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¹ Experimental Therapeutics Program, Division of Medical Oncology, College of Physicians and Surgeons and ² Department of Environmental Health Sciences, Mailman School of Public Health, Columbia University Medical Center, New York, New York and ³ Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol, United Kingdom

Requests for reprints: Robert L. Fine, Division of Medical Oncology, College of Physicians and Surgeons, Columbia University Medical Center, Black Building 20-25, 650 West 168th Street, New York, NY 10032. Phone: 212-305-1168; Fax: 212-305-7348. E-mail: rlf20{at}columbia.edu

PRIMA-1 (p53 reactivation and induction of massive apoptosis) is a chemical compound that was originally identified as a selective mutant p53-dependent growth suppressor by screening a library of low-molecular-weight compounds. However, its mechanism of action is unknown. In this study, we examined toxicity of PRIMA-1 to three premalignant human colorectal adenoma cell lines (RG/C2, BR/C1, and AA/C1) and four colorectal carcinoma cell lines (DLD-1, SW480, LOVO, and HCT116) and its mechanism of action. It selectively induced apoptosis only in the mutant p53 premalignant and malignant colon cell lines, but was not toxic to the wild-type p53 premalignant and malignant colon cell lines. Using stable transfectants of temperature-sensitive p53 mutant Ala¹⁴³ in null p53 H1299 lung cancer cells, we found that PRIMA-1 induced significantly more apoptosis in cells with mutant p53 conformation (37°C) than the wild-type p53 conformation (32.5°C). Cell cycle analysis indicated that its inhibition of cell growth was correlated with induction of G₂ arrest. Western blot analysis showed PRIMA-1 increased p21 and GADD45 expression selectively in the mutant p53 cells. However, Fas, Bcl-2 family proteins, and caspases were not involved in PRIMA-1–induced cell death. The c-Jun-NH₂-kinase (JNK) inhibitor SP 600125, but not p38 mitogen-activated protein kinase inhibitor SB 203580 or extracellular signal-regulated kinase inhibitor PD 98059, blocked PRIMA-1–induced apoptosis. Transfection with a dominant-negative phosphorylation mutant JNK, but not a dominant-negative p38 or wild-type JNK, inhibited PRIMA-1–induced cell death, suggesting that the JNK pathway plays an important role in PRIMA-1–induced apoptosis. PRIMA-1 is a highly selective small molecule toxic to p53 mutant cells and may serve as a prototype for the development of new p53-targeting agents for therapy of premalignant and malignant cells.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

⁴ Y. Li and R.L. Fire, unpublished data.

Received 8/13/04; revised 3/16/05; accepted 4/12/05.