Abstract
Acute lymphoblastic leukemia (ALL) of childhood arises from dysregulated clonal expansion of immature lymphoid precursor cells that fail to differentiate into functional lymphocytes. The cell-cycling status of ALL cells shares many common features with that of normal CD34+ hematopoietic progenitor cells, such as low number of resting G0 and cycling S phase cells even though the growth fraction is high. Thus, ALL cells should be in a long G1 phase. Phosphorylation of the retinoblastoma protein is a crucial step in cell-cycle progression from G0/early G1 to late G1/S phase. We therefore analyzed the G1 distribution of these two immature cell populations by immunostaining and Western blot. Bone marrow samples from children with ALL at diagnosis as well as purified CD34+ cells, before and after in vitro stimulation with cytokines, were investigated for the expression of hypophosphorylated p110RB (early G1 phase), total retinoblastoma protein, statin (G0 phase), bromo-deoxyuridine (S phase), proliferating cell nuclear antigen, and p120 (cycling cells). Compared with unstimulated CD34+ cells (95.8 ± 1.2%) the component of ALL cells containing hypophosphorylated p110RB (16.3 ± 13.2%) was significantly reduced (p = 0.00018), whereas only a minor difference could be detected for the proportion of cycling cells (p = 0.03), and no difference in G0 and S phase cells (p > 0.05). Our results indicate that, as opposed to unstimulated CD34+ cells, the majority of ALL cells are beyond the restriction point and therefore irreversibly committed to DNA replication and mitosis.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- ALL :
-
acute lymphoblastic leukemia
- BrdU :
-
bromo-deoxyuridine
- PCNA :
-
proliferating cell nuclear antigen
- pRB :
-
retinoblastoma protein
- hypo-p110 RB :
-
hypophosphorylated retinoblastoma protein p110RB
- G-CSF :
-
granulocyte colony-stimulating factor
- SCF :
-
stem cell factor
- Epo :
-
erythropoietin
- IP :
-
immunoprecipitates
References
Carroll M, Zhu Y, D'Andrea AD 1995 Erythropoietin-induced cellular differentiation requires prolongation of the G1 phase of the cell cycle. Proc Natl Acad U S A 92: 2869–2873.
Richon VM, Rifkind RA, Marks PA 1992 Expression and phosphorylation of the retinoblastoma protein during induced differentiation of murine erythroleukemia cells. Cell Growth Diff 3: 413–429.
Pardee AB 1989 G1 events and regulation of cell proliferation. Science 246: 603–608.
Hunter T, Pines J 1994 Cyclins and cancer. II: cyclin D and CDK inhibitors come of age. Cell 79: 573–582.
Morgan DO 1995 Principles of CDK regulation. Nature 374: 131–134.
Sherr CJ 1994 G1 phase progression: cycling on cue. Cell 81: 551–555.
Weinberg RA 1995 The retinoblastoma protein and cell cycle control. Cell 81: 323–330.
Riley D, Lee EY, Lee WH 1994 The retinoblastoma protein: more than a tumor suppressor. Annu Rev Cell Biol 10: 1–29.
Goodrich DW, Wang NP, Qian YW, Lee EY, Lee WH 1991 The retinoblastoma gene product regulates progression through the G1 phase of the cell cycle. Cell 67: 293–302.
Weintraub SJ, Prater CA, Dean DC 1992 Retinoblastoma protein switches the E2F site from positive to negative element. Nature 358: 259–261.
Johnson DG, Ohtani K, Nevins JR 1994 Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression. Genes Dev 7: 1514–1525.
La Thangue NB 1994 DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci 19: 108–114.
Hirt A, Antic V, Wang E, Ridolfi Lüthy A, Leibundgut K, von der Weid N, Tobler A, Wagner HP 1997 Acute lymphoblastic leukaemia in childhood: cell proliferation without rest. Br J Haematol 96: 366–368.
Roberts AW, Metcalf D 1995 Noncycling state of peripheral blood progenitor cells mobilized by granulocyte colony-stimulating factor and other cytokines. Blood 86: 1600–1605.
Leitner A, Strobl H, Fischmeister G, Kurz M, Romanakis K, Haas OA, Printz D, Buchinger P, Bauer S, Gadner H, Fritsch G 1996 Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood. Br J Haematol 92: 255–262.
Leibundgut K, von Rohr A, Brülhart K, Hirt A, Ischi E, Jeanneret C, Muff J, Ridolfi Lüthy A, Wagner HP, Tobler, A 1995 The number of circulating CD34+ blood cells predicts the colony-forming capacity of leukapheresis products in children. Bone Marrow Transplant 15: 25–31.
To LB, Haylock DN, Dowse T, Simmons PJ, Trimboli S, Ashman LK, Juttner CA 1994 A comparative study of the phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells. Blood 84: 2930–2939.
Williams CD, Linch DC, Watts MJ, Thomas NSB 1997 Characterization of cell cycle status and E2F complexes in mobilized CD34+ cells before and after cytokine stimulation. Blood 90: 194–203.
Wang E 1989 Statin, a nonproliferation-specific protein, is associated with the nuclear envelop and is heterogeneously distributed in cells leaving quiescent state. J Cell Physiol 140: 418–426.
Ito M, Tsurusawa M, Zha Z, Kawai S, Takasaki Y, Fujimoto T 1992 Cell proliferation in childhood acute leukemia: comparison of Ki-67 and proliferating cell nuclear antigen immunocytochemical and DNA flow cytometric analysis. Cancer 69: 2176–2182.
Freeman JW, Busch RK, Gyorkey F, Ross BE, Busch H 1988 Identification and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120 000 expressed in early G1 phase. Cancer Res 48: 1244–1251.
Moir DJ, Ghosh AK, Abdulaziz Z, Knight PM, Mason DY 1983 Immunoenzymatic staining of haematological samples with monoclonal antibodies. Br J Haematol 55: 395–410.
Shao Z, Ruppert S, Robbins PD 1995 The retinoblastoma-susceptibility gene product binds directly to human TATA-binding protein-associated factor TAFII250. Proc Natl Acad Sci U S A 92: 3115–3119.
Zarkowska TUS, Harlow E, Mittnacht S 1997 Monoclonal antibodies specific for underphosphorylated retinoblastoma protein identify a cell cycle regulated phosphorylation site targeted by CDKs. Oncogene 14: 249–254.
Ludlow JW, Shon J, Pipas JM, Livingston DM, DeCaprio JA 1990 The retinoblastoma susceptibility gene product undergoes cell cycle-dependent dephosphorylation and binding to and release from SV40 large T. Cell 60: 387–396.
Ahuja HG, Jat PS, Foti A, Bar-Eli M, Cline MJ 1991 Abnormalities of the retinoblastoma gene in the pathogenesis of acute leukemia. Blood 78: 3259–3268.
Kornblau SM, Xu H-J, Hu S-H, Beran M, Smith TL, Hester J, Estey E, Benedict WF, Deisseroth AB 1994 Levels of retinoblastoma protein expression in newly diagnosed acute myelogenous leukemia. Blood 84: 256–261.
Hirt A, Leibundgut K, Ridolfi Lüthy A, von der Weid N, Wagner HP 1997 Cell birth and death in childhood acute lymphoblastic leukaemia: how fast does the neoplastic clone expand? Br J Haematol 98: 999–1001.
Acknowledgements
The authors gratefully acknowledge the skilled technical assistance of Christine Steffen.
Author information
Authors and Affiliations
Additional information
Supported by the Swiss National Science Foundation (32-46838.96), the Bernese Cancer League, and the Foundation for Clinical and Experimental Cancer Research, Bern.
Rights and permissions
About this article
Cite this article
Leibundgut, K., Schmitz, N., Tobler, A. et al. In Childhood Acute Lymphoblastic Leukemia the Hypophosphorylated Retinoblastoma Protein, p110RB, Is Diminished, as Compared with Normal CD34+ Peripheral Blood Progenitor Cells. Pediatr Res 45 (Suppl 5), 692–696 (1999). https://doi.org/10.1203/00006450-199905010-00015
Received:
Accepted:
Issue date:
DOI: https://doi.org/10.1203/00006450-199905010-00015
This article is cited by
-
Differentiation resistance through altered retinoblastoma protein function in acute lymphoblastic leukemia: in silico modeling of the deregulations in the G1/S restriction point pathway
BMC Systems Biology (2016)
-
CDK2 catalytic activity and loss of nuclear tethering of retinoblastoma protein in childhood acute lymphoblastic leukemia
Leukemia (2005)
