Role of Acinus in regulating retinoic acid-responsive gene pre-mRNA splicing.

Acinus-S' is a corepressor for retinoic acid receptor (RAR)-dependent gene transcription and has been suggested to be involved in RNA processing. In this study, the role of Acinus isoforms in regulating pre-mRNA splicing was explored using in vivo splicing assays. Both Acinus-L and Acinus-S', with the activity of Acinus-L higher than that of Acinus-S', increase the splicing of a retinoic acid (RA)-responsive minigene containing a weak 5' splice site but not a RA-responsive minigene containing a strong 5' splice site. RA treatment further enhances the splicing of the weak 5' splice site by Acinus in a dose- and time-dependent manner, suggesting a RA-dependent activity in addition to a RA-independent activity of Acinus. The RA-independent effect of Acinus occurs to varying degrees using minigene constructs containing several different promoters, while the RA-dependent splicing activity of Acinus is specific for transcripts derived from the minigene driven by a RA response element (RARE)-containing promoter. This suggests that the ligand-dependent splicing activity of Acinus is related to the RA-activated RAR bound to the RARE. The RRM domain is necessary for the RA-dependent splicing activity of Acinus and the RA-independent splicing activity of Acinus is repressed by RNPS1. Importantly, measurement of the splicing of endogenous human RARß and Bcl-x in vivo demonstrates that Acinus stimulates the use of the weaker alternative 5' splice site of these two genes in a RA-dependent manner for RARß and a RA-independent manner for Bcl-x. Taken together, these studies demonstrate that Acinus functions in both RAR-dependent splicing and RAR-dependent transcription.

The SAP motif and C-terminal RS- and RD/E-rich region influences the sub-nuclear localization of Acinus isoforms.

Acinus has been reported to function in apoptosis, RNA processing and regulation of gene transcription including RA-dependent transcription. There are three different isoforms of Acinus termed Acinus-L, Acinus-S', and Acinus-S. The isoforms of Acinus differ in their N-terminus while the C-terminus is consistent in all isoforms. The sub-nuclear localization of Acinus-L and Acinus-S' was determined using fluorescence microscopy. Acinus-S' colocalizes with SC35 in nuclear speckles while Acinus-L localizes diffusely throughout the nucleoplasm. RA treatment has little effect on the sub-nuclear localization of Acinus-L and Acinus-S'. The domains/regions necessary for the distinct sub-nuclear localization of Acinus-L and Acinus-S' were identified. The speckled sub-nuclear localization of Acinus-S' is dependent on its C-terminal RS- and RD/E-rich region but is independent of the phosphorylation status of Ser-453 and Ser-604 within this region. The unique N-terminal SAP motif of Acinus-L is responsible for its diffuse localization in the nucleus. Moreover, the sub-nuclear localization of Acinus isoforms is affected by each other, which is determined by the combinatorial effect of the more potent SAP motif of Acinus-L and the C-terminal RS- and RD/E-rich region in all Acinus isoforms. The C-terminal RS- and RD/E-rich region of Acinus mediates the colocalization of Acinus isoforms as well as with its interacting protein RNPS1. In conclusion, the SAP motif is responsible for the difference in the nuclear localization between Acinus-L and Acinus-S'. This difference in the nuclear localization of Acinus-S' and Acinus-L may suggest that these two isoforms have different functional roles.

Role of COUP-TFI during retinoic acid-induced differentiation of P19 cells to endodermal cells.

Retinoic acid (RA) is a positive regulator of P19 cell differentiation. Silencing of pre-B cell leukemia transcription factors (PBXs) expression in P19 cells (AS cells) results in a failure of these cells to differentiate to endodermal cells upon RA treatment. Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) is an orphan member of the steroid-thyroid hormone superfamily. RA treatment of wild type P19 cells results in a dramatic increase in the expression of COUP-TFI; however, COUP-TFI mRNA levels fail to be elevated upon RA treatment of AS cells indicating that PBX expression is required for elevation in COUP-TFI expression. To study the role of COUP-TFI during RA-dependent differentiation of P19 cells, AS cells that inducibly express various levels of COUP-TFI were prepared. Exogenous expression of COUP-TFI in AS cells, in a dose-dependent fashion, leads to growth inhibition, modest cell cycle disruption, and early apoptosis. Furthermore, AS cells can overcome the blockage in RA-dependent differentiation to endodermal cells when either pharmacological levels of COUP-TFI are expressed or a combination of both the expression of physiological levels of COUP-TFI and RA treatment. Additionally, the mRNA level of several pluripotency associated genes including OCT-4, DAX-1, and SF-1 in the COUP-TFI expressing AS cells are reduced. Moreover, analysis of the expression of primary RA response genes indicates that COUP-TFI is involved in the regulatory modulation of the expression of at least two genes, CYP26A1 and HoxA1. These studies demonstrate that COUP-TFI functions as a physiologically relevant regulator during RA-mediated endodermal differentiation of P19 cells.

Role of SF-1 and DAX-1 during differentiation of P19 cells by retinoic acid.

Retinoic acid (RA) is critical for embryonic development and cellular differentiation. Previous work in our laboratory has shown that blocking the RA-dependent increase in pre-ß cell leukemia transcription factors (PBX) mRNA and protein levels in P19 cells prevents endodermal and neuronal differentiation. Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (DAX-1) and steroidogenic factor (SF-1) were found by microarray analysis to be regulated by PBX in P19 cells. To determine the roles of DAX-1 and SF-1 during RA-dependent differentiation, P19 cells that inducibly express either FLAG-DAX-1 or FLAG-SF-1 were prepared. Unexpectedly, overexpression of DAX-1 had no effect on the RA-induced differentiation of P19 cells to either endodermal or neuronal cells. However, SF-1 overexpression prevented the RA-dependent loss of OCT-4, DAX-1 and the increase in COUP-TFI, COUP-TFII, and ETS-1 mRNA levels during the commitment stages of both endodermal and neuronal differentiation. Surprisingly, continued expression of SF-1 for 7 days caused the RA-independent loss of OCT-4 protein and RA-dependent loss of SSEA-1 expression. Despite the loss of well-characterized pluripotency markers, these cells did not terminally differentiate into either endodermal or neuronal cells. Instead, the cells gained the expression of many steroidogenic enzymes with a pattern consistent with adrenal cells. Finally, we found evidence for a feedback loop in which PBX reduces SF-1 mRNA levels while continued SF-1 expression blocks the RA-dependent increase in PBX levels. Taken together, these data demonstrate that SF-1 plays a dynamic role during the differentiation of P19 cells and potentially during early embryogenesis.

Expression of homeobox genes in oral squamous cell carcinoma cell lines treated with all-trans retinoic acid.

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on day 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling.

HACE1: A novel repressor of RAR transcriptional activity.

The diverse biological actions of retinoic acid (RA) are mediated by RA receptors (RARs) and retinoid X receptors (RXRs). While the coregulatory proteins that interact with the ligand-dependent AF-2 in the E region are well studied, the ligand-independent N-terminal AF-1 domain-interacting partners and their influence(s) on the function of RARs are poorly understood. HECT domain and Ankyrin repeat containing E3 ubiquitin-protein ligase (HACE1) was isolated as a RARbeta(3) AB region interacting protein. HACE1 interacts with RARbeta(3) both in in vitro GST pull-down and in cell-based coprecipitation assays. The interaction sites map to the N terminus of RARbeta(3) and the C terminus of HACE1. HACE1 functionally represses the transcriptional activity of RARalpha(1), RARbeta isoforms 1, 2, and 3, but not RARgamma(1) in luciferase reporter assays. In addition, HACE1 represses the endogenous RAR-regulated genes CRABP II, RIG1 and RARbeta(2), but not RAI3 in CAOV3 cells. Mutation of the putative catalytic cysteine (C876 of LF HACE1), which is indispensable for its E3 ubiquitin ligase activity, does not alter the repressive effect of HACE1 on the transcriptional activity of RARbeta(3). On the other hand, HACE1 inhibits the RA dependent degradation of RARbeta(3). It is possible that the repression of RAR-regulated transcription by HACE1 is due to its ability to inhibit the RA-dependent degradation of RARs.

Insulin receptor substrate-1 is an important mediator of ovarian cancer cell growth suppression by all-trans retinoic acid.

There is a need to identify more effective drugs for the treatment of ovarian cancer as it is the leading cause of death among gynecologic tumors. All-trans retinoic acid (ATRA), a natural retinoid, arrests the growth of CA-OV3 ovarian carcinoma cells in G(0)-G(1). Because the insulin-like growth factor-I receptor has been implicated in the proliferation of various tumors, we investigated its potential role in the suppression of ovarian cancer cell growth by ATRA. Our studies revealed that insulin receptor substrate-1 (IRS-1) protein levels decrease in CA-OV3 cells on ATRA treatment, whereas no differences in IRS-1 levels were seen in the ATRA-resistant SK-OV3 cells. Moreover, CA-OV3 clones overexpressing IRS-1 were growth inhibited less by ATRA, whereas SK-OV3 clones in which levels of IRS-1 were reduced by expression of antisense IRS-1 became sensitive to growth inhibition by ATRA treatment. Studies to determine the mechanism by which ATRA reduced IRS-1 expression showed that ATRA altered steady-state levels of IRS-1 mRNA and the stability of IRS-1 protein. Finally, the role of IRS-1 as a potential molecular target of ATRA in ovarian tumors was assessed by immunohistochemistry in an ovarian cancer tissue array. Compared with normal ovary, the majority of malignant epithelial ovarian tumors overexpressed IRS-1. Thus, there seems to be a correlation between IRS-1 expression and malignancy in ovarian tumors. Our results suggest that IRS-1 is in fact an important growth-regulatory molecule that can be a potential effective target for chemotherapeutic intervention with growth-suppressive agents, including retinoids.

Characterization of alterations of Rb2/p130 tumor suppressor in all-trans-retinoic acid resistant SK-OV3 ovarian carcinoma cells.

Rb2/p130 tumor suppressor protein regulates cell cycle progression primarily through interactions with members of the E2F family of transcription factors and repression of the transactivation of E2F target genes. In ATRA sensitive ovarian carcinoma CA-OV3 cells, a dramatic increase in Rb2/p130 protein mediates growth arrest at G0/G1. However, although Rb2/p130 is expressed at high levels in SK-OV3 cells, they fail to growth arrest in response to ATRA treatment. We show that the functional activity of Rb2/p130 in SK-OV3 cells is reduced when compared to CA-OV3 cells. To determine the basis for the reduced functional activity, we characterized the Rb2/p130 protein in SK-OV3 cells and investigated the possible role of alterations to this molecule in mediating resistance to ATRA growth suppression. Direct sequencing of Rb2/p130 cDNA cloned from SK-OV3 cells revealed the presence of two homozygous missense mutations (T178C and C259G) which result in amino acid changes Ser60 to Pro60 and Pro87 to Ala87 respectively. Unfortunately the same missense mutations were observed in Rb2/p130 cDNA cloned from ATRA sensitive CA-OV3 cells. We next investigated differences in Rb2/p130 protein subcellular localization. While Rb2/p130 was localized in the nucleus in both cell lines, we observed regions of intense staining within the nucleus of SK-OV3 cells. This is suggestive of aggregation and/or subnuclear sequestration of the Rb2/p130 protein. Finally, the PAGE migration pattern of Rb2/p130 suggested that a hyperphosphorylated form of Rb2/p130 accumulated in SK-OV3 cells but not in CA-OV3 cells. It is possible that this hyperphosphorylated form can be responsible for the decreased Rb2/p130 functional activity observed in SK-OV3 cells and may contribute to the resistance of these cells to ATRA mediated growth suppression.

Retinoid targets for the treatment of cancer.

Retinoic acid (RA), the most potent natural retinoid, is essential for normal cell growth and differentiation. The RA signaling pathway is multistep, involving the precise regulation of retinoid levels and the control of RA-dependent gene expression in target cells. Within this complex scheme, there are many different aberrations in the RA signaling pathway of tumor cells that have been found to be associated with abnormal cell growth and tumorigenesis. This article reviews the normal pathways of RA signaling, followed by a discussion of the various sites that have been implicated in tumorigenesis and targeted for drug development. Currently, there are several retinoids and one rexinoid approved for the treatment of specific cancers. Future experimentation in drug discovery will continue to explore the efficacy of retinoids/rexinoids, either alone or in combination with other chemotherapeutic agents and/or chromatin remodeling agents, and the development of agents to modulate RA metabolism within cells. It is likely that different drug treatments will be developed that are specifically tailored to the unique point(s) in the RA signaling pathways that are aberrant in specific types of tumor cells.

Early events in the induction of apoptosis in ovarian carcinoma cells by CD437: activation of the p38 MAP kinase signal pathway.

Retinoids have great potential in the areas of cancer therapy and chemoprevention. 6-[3-(1-admantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a conformationally restricted synthetic retinoid that has been reported to induce growth arrest and apoptosis in ovarian tumor cell lines but the entire mechanism for apoptotic induction has not been fully defined. We set out to identify the early events of CD437-induced apoptosis of the CA-OV-3 cell line and determine if these occur in a CA-OV-3 cell line resistant to CD437 (CA-CD437R). Using inhibitors for the MAP kinase cascade, we determined that MEK and p38 inhibitors could block CD437-induced apoptosis of the CA-OV-3 cell line. Moreover, treatment of CA-OV-3 and CA-CD437R cells with CD437 resulted in increased phosphorylation and activity of p38 independent of caspase-3 activation. Furthermore, p38 induced the phosphorylation of MEF2 in both CA-OV-3 and CA-CD437R cells after CD437 treatment. Finally, GFP-TR3 protein translocated to the cytosol and associated with mitochondria in both cell lines in response to CD437 treatment. This leads to depolarization of mitochondria and subsequent induction of apoptosis only in CA-OV-3 cells. These results identify a number of initial molecular events in the induction of apoptosis by CD437 in CA-OV-3 cells and demonstrate that the alteration in CA-CD437R cells, which results in resistance to CD437 maps downstream of these early events after TR3 translocation but prior to mitochondrial depolarization.

Frequent loss of pRb2/p130 in human ovarian carcinoma.

PURPOSE: RB2/p130, a member of the retinoblastoma gene family, maps to human chromosome 16q12.2, a region in which deletions have been found in several human neoplasms including breast, prostatic, and ovarian carcinoma. We sought to evaluate pRb2/p130 protein expression and function in ovarian carcinoma. EXPERIMENTAL DESIGN: pRb2/p130 expression was detected by immunohistochemical and Western blot analyses in 45 primary ovarian carcinoma samples. RESULTS: Immunohistochemical analysis revealed loss or decrease of pRb2/p130 expression in 18 cases (40%). pRb2/p130 expression was mostly nuclear and inversely correlated to the tumor grade (P < 0.05). Western blot analysis correlated with immunohistochemical expression. Reverse transcription-PCR followed by Southern blot analysis was performed on a representative set of 20 ovarian carcinomas. RB2/p130 mRNA levels were consistent with protein expression. We found a significant increase in the percentage of G(1)-phase-arrested cells in CAOV3 and A2780 ovarian carcinoma cell lines after transduction with an adenovirus carrying the RB2/p130 gene (Ad-CMV-RB2/p130). CONCLUSIONS: These data indicate that loss or decrease of pRb2/p130 expression is a frequent event in ovarian carcinoma and is regulated mostly at the transcriptional level. Moreover, pRb2/p130 overexpression is able to arrest cell growth in ovarian carcinoma cells, suggesting the putative role of pRb2/p130 as a tumor suppressor in this malignancy.

Novel retinoic acid metabolism blocking agents endowed with multiple biological activities are efficient growth inhibitors of human breast and prostate cancer cells in vitro and a human breast tumor xenograft in nude mice.

Novel retinoic acid metabolism blocking agents (RAMBAs) have been synthesized and characterized. The synthetic features include introduction of nucleophilic ligands at C-4 of all-trans-retinoic acid (ATRA) and 13-cis-retinoic acid, and modification of terminal carboxylic acid group. Most of our compounds are powerful inhibitors of hamster liver microsomal ATRA metabolism enzyme(s). The most potent compound is methyl (2E,4E,6E,8E)-9-(3-imidazolyl-2,6,6-trimethylcyclohex-1-enyl)-3,7-dimethylnona-2,4,6,8-tetraenoate (5) with an IC(50) value of 0.009 nM, which is 666,667 times more potent than the well-known RAMBA, liarozole (Liazal, IC(50) = 6000 nM). Quite unexpectedly, there was essentially no difference between the enzyme inhibitory activities of the two enantiomers of compound 5. In MCF-7 cell proliferation assays, the RAMBAs also enhance the ATRA-mediated antiproliferative activity in a concentration dependent manner. The novel atypical RAMBAs, in addition to being highly potent inhibitors of ATRA metabolism in microsomal preparations and in intact human cancer cells (MCF-7, T47D, and LNCaP), also exhibit multiple biological activities, including induction of apoptosis and differentiation, retinoic acid receptor binding, and potent antiproliferative activity on a number of human cancer cells. Following subcutaneous administration to mice bearing human breast MCF-7 tumor xenografts, 6 (VN/14-1, the free carboxylic acid of 5) was well-tolerated and caused significant tumor growth suppression ( approximately 85.2% vs control, p = 0.022). Our RAMBAs represent novel anticancer agents with unique multiple mechanisms of action. The most potent compounds are strong candidates for development as therapeutic agents for the treatment of a variety of cancers.

Acinus-S' represses retinoic acid receptor (RAR)-regulated gene expression through interaction with the B domains of RARs.

The diverse biological actions of retinoic acid (RA) are mediated by RA receptors (RARs) and retinoid X receptors (RXRs). Modulation of transcription by RARs/RXRs is achieved through two activation functions, ligand-independent AF-1 and ligand-dependent AF-2, located in the A/B and E domains, respectively. While the coregulatory proteins that interact with the E domain are well studied, the A/B domain-interacting partners and their influence(s) on the function of RARs are poorly understood. Acinus-S' is an ubiquitous nuclear protein that has been implicated in inducing apoptotic chromatin condensation and regulating mRNA processing. Our data demonstrate that Acinus-S' can specifically repress ligand-independent and ligand-dependent expression of a DR5 RA response element(RARE)-dependent reporter gene and several endogenous RAR-regulated genes in a dose-dependent and gene-specific manner. Chromatin immunoprecipitation assays show that Acinus-S' associates with RAREs within the promoters of endogenous genes independent of RA treatment. Furthermore, the C-terminal end of Acinus-S' and the B domain of RARbeta interact independently of ligand, and the C-terminal end of Acinus-S' is sufficient for the repression of RAR-regulated gene expression. Finally, histone deacetylase activity only partially accounts for the repressive effect of Acinus-S' on RAR-dependent gene expression. These findings identify Acinus-S' as a novel RAR-interacting protein that regulates the expression of a subset of RAR-regulated genes through direct binding to the N-terminal B domains of RARs.

GADD45A is a mediator of CD437 induced apoptosis in ovarian carcinoma cells.

Ovarian cancer is one of the leading causes of cancer death in women. A number of studies have suggested that synthetic retinoids may play an important role as an ovarian cancer chemotherapeutic agent. The synthetic retinoid CD 437 induces apoptosis in ovarian tumor cells by a mechanism that is not completely understood. In this study we demonstrate that CD437 treatment leads to an increase in GADD45A and GADD45B mRNA expression in CA-OV3 cells but not in CA-CD437R cells, a cell line which is resistant to CD437. This induction is specific to CD437 since no change in expression of either GADD45A or GADD45B was observed with either all-trans-RA or 4-HPR treatment. Western blot analysis demonstrated that the induction of GADD45A mRNA in the CA-OV3 cell line by CD437 was accompanied by an increase in GADD45A protein. Upregulation of GADD45A by CD437 is regulated at least in part at the post-transcriptional level. In contrast, CD437 regulates GADD45B expression by different mechanisms. The importance of GADD45A induction in mediating apoptosis was demonstrated in CA-OV3 cells overexpressing GADD45A antisense RNA (GADD45A-AS cells). Our results suggest that induction of GADD45A expression might play a role in mediating the apoptotic response of ovarian cancer cells to the synthetic retinoid CD437.

Role of retinoic acid in the differentiation of embryonal carcinoma and embryonic stem cells.

Retinoic acid (RA), the most potent natural form of vitamin A, plays an important role in many diverse biological processes such as embryogenesis and cellular differentiation. This chapter is a review of the mechanism of action of RA and the role of specific RA-regulated genes during the cellular differentiation of embryonal carcinoma (EC) and embryonic stem (ES) cells. RA acts by binding to its nuclear receptors and inducing transcription of specific target genes. The most studied mouse EC cell lines include F9 cells, which can be induced by RA to differentiate into primitive, parietal, and visceral endodermal cells; and P19 cells, which can differentiate to endodermal and neuronal cells upon RA treatment. ES cells can be induced to differentiate into a number of different cell types; many of which require RA treatment. Over the years, many RA-regulated genes have been discovered in EC and ES cells using a diverse set of techniques. Current research focuses on the elucidation how these genes affect differentiation in EC and ES cells using a variety of molecular biology approaches. However, the exact molecule events that lead from a pluripotent stem cell to a fully differentiated cell following RA treatment are yet to be determined.

Retinoids in biological control and cancer.

More than 80 years ago, Wolbach and Howe provided the first evidence suggesting a link between alterations within human cells that lead to malignancies and vitamin A deficiencies (Wolbach and Howe 1925 Nutr. Rev. 36: 16-19). Since that time, epidemiological, preclinical and clinical studies have established a causative relationship between vitamin A deficiency and cancer. Laboratory research has provided insight into the intracellular targets, various signaling cascades and physiological effects of the biologically-active natural and synthetic derivatives of vitamin A, known as retinoids. Collectively, this body of research supports the concept of retinoids as chemopreventive and chemotherapeutic agents that can prevent epithelial cell tumorigenesis by directing the cells to either differentiate, growth arrest, or undergo apoptosis, thus preventing or reversing neoplasia. Continued refinement of the retinoid signaling pathway is essential to establishing their use as effective therapeutics for tumor subtypes whose oncogenic intracellular signaling pathways can be blocked or reversed by treatment with retinoids.

Interaction of PP2A catalytic subunit with Rb2/p130 is required for all-trans retinoic acid suppression of ovarian carcinoma cell growth.

All-trans retinoic acid (ATRA) treatment causes CAOV3 ovarian carcinoma cells to growth arrest in the G0/G1 phase and to elevate the level of Rb2/p130 protein. PP2A, a serine/threonine phosphatase, binds and dephosphorylates Rb2/p130, thereby increasing the half-life of Rb2/p130 in the cell. In order to further characterize the interaction between Rb2/p130 and PP2A upon ATRA treatment, we examined the posttranslational modification of PP2A. ATRA treatment leads to hypophosphorylation of PP2A catalytic subunit (PP2Ac) that correlates with increased PP2A activity. In addition, the N-terminus of PP2Ac binds directly to NLS sequences located in the C-terminus of Rb2/p130. Furthermore, CAOV3 cells transfected with a truncated Rb2/p130 construct consisting of only the wt C-terminus grew more aggressively and were less sensitive to ATRA treatment when compared to parental CAOV3 cells. In contrast, CAOV3 cells transfected with a truncated Rb2/p130 construct consisting of only the C-terminus in which the NLS sites were mutated and which could not interact with PP2A, were as sensitive to ATRA treatment as parental CAOV3 cells. These studies suggest that ATRA treatment suppresses the growth of CAOV3 cells via a novel posttranscriptional mechanism involving PP2A.

Retinoic acid induced repression of AP-1 activity is mediated by protein phosphatase 2A in ovarian carcinoma cells.

In previous studies we have shown that all-trans retinoic acid (atRA)-treatment of the atRA-sensitive ovarian carcinoma cell line CA-OV3 repressed AP-1 activity by about 50%, while a similar effect was not observed in the atRA-resistant ovarian carcinoma cell line, SK-OV3. These results suggested that the repression of AP-1 activity may be one of the mechanisms by which atRA inhibits the growth of atRA-sensitive CA-OV3 cells. In the present studies, we investigated further the molecular mechanism by which AP-1 activity is repressed by atRA. We show that the repression of AP-1 activity correlates with an increase in JunB protein expression and a decrease in N-terminal phosphorylation of c-Jun. The decrease in N-terminal phosphorylation of c-Jun does not appear to be modulated by JNK or ERK, since their protein expression patterns and kinase activity do not correlate with the repression of AP-1 activity following treatment with atRA. However, the activity of the protein phosphatase PP2A was found to increase 24 h following atRA treatment in CA-OV3 cells. Moreover, the catalytic subunit of PP2A was found to associate with c-Jun in vivo following atRA treatment. Since the inhibition of AP-1 activity following atRA treatment of CA-OV3 cells was abolished in the presence of specific PP2A inhibitors, it is likely that PP2A plays an important role in the atRA-induced repression of AP-1.

Pharmacological doses of some synthetic retinoids can modulate both the aryl hydrocarbon receptor and retinoid receptor pathways.

Retinoids have been demonstrated to have pharmacological application in the areas of dermatology and oncology. In addition to the natural retinoids such as all-trans-retinoic acid and 9-cis-retinoic acid, many new potential retinoid drugs have been synthesized, including retinoic acid receptor (RAR)-subtype selective agonists, retinoid X receptor (RXR)-selective agonists, RAR-selective antagonists, anti-AP1-specific retinoids and retinoids that induce apoptosis. Recent studies demonstrate that some retinoids, in addition to modulating the RAR/RXR pathway, are also capable at pharmacological concentrations of binding to aryl hydrocarbon receptor (AhR) and activating the AhR/AhR nuclear translocator pathway. Future studies are necessary to ascertain the consequences, if any, of activation of the AhR signaling pathway by pharmacological doses of specific retinoids.

Retinoic acid receptors and cancer.

Retinoids have been shown to inhibit the growth of many human tumor cells. Although the exact molecular mechanism of retinoid-mediated growth suppression remains known, the importance of the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) has been in established a number of tumor cell models. We wanted to determine if modulation of RAR/RXR function would alter the retinoid sensitivity of oral squamous carcinoma cells (SCCs). Growth of SCCs was significantly suppressed by treatment with either all-trans retinoic acid (RA) or the synthetic, conformationally restricted RAR-gamma-selective retinoids SR 11254 and SR 11389. In contrast, stable oral SCC clones that constitutively overexpressed the mouse dominant negative mutant, RAR-beta (R269Q), were shown to exhibit reduced RAR/RXR transcriptional transactivation function and reduced sensitivity to growth inhibition by RA, SR 11254 and SR 11389. Likewise, the RAR-gamma antagonist SR 11253 was found to block the ability of SR 11254 and SR 11389 to inhibit SCC growth. These results indicate that modulation of RAR function through the use of either an RAR-gamma-selective antagonist or a pan-RAR dominant negative mutant significantly alters the growth inhibitory response of oral SCCs to retinoids.