Journal of Dermatological Science 26 (2001) 67 – 78 Abnormal NF-B signaling pathway with enhanced susceptibility to apoptosis in immortalized keratinocytes Vijaya Chaturvedi a , Jian-Zhong Qin a , Mitchell F. Denning a , Divaker Choubey b , Manuel O. Diaz c , Brian J. Nickoloff a, * a Department of Pathology, Loyola Uniersity Medical Center, Skin Cancer Research Laboratories, Cardinal Bernardin Center, Building c112, 2160 South First Aenue, Maywood, IL 60153, USA b Department of Radiation Oncology, Loyola Uniersity Medical Center, Skin Cancer Research Laboratories, Cardinal Bernardin Center, Building c112, 2160 South First Aenue, Maywood, IL 60153, USA c Department of Medicine, Loyola Uniersity Medical Center, Skin Cancer Research Laboratories, Cardinal Bernardin Center, Building c112, 2160 South First Aenue, Maywood, IL 60153, USA Received 26 June 2000; received in revised form 11 October 2000; accepted 11 October 2000 Abstract The transcriptional activation and proper regulation of NF-B is known to be important to the apoptotic resistant phenotype of epidermal-derived keratinocytes. By comparing and contrasting the responses of normal foreskin- derived keratinocytes versus an immortalized skin-derived keratinocyte cell line (i.e. HaCaT cells), several molecular defects involving NF-B signaling pathway were delineated in the immortalized keratinocytes. While exposure to IFN- plus TPA produces growth arrest in both normal and immortalized keratinocytes, with rapid phosphorylation of MEKKI and recruitment of distinctive protein kinase C isoforms into the signalosome complex, subsequent molecular events necessary for NF-B activation were abnormal in HaCaT cells. This disrupted NF-B activation in HaCaT cells was accompanied by enhanced susceptibility to UV-light induced apoptosis, which was associated with elevated levels of E2F-1 and decreased TRAF1/TRAF2 levels. Additional defects in HaCaT cells included markedly diminished levels of IKK (and lack of induction of kinase activity) in response to inflammatory stimuli, a failure of p21 WAF1/CIP1 to associate with CDK2, and a decreased association between p65 and p300. These studies suggest caution in using HaCaT cells as a substitute for normal keratinocytes to study apoptosis in the skin. Thus, it appears that while the immortalized cells can escape cell cycle checkpoints by elevated levels of E2F-1, an adverse biological consequence of such dysregulated cell cycle control is the inability to activate the anti-apoptotic NF-B signaling pathway. Therefore, exploiting this apoptosis vulnerability in pre-malignant, or immortalized cells, prior to acquiring a death-defying phenotype characteristic of more advanced malignant cell types, provides the basis for an early interventional therapeutic strategy for cutaneous oncologists. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: NF-B; Keratinocytes; Apoptosis; HaCaT cells www.elsevier.com/locate/jdermsci * Corresponding author. Tel.: +1-708-3273241; fax: +1-708-3273239. E-mail address: bnickol@lumc.edu (B.J. Nickoloff). 0923-1811/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII:S0923-1811(00)00157-2