Apoptosis and Restriction of G 1 /S Cell Cycle by Fenretinide in Burkitt’s Lymphoma Mutu I Cell Line Accessed with bcl-6 Down-Regulation Tze-chen Hsieh and Joseph M. Wu 1 Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595 Received August 10, 2000 Fenretinide (4-HPR) is a synthetic retinoid with cancer chemopreventative potential and clinically manageable side effects, compared to the prototype retinoid, all-trans retinoic acid (RA). 4-HPR has been shown to modulate cell proliferation and in- duce apoptosis in a variety of human tumor cell types, but its effects on B-cell non-Hodgkin’s lym- phomas (NHL-B) have not been explored. Treatment of Burkitt’s lymphoma Mutu I cells with 3 M 4-HPR is accompanied by growth arrest, induction of apoptosis, and restricted progression of the cell cy- cle at the G 1 /S checkpoint. We also observed that 4-HPR elicited a reduced expression of bcl-6 in these cells, which supports the proposed role of bcl-6 as an anti-apoptotic gene. While 4-HPR treatment had no effect on total Rb gene expression, it significantly reduced the state of hyperphosphorylation of Rb, resulting in the predominant existence of Rb in the underphosphorylated state. © 2000 Academic Press 4-HPR, a synthetic retinoid in which an N-4- hydroxyphenyl group replaces the carboxyl end of the parent molecule RA, demonstrated chemopreventive properties when tested in chemically induced tumors of the bladder, prostate, and skin in rat and mouse mod- els (1–3). In the late 1980’s, 4-HPR entered clinical trials in patients with advanced cancers. These studies produced a range of outcomes, the full significance and clinical importance of which remains to be elucidated. For example, while 4-HPR alone did not affect remis- sion in patients with advanced breast cancer (4), it augmented the efficacy of tamoxifen (5). In prostate cancer patients, treatment with 4-HPR showed effec- tiveness in patients diagnosed with early prostate can- cer (6). In breast, head and neck, and prostate patients receiving surgery as the first-line therapy, post- surgical treatment with 4-HPR prevented the forma- tion of second cancers (7–9). Compared to its parent compound RA, 4-HPR produced more tolerant side ef- fects, particularly hepatotoxicity due to lack of its ac- cumulation in the liver (10). Because it offers promise as a therapeutic agent, in vitro studies have been performed to probe the mech- anism by which 4-HPR may be mediating the observed effects. In a variety of human tumor cell types, 4-HPR not only inhibits cell proliferation but also induces programmed cell death or apoptosis, including cell lines derived from head and neck, non-small lung can- cer, melanoma, prostate, bladder, neuroblastoma, leu- kemia, and cervical carcinoma (11–18). However, the effects of 4-HPR on B-cell non-Hodgkin’s lymphomas (NHL-B) as yet have not been explored. This commu- nication reports results of studies aimed at investigat- ing the cellular effects of 4-HPR in Burkitt’s lymphoma Mutu I cells. These cells have previously been used to study the regulation of bcl-6 (19 –21), a frequently translocated proto-oncogene implicated in the etiology of non-Hodgkin, diffuse large B-cell lymphomas (22– 26). Additional biological functions proposed for bcl-6 include: differentiation (27), tissue and organ develop- ment (28 –31), immune cell activation/maturation (32– 37), transcriptional regulation (20, 38 – 40), and sup- pression of apoptosis (30, 41, 42). Results of our experiments show that cells treated with 4-HPR were growth arrested, exhibited significant apoptosis, and also were characterized by restricted cell cycle progres- sion, at the G 1 /S checkpoint. In support of the proposed role of bcl-6 as an anti-apoptotic gene, exposure of Mutu I cells to 4-HPR also resulted in reduced expres- sion of bcl-6. MATERIALS AND METHODS 4-HPR (supplied by the Johnson Pharmaceutical Research Insti- tute) and all-trans retinoic acid (RA) (purchased from Sigma Chem- icals Co.) were dissolved in absolute ethanol, as 1 and 10 mM stock, respectively. Cell culture and treatment with retinoids. The Mutu I cells were gifts of Dr. Chih-Chao Chang, Columbia University (originally from 1 To whom reprint requests should be addressed. Biochemical and Biophysical Research Communications 276, 1295–1301 (2000) doi:10.1006/bbrc.2000.3617, available online at http://www.idealibrary.com on 1295 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.