Peter R. Reczek

4HPR triggers apoptosis but not differentiation in retinoid sensitive and resistant human embryonal carcinoma cells through an RARγ independent pathway

Retinoids signal biological effects through retinoic acid receptors (RAR) and retinoid X receptors (RXR) and their co-regulators. We previously reported that all-trans retinoic acid (RA) triggers terminal differentiation in the human embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1), through an RARγ dependent pathway. RARγ repression in NT2/D1-R1 cells accounts for RA resistance in this line. This report finds RARγ repression is due to selective repression of RARγ but not RARβ transcription in NT2/D1-R1 cells. The repression is neither due to mutations in RARγ nor its promoter containing the RA response element. Prior work was confirmed and extended by demonstrating that an RARγ selective agonist preferentially signals differentiation of NT2/D1 cells, while RARα/β, RARβ, RXR agonists and an RAR pan-antagonist do not even when NT2/D1 cells are treated with these retinoids at 10 μM dosages. None of these examined retinoids induced differentiation of the RA resistant NT2/D1-R1 cells. In contrast, N-(4-hydroxyphenyl)retinamide (4HPR), a reported transcriptional activator of RARγ was shown to potently induce growth inhibition and apoptosis in both NT2/D1 and NT2/D1-R1 cells. 4HPR-induced apoptosis was unaffected by co-treatment of both cell lines with equimolar RAR antagonist. Semi-quantitative reverse transcription-polymerase chain reaction (RT – PCR) assays of total RNA from 4HPR-treated NT2/D1 and NT2/D1-R1 cells did not reveal RARγ induction. Since 4HPR signals in RA-resistant NT2/D1-R1 cells having an RARγ transcriptional block, these results indicate that 4HPR triggers apoptosis but not differentiation through an RARγ independent pathway. Taken together, these findings implicate a therapeutic role for 4HPR mediated apoptosis in germ cell tumors even when a maturation block is present.

The Tetramerization Region of the Retinoid X Receptor Is Important for Transcriptional Activation by the Receptor

The retinoid X receptor (RXR), a member of the superfamily of hormone nuclear receptors, is a ligand-inducible transcription factor that is activated by the vitamin A derivative 9-cis-retinoic acid. We previously showed that RXR self-associates into tetramers with a high affinity and that ligand binding induces rapid dissociation of receptor tetramers to smaller species. Here, the RXR region that is responsible for mediating tetramer formation is identified. It is shown that this interface, which we term the “tetramerization domain,” critically contains two consecutive phenylalanine residues located at the C-terminal region of the receptor. Mutation of these residues is sufficient to disrupt RXR tetramers without affecting the overall fold of the protein or interfering with ligand binding, dimer formation, or DNA binding by the receptor. Nevertheless, the tetramer-impaired mutant was found to be transcriptionally defective. The newly characterized tetramerization domain and the previously identified main dimerization interface of RXR act autonomously to affect separate intersubunit interactions that, overall, lead to formation of tetramers. Protein-protein interactions mediated by the tetramerization domain, but not those that involve the dimerization interface, are disrupted following ligand binding by RXR. Overall, these data attest to the specificity of the interaction and implicate the tetramerization interface in playing a direct role in regulating transcriptional activation by RXR.

Retinoid‐Mediated Suppression of Tumor Invasion and Matrix Metalloproteinase Synthesis

ABSTRACT: Cancer mortality usually results from the tumor invading the local environment and metastasizing to vital organs, e.g. liver, lung, and brain. Degradation of the extracellular matrix is, therefore, the sine qua non of tumor cell invasion. this degradation is mediated mainly by MMPs, and thus, inhibition of MMP synthesis is a target for anticancer agents. Tumor cells must traverse both the basement membrane (type IV collagen) and the interstitial stroma (type I collagen). Therefore, we used scanning electron microscopy to examine the invasive behavior of several aggressive tumor cell lines, A2058 melanoma cells, and SCC and FaDu squamous cell carcinomas through these matrices; and we monitored the ability of all‐trans retinoic acid and several RAR‐specific ligands to block invasion. We demonstrate that several retinoids, which are specific RAR α, β, or γ agonists/antagonists, selectively inhibited MMP synthesis in the three tumor cell lines. However, there was not a common pattern of MMP inhibition by a particular retinoid. For instance, a RAR α antagonist suppressed MMP‐1 and MMP‐2 synthesis in the melanoma cell line, but not in the FaDu or SCC‐25 cells. On the other hand, synthesis of MMP‐1 and MMP‐9 by the FaDu cells was affected hardly at all, while a RARγ antagonist reduced the levels of MMP‐2. Only all‐trans retinoic acid reduced MMP‐1 synthesis in these cells. We postulate that the differences may be related to a differential pattern of RAR expression in each of these cells, and that the RARs expressed by each cell line may not be targets of these RAR specific compounds. All‐trans retinoic acid is a pan ligand, binding to all three RARs and, therefore, may modulate gene expression more generally. We conclude that the power of these new ligands lies in their specificity, which can be directed towards modulating expression of certain RARs and, thus, of certain MMPs. By blocking MMP synthesis, retinoids may be effective in cancer therapy by decreasing tumor invasiveness.

Role of Retinoic Acid Receptor Gamma in the Rhino Mouse and Rabbit Irritation Models of Retinoid Activity

The three retinoic acid receptors (RAR alpha, RAR beta and RAR gamma) are known to modulate the transcription of target genes through interaction of the individual receptors with their naturally occurring ligand, retinoic acid (RA). Since RA has multiple effects in vivo, considerable effort has recently been devoted to finding selective compounds to elucidate the functions of individual receptors and to relate these functions to specific in vivo effects. The racemic synthetic retinoid 6-[(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)hydroxy-methyl]-2- naphthalene carboxylic acid has recently been identified as an RAR gamma-selective agonist. A synthetic method involving lipase-mediated transformation has been developed to prepare the individual enantiomers. Discrimination between the two enantiomers is seen in both transcriptional activity and binding to recombinant receptors with the (S)-enantiomer being the more active. Differences between the two compounds are also seen in the Rhino mouse utriculi reduction assay and the rabbit irritation model. In both animal models, the (S)-enantiomer consistently gave a greater response. Taken together, these results suggest that the activity and irritation seen with RA and related compounds is receptor mediated. Further, the strong selectivity of the compounds reported here for RAR gamma suggests that this receptor plays an important role in these in vivo biological activities. The discrimination between these enantiomers may be useful in the design of novel retinoids with uniquely defined biological properties.

Structural modifications of 6-naphthalene-2-carboxylate retinoids

Abstract The keto linker of 2-naphthoate retinoid 1 has been found nonessential for RAR transactivation activity and can be replaced with heteroatoms such as S, O, N without significant reduction of the activity. On the other hand, substitutions on the aromatic rings of retinoids 1 and 2 resulted in analogs with reduced potentcy and RAR selectivity.

APPLICATION OF THE HECK REACTION IN THE SYNTHESIS OF TRUNCATED NAPHTHOIC ACID RETINOIDS

Abstract A series of truncated naphthoic acid retinoids have been prepared using the Heck reaction. These retinoids were evaluated in the RAR transactivation assay in vitro and in the utriculi reduction assay in vivo. It has been found that the naphthalene ring of the retinoids is crucial for their retinoid activity and receptor selectivity.

Synthesis of Nitrone Analogues of Rar α Selective Retinoid AM580

Abstract Synthesis of nitrone analogues of RAR α-selective retinoid Am 580 in which the amide linker is replaced with a nitrone moiety is discribed. The nitrone segment was constructed by oxidizing the corresponding amine using MCPBA or dimethyldioxirane. The resulting nitrone derivatives were found unstable in acidic and basic conditions. The stability limitations of using this nitrone moiety as an amide surrogate are briefed.