Synergistic Effects of Retinoic Acid and 8-Chloro-Adenosine 3,5- Cyclic Monophosphate on the Regulation of Retinoic Acid Receptor  and Apoptosis: Involvement of Mitochondria Rakesh K. Srivastava, 1, 2 Aparna R. Srivastava, Yoon S. Cho-Chung, and Dan L. Longo Laboratory of Immunology, National Institute on Aging, NIH, Baltimore, Maryland 21224-6825 [R. K. S., D. L. L.], and Medicine Branch [A. R. S.] and Cellular Biochemistry Section [Y. S. C-C.], Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, Maryland 20892 ABSTRACT In advanced or recurrent malignant diseases, retinoic acid (RA) is not effective, even at doses that are toxic to the host. In late stages of breast cancer, patients do not respond to RA because the expression of RA receptor  (RAR) is lost. In the present study, the intracellular mechanism(s) of synergistic effects of RA and a site-selective cyclic AMP (cAMP) analogue, 8-chloro-adenosine 3,5-cyclic mono- phosphate (8-Cl-cAMP), on growth inhibition and apoptosis in breast cancer cells was examined. Our data demonstrated that hormone-dependent MCF-7 cells, but not hormone- independent MDA-MB-231 cells, are sensitive to RA-in- duced growth inhibition and apoptosis. Introduction of the RAR gene into MDA-MB-231 cells resulted in a gain of RA sensitivity. 8-Cl-cAMP acted synergistically with all- trans-RA in inducing and activating RAR gene expression that correlates with the reduction in mitochondrial mem- brane potential, redistribution of cytochrome c, activation of caspases, cleavage of poly(ADP-ribose) polymerase and DNA-dependent protein kinase (catalytic subunit), and in- duction of apoptosis. Mutations in the cAMP response ele- ment-related motif within the RAR promoter resulted in loss of synergy in RAR transcription. In addition, inhibi- tion of RAR expression by an antisense construct also blocked the antitumor effects of RA  8-Cl-cAMP. Thus, RAR can mediate RA and/or cAMP action in breast cancer cells by promoting apoptosis. Therefore, loss of RAR ex- pression may contribute to the tumorigenicity of human mammary epithelial cells. These findings suggest that RA and 8-Cl-cAMP act in a synergistic fashion and may have potential for combination biotherapy for the treatment of malignant diseases. INTRODUCTION Chemoprevention by agents that delay, reverse, or block cancer development is a promising approach to the cancer problem (1). Retinoids (natural and synthetic) are known to possess antiproliferative, differentiative, and immunomodula- tory properties (2). A growing body of evidence from clinical research supports the concept that retinoids are useful sub- stances in the prevention and treatment of cancer. Retinoids, either alone or in combination with biological response modi- fiers or chemotherapy, have proven to be effective against skin diseases including some cancers of the skin, acute promyelo- cytic leukemia, cervical cancer, and other malignancies (2). Furthermore, it is believed that physiological levels of retinoids guard the organism against the development of premalignant and malignant lesions. Retinoid therapy has been shown to prevent the development of second primary cancers among patients with head and neck cancer and lung cancer (3, 4). Retinoids exert their modulatory effects on cell growth by binding to the retinoid receptor nuclear proteins, of which there are two classes (the RARs 3 and the RXRs), each of which has three subtypes (, , and ; Refs. 1 and 2). These receptors display distinct patterns of expression during development and differentiation (1, 2), suggesting that each of them may have distinct and specific functions. All-trans-RA specifically binds and activates RARs, whereas 9-cis-RA binds and activates both RARs and RXRs. In DNA binding and transcriptional activation by ligand, retinoid receptors function as heterodimers of RXR and RAR or as RXR homodimers (2, 5). The retinoid receptors may influence gene transcriptional activation by binding to specific DNA sequences (RAREs and retinoid X response ele- ments; Ref. 6). One of the target genes of retinoid receptors is the gene encoding RAR (5). In its promoter region, a DR5 RARE named -RARE was identified that mediates RA-induced RAR gene expression in many different cell types (7). Auto- Received 12/4/98; revised 3/17/99; accepted 3/18/99. 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. 1 R. K. S. is a recipient of a National Research Council Fellowship. 2 To whom requests for reprints should be addressed, at Laboratory of Immunology, National Institute on Aging, NIH, Box 28, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825. Phone: (410) 558-8480; Fax: (410) 558-8284. 3 The abbreviations used are: RAR, retinoic acid receptor; RA, retinoic acid; PKA, cAMP-dependent protein kinase; MTT, 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide; PARP, poly(ADP-ribose) polymerase; DNA-PK, DNA-dependent protein kinase; DiOC 6 (3), 3,3'- dihexyloxacarbocyanine iodide; 8-Cl-cAMP, 8-chloro-adenosine 3',5'- cyclic monophosphate; TTNPB, (E)-4-[2-(5,5,8,8-tetramethyl-5,6,7,8- tetrahydro-2-naphthalenyl)-1-propenyl]benzoic acid; cAMP, cyclic AMP; CRE, cAMP response element; CREB, cAMP-responsive ele- ment binding protein; RXR, retinoid X receptor; RARE, retinoic acid response element; CAT, chloramphenicol acetyltransferase; PMSF, phenylmethylsulfonyl fluoride; DNA-PKcs, DNA-PK catalytic subunit; MPT, mitochondrial permeability transition. 1892 Vol. 5, 1892–1904, July 1999 Clinical Cancer Research Research. on November 25, 2021. © 1999 American Association for Cancer clincancerres.aacrjournals.org Downloaded from