liver. (b) Immunoblot of lysates prepared by direct lysis of fetal liver, bone marrow and spleen in 2× Laemmli buffer. (c) In vitro translation of pRb in TNT rabbit reticulocyte lysates in the presence of exogenously added caspase 1 (lane 2) or caspase 2 (lane 5), caspase inhibitors (YVAD, lanes 3, 4; XVDAD, lanes 6, 7). (d) PCR‐mediated point mutagenesis carried out to mutate specific methionine residues (M1, M107, M202 and M213) to alanine and to introduce a 2‐base‐pair frameshift mutation (FSM) between ATG1 and ATG2 at the KpnI site. Sequence analysis (lower panel) reveals that ATG5 lies within a particularly good Kozak consensus sequence for translational initiation. (e) pRb point mutants were transiently expressed in Rb‐deficient Saos2 osteosarcoma tumour cells and lysates were harvested for immunoblotting to examine pRb expression. (f) Diagrammatic representation of pRb, point mutations introduced and the antibodies used in immunoprecipitations. (g) Immunoprecipitation of endogenous pRb and ΔRb‐p70 with antibodies to E2fs (lanes 2–5) or with domain‐specific anti‐pRb antibodies (lanes 1, 6–9) followed by Western blotting with G3‐245 to pRb.

Figure 3

ΔRb is down‐regulated during myeloid differentiation. (a) Immunoblot of pRb expression in human tumour cell lines: U937 myelomonocytic leukaemia (lane 1), Jurkat T cells (lane 2), MCF7 breast tumour (lane 3), Rb‐deficient MDA‐468 breast tumour (lane 4), HL60 monocytic leukaemia (lane 5), p53‐deficient MDA‐231 breast tumour (lane 6), U2OS osteosarcoma (lane 7), NB7 neuroblastoma (lane 8) and L8057 megakaryoblast (lane 9). (b) Immunoblot analysis of pRb expression in U937s induced to differentiate with 10 nm phorbol 12‐myristate 13‐acetate (PMA). (c) Flow cytometric analysis of Mac‐1 (myeloid‐specific) and F4‐80 (macrophage‐specific) expression in differentiating U937s. (d) Immunoprecipitation with the indicated antibodies from undifferentiated U937s and immunoblotting with G3‐245 antibody for pRB. (e) Electrophoretic mobility shift assays were used to examine E2f and pRB binding in nuclear lysates from undifferentiated U937s (lanes 1–7) or from U937s, induced to differentiate for 24 h with PMA (lanes 8–14).

Figure 4

ΔRb is expressed in tumour‐associated macrophages. (a) Immunoblotting of whole cell lysates from primary human gastric tumours (T) and normal tissue controls (N). (b) Immunoblotting of whole cell lysates from six representative primary human breast tumours with the G3‐245 antibody or the C15 antibody. (c) Immunoblotting of primary tumour and U937 whole cell lysates with the G3‐245 antibody and with an antibody specific to ΔRb‐p70 (Imgenex, IMG‐395). Immunohistochemistry on sections of primary human breast tumours using antibodies specific to: (d) pRb (G3‐245), (e) the proliferation antigen Ki‐67, (f) ΔRb‐p70 (IMG‐395), and (g) myeloperoxidase. All tumours used in this study were provided by the Medical Research Council UK/Cancer Research UK Tissue Bank and their use was approved by the Regional Ethics Committee, Ninewells Hospital, Tayside.