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Id3 induces a caspase-3- and -9-dependent apoptosis and mediates UVB sensitization of HPV16 E6/7 immortalized human keratinocytes

Oncogene volume 25, pages 3649–3660 (2006)Cite this article

Abstract

Inhibitor of differentiation/DNA binding (Id) proteins comprise a class of helix–loop–helix transcription factors involved in proliferation, differentiation, apoptosis, and carcinogenesis. We have shown that while Id2 is induced by UVB in primary keratinocytes, Id3 is upregulated only in immortalized cells. We have now determined that the consequences of ectopic expression of Id3 protein are strikingly different between immortalized and primary keratinocytes. Overexpression of Id3 induces a significant increase in apoptotic cells as revealed by Annexin V positivity as well as proteolytic processing of caspase-3 in immortalized, but not in primary keratinocytes. Id3-green fluorescent protein (GFP)-positive cells exhibited a fivefold increase in apoptotic nuclear fragmentation compared to Id3-GFP-negative cells. These apoptotic responses were accompanied by activation of caspase-3, as shown by immunocytochemical staining with antibodies to active caspase-3. Immunostaining with antibodies to the active form of caspase-9 as well as to the active form of Bax further revealed that induction of apoptosis in Id3-overexpressing keratinocytes occurred via a mitochondrial-caspase-9-mediated pathway. Coexpression of dominant-negative caspase-9 with Id3 significantly suppressed apoptotic nuclear fragmentation, indicating that caspase-9 activation is essential for Id3-induced cell death. This response was also markedly attenuated by coexpression with the Bax antagonist antiapoptotic protein Bcl2, confirming a role for Bax activation in this apoptotic response. Id3-induced Bax activation may result from increased expression of Bax protein. Furthermore, reduction of Id3 expression by small interfering RNAs abrogated the UVB-induced proteolytic activation of caspase-3 in these cells. These data together suggest that UVB-induced apoptosis of immortalized keratinocytes is at least in part due to Id3 upregulation in these cells.

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Abbreviations

AK:

actinic keratosis (es)

DN:

dominant-negative

GFP:

green fluorescent protein

HLH:

helix–loop–helix

HPV:

human papillomavirus

Id:

inhibitor of differentiation/DNA binding

KC:

keratinocyte(s)

RT–PCR:

reverse transcription–polymerase chain reaction

SCC:

squamous cell carcinoma(s)

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Acknowledgements

We thank Yuri Lazebnik (Cold Spring Harbor, NY, USA) for the pUC18-caspase-9-DN, and Richard Schlegel (Department of Oncology, Georgetown University) for the HPV16-E6/7 retroviral vectors. Bcl2 expression constructs were a kind gift from Dr Richard Youle, NIH. This work was supported in part by the National Cancer Institute Grant 1RO1 CA100443-01A1 and the US Army Medical Research and Materiel Command contract DAMD17-00-C-0026 (to DSR).

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  1. Department of Biochemistry and Molecular Biology, Georgetown University School of Medicine, Washington, DC, USA

    C M Simbulan-Rosenthal, A Daher, V Trabosh, W-C Chen, D Gerstel, E Soeda & D S Rosenthal

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  1. C M Simbulan-Rosenthal
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  2. A Daher
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Correspondence to D S Rosenthal.

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Simbulan-Rosenthal, C., Daher, A., Trabosh, V. et al. Id3 induces a caspase-3- and -9-dependent apoptosis and mediates UVB sensitization of HPV16 E6/7 immortalized human keratinocytes. Oncogene 25, 3649–3660 (2006). https://doi.org/10.1038/sj.onc.1209407

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