Immortalization of primary human keratinocytes by the helix–loop–helix protein, Id-1

Immortalization of primary human keratinocytes by the helix–loop–helix protein, Id-1

^*Department of Pathology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115; ^†Department of Dermatology, Harvard Medical School, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114; and ^¶Department of Neurosurgery, Preuss Laboratory of Molecular Neurooncology, University of California, San Francisco, CA 94143

Edited by Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, and approved June 22, 1999 (received for review February 11, 1999)

Abstract

Basic helix–loop–helix (bHLH) DNA-binding proteins have been demonstrated to regulate tissue-specific transcription within multiple cell lineages. The Id family of helix–loop–helix proteins does not possess a basic DNA-binding domain and functions as a negative regulator of bHLH proteins. Overexpression of Id proteins within a variety of cell types has been shown to inhibit their ability to differentiate under appropriate conditions. We demonstrate that ectopic expression of Id-1 leads to activation of telomerase activity and immortalization of primary human keratinocytes. These immortalized cells have a decreased capacity to differentiate as well as activate phosphorylation of the retinoblastoma protein. Additionally, these cells acquire an impaired p53-mediated DNA-damage response as a late event in immortalization. We conclude that bHLH proteins play a pivotal role in regulating normal keratinocyte growth and differentiation, which can be disrupted by the immortalizing functions of Id-1 through activation of telomerase activity and inactivation of the retinoblastoma protein.

Footnotes

↵ ‡ R.M.A. and J.H. contributed equally to this work.
↵ § To whom reprint requests should be addressed at the present address: Cell Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 241, New York, NY 10021. E-mail: r-cole{at}ski.mskcc.org.
This paper was submitted directly (Track II) to the Proceedings Office.
ABBREVIATIONS:

bHLH,

basic helix–loop–helix;

HFK,

human foreskin keratinocyte;

RT,

reverse transcription;

hTERT,

human telomerase reverse transcriptase;

pRb,

retinoblastoma protein