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Ectopic expression of cyclin E allows non-endomitotic megakaryoblastic K562 cells to establish re-replication cycles

Oncogene volume 19, pages 1820–1833 (2000)Cite this article

Abstract

Megakaryocytes become polyploid by entering a truncated cell cycle, consisting of alternate S phases and abortive mitoses. We have investigated the regulation of the G1/S transition by comparing two megakaryoblastic cell lines, HEL and K562, which respectively do or do not become polyploid in response to phorbol esters. A pronounced downregulation of cyclin A, and to a lesser extent of cyclin E, occurred in K562 cells during the first 24 h after TPA treatment, in contrast with re-replicating HEL cells, in which both cyclins were present in individual G2/M cells. Transactivation experiments suggested that the absence of cyclin A in differentiated K562 cells could be due to a TPA-mediated inhibition of its transcription. To investigate the potential role of cyclin E in the establishment of re-replication cycles, we isolated K562 clones constitutively expressing cyclin E. The resulting clones, and also K562 cells transiently expressing cyclin E, entered re-replication cycles when treated with TPA. The transcriptional activity of the cyclin A promoter was not inhibited after TPA treatment, and although the levels of cyclin A fluctuated during further re-replication cycles, they never decreased below S phase levels. We conclude that the presence of cyclin E in megakaryoblastic G2/M cells determines cyclin A expression and allows the entrance into an extra S phase.

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Acknowledgements

We are grateful to Dr Manuel Serrano for crucial suggestions and reagents (p16 expression vector and GST-Rb recombinant protein). We thank Dr Jonathon Pines for polyclonal antibodies against cyclins A and B1, and for the cyclin A expression vector; Dr Steven Reed for cyclin E cDNA; Dr Chris Norbury for cdc2 antibodies; Dr Steven Elledge for p21 cDNA; Dr Joan Massagué for p27 cDNA; and Dr David Mason for anti-glycophorin A (JC159a) and anti-CD61 (Y2/51) monoclonal antibodies. We also thank Anthony Crawford and Carmen Domínguez for technical assistance and Miguel Vidal for critical reading of the manuscript. This work was supported by grants from the Fundación para la Investigación y Formación en Oncología to C Calés, a Wellcome Trust Senior Biomedical Fellowship to J Frampton, a grant from the Association for International Cancer Research to J Frampton, and a joint collaborative grant from the British Council-Ministerio de Educación y Cultura to C Calés and J Frampton. P García was a recipient of a short-term EMBO fellowship.

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  1. Paloma García and Jonathan Frampton: P García and J Frampton contributed equally to this work

Authors and Affiliations

  1. Departamento de Bioquímica, Instituto de Investigaciones Biomédicas ‘A. Sols’, Universidad Autónoma-CSIC, Arturo Duperier, 4. 28029, Madrid, Spain

    Paloma García, Alicia Ballester & Carmela Calés

  2. Institute of Molecular Medicine, John Radcliffe Hospital, Headington, OX3 9DS, Oxford, UK

    Paloma García & Jonathan Frampton

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  1. Paloma García
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García, P., Frampton, J., Ballester, A. et al. Ectopic expression of cyclin E allows non-endomitotic megakaryoblastic K562 cells to establish re-replication cycles. Oncogene 19, 1820–1833 (2000). https://doi.org/10.1038/sj.onc.1203494

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