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Retinoic acid nuclear receptors and tumor promotion: Decreased expression of retinoic acid nuclear receptors by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate
Abstract
During studies to determine the mechanism of tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA), we found that TPA downregulates mouse epidermal retinoic acid nuclear receptors (RAR), a superfamily of nuclear steroid/thyroid receptors implicated in mediating effects of retinoic acid (RA). Application of TPA to mouse skin decreased the binding of [H-3]RA to RAR from mouse epidermal nuclear extracts. In this experiment, 20 nmol of TPA was applied to mouse skin and 3.5 h later binding of [H-3]RA to RAR was analyzed by chromatography on a size-exclusion column. TPA treatment resulted in an similar to 67% decrease in the specific binding of [H-3]RA to RAR. In a more detailed time course, application of 20 nmol of TPA to mouse skin led to 20, 36, 92 and 0% decrease in the binding of [H-3]RA to mouse epidermal RAR at 2, 4, 12 and 72 h after treatment respectively. Okadaic acid, an inhibitor of protein phosphatases 1 and 2A but a mouse skin tumor promoter, also inhibited the binding of RA to RAR. RAR alpha and RAR gamma, but not RAR beta mRNA, could be detected in mouse epidermis. In addition, RA nuclear receptor RXR alpha was also expressed in the mouse epidermis. As determined by Northern blot analysis of total as well as poly(A)(+) RNA, application of 10 mmol of TPA to mouse skin led to decreased expression of RAR alpha, RAR gamma and RXR alpha mRNA at 3.5 h after treatment. The effect of TPA on the attenuation of RAR expression was specific. Specific binding of RA to RAR was decreased when TPA-induced expression of the c-fos, c-jun and ornithine decarboxylase gene was increased. Downregulation of RAR(s) may be an essential component of the mechanism of mouse skin tumor promotion.
... RARy could not be detected in skin treated with TPA or mezerein after acute exposure. A previous report (32) To determine if TPA-and mezerein-promoted tumors may have constitutive alterations in the levels of RARa in addition to those produced by exposure to the promoting agent, nuclear extracts from tumors and from promoter-treated and untreated areas of normal skin were analyzed from mice that had been promoted for 20 weeks and untreated for 30 weeks (Fig. 2B). RARa was detected in untreated skin (from the abdomen) and dorsal skin treated previously with TPA or mezerein. ...
... Certain of the alterations in @ keratinocytes havebeenattributed to thePKCsignal trans duction pathway (19,20), and TPA treatment of cultured keratino cytes and mouse skin attenuated the levels of RA.Ra and RARy proteins. Previously, the application of TPA to mouse skin was shown to decrease the binding of [3H]RA to RARs and transiently reduce the steady-state level of mRNA for RARa and y (32). This suggests that PKC activation could regulate expression of retinoid receptors in skin. ...
Retinoic acid receptor transcripts (RARα and RARγ) are decreased in benign mouse epidermal tumors relative to normal skin and are almost absent in carcinomas. In this report, the expression of RARα and RARγ proteins was analyzed by immunoblotting in benign skin tumors induced by two different promotion protocols designed to yield tumors at low or high risk fur malignant conversion. RARα was slightly reduced in papillomas promoted with 12-O-tetradecanoylphorbol-13-acetate (low risk) and markedly decreased or absent in papillomas promoted by mezerein (high risk). However, mezerein also caused substantial reduction of RARα in nontumorous skin. RARγ was not detected in tumors from either protocol and was greatly reduced in skin treated by either promoter. Both RARα and RARγ proteins were decreased in keratinocytes overexpressing an oncogenic v-ras(Ha) gene, and RARα was underexpressed in a benign keratinocyte cell line carrying a mutated c- ras(Ha) gene. Introduction of a recombinant RARα expression vector into benign keratinocyte tumor cells reduced the S-phase population and inhibited [3H]thymidine incorporation in response to retinoic acid. Furthermore, transactivation of B-RARE-tk-LUC by retinoic acid was markedly decreased in keratinocytes transduced with the v-ras(Ha) oncogene (v-ras(Ha)- keratinocytes). Blocking protein kinase C function in v-ras(Ha)-keratinocytes with bryostatin restored RARα protein to near normal levels, reflecting the involvement of protein kinase C in RARα regulation. Both RARα and RARγ are down-regulated in cultured keratinocytes by 12-O-tetradecanoylphorbol-13- acetate, further implicating PKC in the regulation of retinoid receptors. Our data suggest that modulation of RARs could contribute to the neoplastic phenotype in mouse skin carcinogenesis and may be involved in the differential promoting activity of mezerein and 12-O-tetradecanoylphorbol- 13-acetate, particularly for selecting tumors at high risk for malignant conversion.
... Tukey analysis also showed that only 0.1 and 1.0 M Am80 gave significant inhibition. ANOVA [F (7,37) ϭ 33.45, p Ͻ 0.0001] indicated significant differences between MM11254 concentrations on growth inhibition, and Tukey tests indicated that all concentrations significantly inhibited ODC activity compared to the control. Only 1.0 M Ro41-5253 and 0.1 and 1.0 M MM11253 produced significant inhibition of ODC activity. ...
... Further work is required to establish whether metabolic and pharmacokinetic differences after repeated TPA and retinoid treatments cause this effect in the epidermis or whether RAR␣ begins to have a more important anti-proliferative function with time. Kumar et al. 37 observed that RAR expression declines in the epidermis during TPA promotion, and Darwiche et al. 61 observed a decrease in RAR␥ levels in papillomas obtained from the 2-stage tumor promotion model. Our results suggest that if both ODC and AP-1 pathways have comparable importance in epithelial cell transformation and proliferation 11 , retinoid transcriptional agonists would be the more effective agents in cancer prevention. ...
Evaluation of retinoic acid receptor (RAR) subtype-selective alpha and gamma agonists and antagonists and a retinoid X receptor (RXR) class-selective agonist for efficacy at inhibiting both induction of ornithine decarboxylase (ODC) by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse epidermis and rat tracheal epithelial cells and the appearance of papillomas in mouse epidermis treated in the 2-stage tumor initiation-promotion model indicated that (i) RXR class-selective transcriptional agonists, such as MM11246, were not involved in ODC inhibition; (ii) RAR-selective agonists that induce gene transcription from RA-responsive elements (RAREs) were active at low concentrations; (iii) RAR-selective antagonists that bind RARs and inhibit AP-1 activation on the collagenase promoter but do not activate RAREs to induce gene transcription were less effective inhibitors; and (iv) RARgamma-selective retinoid agonists were more effective inhibitors of TPA-induced ODC activity than RARalpha-selective agonists. These results suggest that RARE activation has a more important role in inhibition of ODC activity than RXR activation or AP-1 inhibition and that RARgamma-selective agonists would be the most useful inhibitors of epithelial cell proliferation induced by tumor promoters. The natural retinoid all-trans-RA induced expression of transcription factor ZBP-89, which represses activation of the GC box in the ODC promoter by the transcription factor Sp1.
... RXR is an NR family member that has been implicated in cancer chemoprevention (17,18). RXRα expression is decreased in mouse skin tumors (19), whereas RXRβ expression is increased in non-small cell lung tumors (20) compared with healthy tissue. However, the clinical significance of RXR in colorectal cancer remains unclear. ...
Cholesterol increases the risk of colorectal cancer. Liver X receptor (LXR), retinoid X receptor (RXR)« and sterol regulatory element binding protein (SREBP)-lc are transcriptional regulators of lipid metabolism. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) serves an essential role in angiogenesis and development, but its role in cancer is controversial. The expression of COUP-TFII, LXR, RXR« and SREBP-lc in colorectal cancer, as well as their association with clinicopathologic features, was assessed, and their utility as prognostic indicators in colorectal cancer evaluated. Colorectal cancer samples (n=707 patients) were analyzed for COUP-TII, LXR, RXR« and SREBP-lc expression by immunohistochemistry. Overall survival curves of patients with tumors expressing different levels of these proteins were produced and risk factors were assessed. Of the 707 patients, 32.7, 50.9, 56.4, and 41.7% were positive for COUP-TFII, LXR, RXR«, and SREBP-lc, respectively. The lack of COUP-TFII or LXR expression was associated with lower overall survival rates (P=0.0154 for COUP-TFII, and 0.0113 for LXR). Following adjustment for other clinical risk factors (age, sex, tumor size, grade, vascular invasion, and 'Rimor-Node-Metastasis stage), the lack of COUP-TFII or LXR expression was a negative independent prognostic factor for survival. The expression of COUP-TFII and LXR alone or in combination may be biomarkers to indicate a positive prognosis in patients with colorectal cancer.
... UV irradiation, the major cause of skin cancer and photo-aging, leads to a functional vitamin A deficiency of the skin due to reduced amounts of RARγ and RXRα [215]. Reduced retinoid receptor levels are found in skin exposed to tumor promoters [216,217] and frequently also in squamous cell carcinomas [214,218,219]. Furthermore, a recent study reported progressive suppression of RARs/RXRs from normal skin to premalignant actinic keratosis to invasive squamous cell carcinoma [166]. ...
... Both keratinocytes and melanocytes downregulated expression of RARs when subjected to UV exposure, potentially initiating cellular responses to ionizing radiation through the removal of repressive transcriptional controls131132133 . A similar downregulation of RAR in mouse skin was seen after application of TPA, as well as when normal human skin progresses through premalignant actinic keratosis to invasive SCC134135136 . A loss of RARc expression exacerbated carcinogenic effects of tumor promoters and enhances malignant transformation [137,138]. ...
Skin homeostasis is maintained, in part, through regulation of gene expression orchestrated by type II nuclear hormone receptors in a cell and context specific manner. This group of transcriptional regulators is implicated in various cellular processes including epidermal proliferation, differentiation, permeability barrier formation, follicular cycling and inflammatory responses. Endogenous ligands for the receptors regulate actions during skin development and maintenance of tissue homeostasis. Type II nuclear receptor signaling is also important for cellular crosstalk between multiple cell types in the skin. Overall, these nuclear receptors are critical players in keratinocyte and melanocyte biology and present targets for cutaneous disease management.
... Using various detection techniques, previous reports have demonstrated the ability of PMA to down-regulate retinoid receptors in keratinocytes in vivo [40] and in vitro [38,41]. We expand this knowledge by showing that exposure to PMA results in a time-dependent decrease in retinoid receptor protein and transcript levels, suggesting that the effect of PMA on retinoid receptor levels is translational or post-translational. ...
Retinoids influence keratinocyte proliferation and differentiation via binding to nuclear retinoic acid receptors (RARalpha, -gamma) and retinoid X receptor alpha (RXRalpha). The effect of keratinocyte differentiation on expression of nuclear retinoid receptors and on the conversion of retinol into retinoic acid has not been examined earlier in depth.
Our aim was to examine the expression of retinoid receptors and a retinoid-regulated gene CRABPII, as well as the metabolism of exogenous [(3)H]retinol in cultured human keratinocytes induced to differentiate by exposure to either calcium, phorbol 12-myristate 13-acetate (PMA), or interferon-gamma (IFNgamma).
Normal human keratinocytes were cultured and exposed to differentiation-inducing agents. The mRNA and protein expression of retinoid receptors were examined using real-time PCR and Western blot. [(3)H]Retinol uptake and metabolism was monitored by HPLC with on-line radioactivity detection.
In calcium-exposed cells, increased expression of RARgamma and RXRalpha, enhanced metabolism of [(3)H]retinol to 3,4-didehydro-RA (ddRA), and an induction of CRABPII mRNA and protein was noted. In contrast, treatment with PMA and IFNgamma reduced the RARgamma and RXRalpha protein expression (preventable by the proteasome inhibitor MG132), increased the accumulation of [(3)H]RA and/or [(3)H]ddRA in the cells, and changed the CRABPII transcription.
Retinoid signalling is profoundly altered upon differentiation of keratinocytes and the effects depend on how cellular differentiation is initiated.
... Inhibition of AP-1-dependent gene expression seems to be mediated selectively through RARp and RARy receptors (Nagpal et al, 1995). Treatment with TPA down-regulates epidermal RARs in mice, which may be an essential component of the mechanism of skin tumor promotion (Kumar et al, 1994). Interestingly, in promyelocytic leukemia, which is caused by translocation of the RARa and fusion with the PML gene, PML-RARa cooperates with c-Jun and c-Fos to stimulate transcription (Doucas et al, 1993). ...
In 1987, a tremendous boost in our understanding of the action of dietary vitamin A occurred with the discovery and characterization of nuclear receptors for retinoic acid, the active form of the vitamin, in the laboratories of P. Chambon and R. Evans. They have shown that the nuclear receptors are ligand-activated transcription factors capable of specific gene regulation. Since that discovery, it has been determined that there are at least six retinoic acid receptors belonging to two families, RARs and RXRs, that they are differentially expressed in various mammalian tissues, and that they act as homo- and heterodimers interacting with other ligand-activated nuclear receptors. The domain structure of the receptors has been described, and their DNA-binding, ligand-binding, dimerization, and transcriptional activation regions characterized. Among the most important retinoid-regulated genes are the homeobox proteins, regulatory transcription factors which are responsible for body axis formation, patterning, limb formation, and other crucial processes during development. Retinoic acid and its receptors also regulate many differentiation markers which are particularly important in stratified epithelia, such as skin and oral epithelia. Our increased understanding led to improved therapy of a large number of skin disorders, ranging from acne to wrinkles and including epidermal and oral carcinomas.
... The specific binding of retinoic acid to RAR was decreased when PMA induced expression of ODC ; it has been suggested that downregulation of RAR(s) may be an essential component of the mechanism of mouse skin tumour promotion [42]. Treatment of fibroblasts from human skin with retinoids has been shown to up-regulate RARs [43,44], which may lead to inhibition of tumour promoter effects in the skin and chemoprevention. ...
Several studies have suggested that murine and human keratinocytes respond differently to phorbol 12-myristate 13-acetate (PMA). Using an in vitro assay, we found that in contrast to its effect on murine skin, PMA did not induce ornithine decarboxylase (ODC) activity in human skin biopsies. To explore the signalling induced by PMA and to determine whether an in vitro culture system could be used to predict biological activity of retinoids in human keratinocytes, we studied a simian virus 40 (SV40)-transformed human keratinocyte cell line. Epidermal growth factor (EGF) stimulates ODC activity and increases the steady-state level of ODC mRNA in a dose- and time-dependent manner in these cells [Prystowsky, Clevenger and Zheng (1993) Exp. Dermatol. 2, 125-132]. In this report, 10(-10) M-10(-7) M PMA induced ODC mRNA and enzyme synthesis at 7 h, but did not significantly induce ODC activity and inhibited the EGF induction of ODC activity. To explore the mechanism whereby PMA interfered with EGF signalling, the effect of PMA on EGF binding to its cell-surface receptor was studied; acute treatment with PMA (within 7 h) decreased EGF binding to 41-57% of the baseline level. In contrast, chronic treatment with PMA (24 h) increased EGF binding to 156% of the baseline level and was associated with an increase in quantity of EGF receptor protein. Protein kinase C (PKC) activation correlated with the acute decrease in EGF binding following PMA treatment. In summary, PMA induced ODC mRNA and ODC enzyme synthesis, while steady-state levels of immunoprecipitable ODC enzyme protein and ODC activity were not increased, demonstrating possible increased turnover of ODC enzyme protein. Additionally, PMA inhibited the induction of ODC by EGF through decreased EGF binding, possibly mediated by PKC activation. Finally treatment of the keratinocytes with retinoids including etretinate, Ro13-7410, etarotene, Ro40-8757, 13-cisretinoic acid, and acitretin blocked the PMA induction of ODC mRNA, suggesting this in vitro model could be a valuable screening assay for predicting biological activity in humans.
... 89, No.9, May 7, 1997 retinoic acid signaling through the RARI3 system and then loses this control by the development of an abnonnality in RAR expression. The questions are as follows: Is this change irreversible (e.g., did it result from a genetic change) or was it the result of reversible exposure to a tumor promoter such as has been noted in experimental models (36)? When did the failure of RARI3 expression occur during preneoplastic pathogenesis? ...
... Proliferation of normal cells is dependent on more than one growth factor, and one growth factor activates multiple Application of the tumour promotor TPA to the skin decreased expression of RARs with a concomitant decrease intracellular signalling pathways. Genetic knockout experiments have established that, if a particular growth factor-of retinoic acid binding to RARs [60]. Decreased expression of retinoic acid receptors may be associated with develop-signalling pathway is inactivated, an alternative pathway takes over [65,67]. ...
The concept that cancer can be prevented, or its onset postponed, by certain diet-derived substances is currently eliciting considerable interest. Agents which interfere with tumour development at the stage of promotion and progression in particular are of potential clinical value. As chemopreventive agents have to be administered over a long period of time in order to establish whether they possess efficacy in humans, it is of paramount importance to establish their lack of toxicity. The desire to select the best chemopreventive drug candidates for clinical trial, and the necessity to monitor efficacy in the short and intermediate term, render the identification of specific mechanism-based in vivo markers of biological activity a high priority. Antioxidation, inhibition of arachidonic acid metabolism, modulation of cellular signal transduction pathways, inhibition of hormone and growth factor activity and inhibition of oncogene activity are discussed as mechanisms by which the soya constituent genistein, the curry ingredient curcumin and the vitamin A analogue 13-cis retinoic acid exert tumour suppression. A better understanding of these mechanisms will help the establishment of screens for the discovery of new and better chemopreventive agents and the identification of surrogate markers to assess the outcome of clinical chemoprevention trials.
... Previous studies have demonstrated that trans-RA could effectively counteract TPA effects (20,25,26,43). trans-RA could prevent transformation of JB6 mouse epidermal cells promoted by TPA (43) and counteract the effect of TPA on expression of fibronectin gene in fibroblasts (44), transglutaminase 1 gene in keratinocytes (45), as well as collagenase (46), stromelysin (47), and ornithine decarboxylase (48). In this study, we found that trans-RA exerted different effects on TPA activities in different cell lines. ...
Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.
... In situ hybridization is very sensitive because it can detect mRNA in individual cells. . The expression of RAR-␣, RAR-␥, and RXR-␣ mRNA in mouse skin was found to decrease a few hours after exposure of the skin to the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (22), and RAR-␣ and -␥ were progressively lost during mouse skin carcinogenesis (23). The profound loss we report here in human skin SCC of expression of all retinoid receptors found in normal skin has not been reported for any other cancer, including non-cutaneous SCCs. ...
Retinoids are essential for normal skin growth, differentiation, and apoptosis and are active pharmacologically in the prevention and treatment of skin cancers and other lesions. Retinoid effects are mediated mainly by retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which act as transcription factors to alter gene expression. Using in situ hybridization, we analyzed the expression of RARs and RXRs in normal sun-exposed skin (n = 85), squamous cell carcinoma (SCC; n = 28), and actinic keratosis [AK (a precursor to SCC); n = 38]. The expressions of five receptors (RAR-alpha and -gamma and RXR-alpha, -beta, and -gamma) were moderate to very strong in normal skin, with higher expressions in spinous and granular layers than in the basal layer. RAR-beta expression was weak or absent in normal and lesion samples. All five receptors expressed in the skin were suppressed progressively from normal skin to premalignant skin (AK) to invasive skin SCC. Specific receptor decreases in lesions relative to normal skin ranged from 75% (RXR-beta) to 96% (RAR-alpha) in SCC and from 37% (RAR-gamma) to 68% (RXR-beta) in AK. The degree of suppression of RXR-alpha and RAR-gamma, the two predominant retinoid receptors in skin, was relatively less for RXR-alpha (58% versus 86%; P = 0.015) and relatively greater for RAR-gamma (37% versus 89%; P = 0.0001) between AK and SCC, suggesting that suppression of RXR-alpha may be an earlier event and expression of RAR-gamma may be a later event of multistep squamous skin carcinogenesis. Our results indicate that suppressed expression of retinoid receptors occurs early (in AK) and is associated with progression of squamous skin carcinogenesis to SCC.
... Current address: BioLactis, Laval, Quebec, Canada Oncogene (2004) 23, 5350–5359 Treatment with certain tumor promoters, such as TPA (12-O-tetradecanolphorbol-13-acetate) or merazein, or exposure to ultraviolet radiation, the major causative factor of nonmelanoma skin cancers (Marks, 1995), leads to a decrease in the expression of both RARa and RARg in the epidermis (Kumar et al., 1994; Darwiche et al., 1995; Wang et al., 1999). Expression of these receptors is also gradually lost with progression of skin tumors from a benign lesion to malignant squamous cell carcinoma (SCC) (Darwiche et al., 1995; Xu et al., 2001). ...
All-trans retinoic acid (RA), the principle biologically active form of vitamin A, is essential for many developmental process as well as homeostasis in the adult. Many lines of evidence also suggest that RA, acting through the RA receptors (RARs), can also suppress growth of tumors of diverse origin. To assess directly the role of the RARs in a model of epidermal tumorigenesis, we investigated the incidence of tumor formation using keratinocytes lacking specific RAR types. Our data suggest that loss of RARgamma, but not RARalpha, predisposed keratinocytes to v-Ha-Ras-induced squamous cell carcinoma. We also found that ablation of RARgamma, but not RARalpha, abolished RA-induced cell cycle arrest and apoptosis in these keratinocytes. Reconstitution of receptor expression into RAR-null cells restored sensitivity to RA, and reversed the tumorigenic potential of receptor-deficient keratinocytes. These data strongly support a tumor suppressor effect for the RARs, in particular endogenous RARgamma, in murine keratinocytes.
... TPA or mezerein) or physical (e.g. UVB radiation) tumor promoters (30,34,35,39), or upon transduction of a v-ras Ha oncogene in primary keratinocytes (30). ...
The main cause of skin cancer and photo-aging is chronic exposure to ultraviolet B (UVB) radiation. Such damage can be ameliorated by retinoid treatment. UVB-radiation-induced skin carcinogenesis is associated with the induction of activator protein 1 (AP1) signaling and factors, namely FOS and JUN family members. We investigated the effects of several retinoids, all-trans-retinoic acid (tRA), 9-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl)-retinamide (HPR), on UVB-induced damage in primary mouse keratinocytes. In addition, the interplay between UVB radiation, retinoid receptors, and AP1 signaling was assessed using Western blot analysis and ribonuclease protection and gene reporter assays. Exposure of keratinocytes to UVB radiation caused a down-regulation of the retinoid receptor protein levels in a proteasome-mediated manner. In contrast, FOS and JUN proteins were transiently induced shortly after exposure to UVB radiation. Retinoid treatment caused a dose-dependent reduction in the levels of retinoid receptor proteins. When irradiated cells were treated with retinoids, no significant effects on AP1 protein expression were noted. Interestingly, pretreatments with tRA and cRA, but not HPR, suppressed UVB-radiation-induced AP1 activity by more than 50%, whereas post-treatment failed to produce similar effects. Our findings indicate that the inhibition of AP1 activity by retinoids explains, at least in part, the chemopreventive potential of retinoids in UV-radiation-associated epidermal damage.
Retinoids are essential for the maintenance of epithelial differentiation. As such, they play a fundamental role in chemoprevention of epithelial carcinogenesis and in differentiation therapy. Physiological retinoic acid is obtained through two oxidation steps from dietary retinol, i.e. retinol→retinal→retinoic acid. The latter retinal→retinoic acid step is irreversible and eventually marks disposal of this essential nutrient, through cytochrome P450-dependent oxidative steps. Mutant mice deficient in aryl hydrocarbon receptor (AHR) accumulate retinyl palmitate, retinol and retinoic acid. This suggests a direct connection between the AHR and retinoid homeostasis. Retinoids control gene expression through the nuclear retinoic acid receptors (RARs) α, β and γ and 9-cis-retinoic acid receptors α, β and γ, which bind with high affinity the natural ligands all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Retinoids are effective chemopreventive agents against skin, head and neck, breast, liver and other forms of cancer. Differentiation therapy of acute promyelocytic leukemia (APL) is based on the ability of retinoic acid to induce differentiation of leukemic promyelocytes. Patients with relapsed, retinoid-resistant APL are now being treated with arsenic oxide, which results in apoptosis of the leukemic cells. Interestingly, induction of differentiation in promyelocytes and consequent remission of APL following retinoid therapy depends on expression of a chimeric PML–RARα fusion protein resulting from a t(15;17) chromosomal translocation. This protein functions as a dominant negative against the function of both PML and RARs and its overexpression is able to recreate the phenotypes of the disease in transgenic mice. The development of new, more effective and less toxic retinoids, alone or in combination with other drugs, may provide additional avenues for cancer chemoprevention and differentiation therapy.
Retinoic acid (RA), an active metabolite of dietary vitamin A (1,2), is essential to maintain normal growth and differentiation of epithelial cells (3,4). RA and certain of its analogs have also been used in experimental animals to prevent and treat cancers of a variety of epithelial tissues (4-9). The mode of action of RA remains speculative. Accumulating evidence indicate that RA mediates its effects through specific nuclear receptors (10-16). RA nuclear receptors belong to a super-family of steroid, thyroid and vitamin D receptors, the ligand-inducible transcriptional factors (10-12). Two major families of RA nuclear receptors (RAR and RXR) have been identified. The RAR (α,β, γ) are activated by both all trans-RA and 9-cis-RA while RXR (α, β, γ) are activated by 9-cis-RA only (16,17). Thus, RAR and RXR are distinct retinoid receptor subfamilies (16,17). RAR require co-regulators to bind effectively to RA responsive element (RARE) in a target gene. RXR functions as auxiliary receptor for RAR and related receptors (18). RXRI3 forms heterodimers with RAR to increase its DNA binding and transcriptional activity (16,19). Other transcriptional factors such as COUP-TF can also modulate RAR activities (20). We determined the role that RAR plays in the signal transduction pathway leading to the expression of tissue transglutaminase (TG) by RA (21,22). Data indicating that vitamin A nutritional status in rats influences the expression of both RAR and tissue TG in certain tissues will be presented. The results that imply that the levels of RAR determine the magnitude of tissue TG induction by retinoic acid will also be summarized.
Retinoids are a group of structural and functional analogs of vitamin A. They regulate a number of fundamental physiological processes including vision, reproduction, metabolism, differentiation, bone development, and pattern formation during embryogenesis (De Luca 1991; Gudas et al. 1994; Lotan 1995a). Certain retinoids are capable of modulating cell growth, differentiation, and apoptosis and suppressing carcinogenesis in a variety of tissue types (e.g., lung, skin, mammary gland, prostate, bladder) in animal models (Moon et al. 1994; Lotan 1996) and in clinical trials with patients with premalignant or malignant lesions of the oral cavity, cervix, bronchial epithelium, skin, and other organs (Kraemer et al. 1992; Hong and Itri 1994; Lotan 1996; Hong and Sporn 1997; Moon et al. 1997). Some retinoids exhibit antitumor activity against fully malignant cells in vitro as reflected by suppression of proliferation and induction of differentiation or apoptosis (Amos and Lotan 1991; Lotan et al. 1991; Gudas et al. 1994; Lotan 1995a). Consequently, retinoids are considered as potential therapeutic agents as well (Smith et al. 1992). The ability of all-trans-retinoic acid (ATRA) to induce differentiation of acute promyelocytic leukemia cells into granulocytes is the basis for its therapeutic activity in patients and ATRA is currently used for therapy of this type of cancer (Degos 1997; Suooignet et al. 1997; Tallman et al. 1997; see Chap. 11, Sect. III).
Retinoids are essential for the maintenance of epithelial differentiation. As such, they play a fundamental role in chemoprevention of epithelial carcinogenesis and in differentiation therapy. Physiological retinoic acid is obtained through two oxidation steps from dietary retinol, i.e. retinol-->retinal-->retinoic acid. The latter retinal-->retinoic acid step is irreversible and eventually marks disposal of this essential nutrient, through cytochrome P450-dependent oxidative steps. Mutant mice deficient in aryl hydrocarbon receptor (AHR) accumulate retinyl palmitate, retinol and retinoic acid. This suggests a direct connection between the AHR and retinoid homeostasis. Retinoids control gene expression through the nuclear retinoic acid receptors (RARs) alpha, beta and gamma and 9-cis-retinoic acid receptors alpha, beta and gamma, which bind with high affinity the natural ligands all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Retinoids are effective chemopreventive agents against skin, head and neck, breast, liver and other forms of cancer. Differentiation therapy of acute promyelocytic leukemia (APL) is based on the ability of retinoic acid to induce differentiation of leukemic promyelocytes. Patients with relapsed, retinoid-resistant APL are now being treated with arsenic oxide, which results in apoptosis of the leukemic cells. Interestingly, induction of differentiation in promyelocytes and consequent remission of APL following retinoid therapy depends on expression of a chimeric PML-RAR alpha fusion protein resulting from a t(15;17) chromosomal translocation, This protein functions as a dominant negative against the function of both PML and RARs and its overexpression is able to recreate the phenotypes of the disease in transgenic mice. The development of new more effective and less toxic retinoids, alone or in combination with other drugs, may provide additional avenues for cancer chemoprevention and differentiation therapy.
We studied the effect of dietary retinoic acid (RA) in a two-stage protocol of skin carcinogenesis in female SENCAR mice. At 3 weeks of age mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA, 20 micrograms) and promoted with either 12-O-tetradecanoylphorbol-13-acetate (TPA, 2 micrograms) once per week or mezerein (MEZ, 4 micrograms) twice per week for 20 weeks. At the week of DMBA initiation mice were also put on a purified diet containing either 3 (physiological dose) or 30 micrograms (pharmacological dose) of RA/g of diet. High dietary RA significantly inhibited papilloma yield but not incidence in the MEZ-promoted group. Papilloma incidence and yield were also lower in the MEZ- than in the TPA-treated groups. Cumulative carcinoma incidence and yield, and conversion efficiency (= (carcinomas/maximal papillomas) x 100%), were all decreased by high dietary RA in both MEZ- and TPA-treated groups. These results demonstrate that high dietary RA inhibited skin carcinogenesis in MEZ-promoted mice at the stages of tumor promotion and malignant conversion, while this inhibition occurred only at the malignant conversion stage in TPA-promoted mice.
In the two-stage protocol of skin carcinogenesis, the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) is applied to the skin of mice at around seven weeks of age. We previously performed DMBA initiation at three weeks of age to study the effect of pharmacological (30 micrograms/g diet) dietary retinoic acid (RA) on skin carcinogenesis. In this study we asked whether dietary pharmacological RA is equally effective against skin carcinogenesis when mice are initiated with (DMBA) at 7 weeks of age and then subjected to weekly applications of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) or mezerein (MEZ) for 20 weeks. Similar to the three-week initiation protocol, high dietary RA inhibited papilloma incidence and yield in MEZ- but not in TPA-promoted female SENCAR mice. In addition, carcinoma incidence and yield were decreased by high dietary RA in TPA- as well as MEZ-treated mice. These data demonstrate that the high dietary RA diet is as effective in inhibiting papilloma and carcinoma formation when the DMBA is applied at seven weeks of age as at three weeks.
Naturally occurring and synthetic vitamin A metabolites and analogs (retinoids) inhibit tumor development in a variety of cellular, animal, and patient studies. They suppress transformation of cells in vitro and inhibit carcinogenesis in various organs in animal models. In a mouse skin carcinogenesis model, topical retinoids exhibit suppressive effects on tumor promotion, but have no effect on tumor initiation. In other models, retinoids administered in the diet suppress tumor development even in an adjuvant setting after excision of the first tumor. Retinoids suppress carcinogenesis in individuals with premalignant lesions and a high risk to develop cancer of the aerodigestive tract. Likewise, retinoids prevent the development of second primary cancers in head/neck and lung cancer patients who had been treated for the first primary. The mechanisms underlying the anticarcinogenic activity of retinoids appear to be associated with the ability of retinoids to modulate the growth, differentiation, and apoptosis of normal, premalignant, and malignant cells in vitro and in vivo. Most of these effects are mediated by nuclear retinoid receptors, but other mechanisms may also be involved. These studies indicate that retinoids are potentially useful agent for cancer chemoprevention.
Peroxisomal proliferators and retinoids have been reported to interact to regulate lipid metabolism, particularly beta-oxidation of fatty acids. Based on postulated interactions of these agents at the levels of receptors and response elements, we examined whether interactions exist between the peroxisomal proliferator, clofibrate (CLF), and retinoic acid (RA) in modulation of phospholipid turnover in cultured human skin fibroblasts. Treatment of cultured cells with either 25 microM CLF or 1 microM RA alone decreased [14C]ethanolamine incorporation into ethanolamine phosphoglycerides (EPG) by 20-30%, and simultaneous exposure to both agents resulted in additive inhibition. By contrast, [3H]choline incorporation into phospholipid was stimulated 5-30% by incubation with either agent; when CLF and RA were administered together, the stimulatory effects were additive. Different types of pulse-chase studies examining effects on uptake, biosynthesis, and degradation of labelled phospholipids indicated stimulation of EPG degradation and inhibition of phosphatidylcholine degradation by CLF; no effect on catabolism of either phospholipid was observed with RA. Combinations of modifiers of protein kinase activity [4 beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride, N-(2'-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride, bis-indolylmaleimide, staurosporine indicated that beta-TPA-responsive protein kinases were not involved. Accordingly, CLF and RA regulate biosynthesis and degradation of ethanolamine and choline phosphoglycerides in cultured skin fibroblasts by different mechanisms that do not involve classical protein kinase C (PKC) isoforms, even though turnover of phospholipids generating lipid activators of PKC occurs.
Retinoid induction of epidermal hyperplasia was investigated in hairless mice with synthetic ligands for the retinoic acid (RAR) and retinoid X (RXR) nuclear receptors. Induction of hyperplasia by all-trans retinoic acid and the RAR-specific retinoids TTNPB, tazarotene and AGN 190121 varied over a wide range (ED50 = 0.2-100 nmol/animal in three daily applications). Potency of induction was not directly correlated to receptor-binding affinity, but specificity of action could be demonstrated by inhibition with the high-affinity antagonist of the RARs, AGN 193109. Although RAR is functionally complexed with RXR in vivo, RXR-selective compounds have only weak potency in induction of hyperplasia. The ED50 value of the RXR-selective AGN 191701 was 600 nmol/animal compared with an ED50 value of 0.2 nmol for the structurally similar RAR-selective TTNPB. SR11237 and SR11217, also RXR-selective, each have an ED50 value of >1000 nmol. Unlike RAR-specific retinoids, RXR-selective retinoids cause only very mild skin flaking at high doses. Relative potencies for cumulative topical irritation (flaking and abrasion) of both RAR and RXR ligands were well correlated with epidermal hyperplasia. These data are consistent with RXR as a silent partner in the RAR-RXR heterodimer in skin.
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Vitamin A and its physiological metabolites (collectively known as retinoids) are important regulators of embryogenesis, cell growth and differentiation, vision, and reproduction (Hofmann and Eichele, 1994; Eskild and Hansson, 1994; DeLuca, 1991; Wald, 1968; Thompson et al., 1964). Deficiency of vitamin A leads to well-described defects in vision, fertility, and, in animals, increased susceptibility to carcinogenesis (Chambon, 1994; Sporn et al., 1976; Wilson et al., 1953). Retinol (vitamin A) is metabolized by cells to form a number of biologically active compounds (Napoli et al., 1993). These include all-trans retinoic acid, 9-cis retinoic acid, and didehydroretinoic acid. Retinol is first converted to retinal, which is then metabolized by a retinal dehydrogenase leading to production of all-trans retinoic acid (Posch et al., 1992, 1991). The metabolic pathway which generates 9-cis retinoic acid has yet to be elucidated. Didehydroretinoic acid is produced from 3,4-didehydroretinol by a pathway that is similar to that which converts retinol to all-trans retinoic acid (Thaller and Eichele, 1990). Dietary sources of vitamin A are β-carotene from plant sources and retinyl ester from animal sources. Vitamin A from these sources is either stored in the liver as retinyl esters, or packaged with retinol-binding protein together with transthyretin for export to the circulatory system (Blaner, 1989).
We report here that ultraviolet irradiation substantially reduced the mRNA and protein of the two major nuclear retinoid receptors, RAR-gamma and RXR-alpha, in human skin in vivo. Pre-treatment with retinoic acid mitigated this loss of nuclear retinoid receptors. Ultraviolet irradiation caused a near-total loss of retinoic acid induction of two RAR/RXR target genes, cellular retinoic acid binding protein-II and RA 4-hydroxylase, but did not affect 1,25-dihydroxyvitamin D3 induction of the vitamin D receptor/RXR-regulated gene vitamin D 24-hydroxylase. In effect, ultraviolet irradiation causes a functional vitamin A deficiency that may have deleterious effects on skin function, contributing to skin photo-aging and carcinogenesis.
Clinical trials have shown a significant increase in incidence of lung cancer among smokers and asbestos workers supplemented with beta-carotene, suggesting a tumor-promoting activity for this agent. We set out to test possible tumor-promoting and chemopreventive activities of dietary and topical beta-carotene in the two-stage 7,12-dimethylbenz[a]anthracene-12-O-tetradecanoylphorbol 13-acetate (TPA) model of mouse skin carcinogenesis. In the first study, the effects of three levels of dietary beta-carotene (6, 60, and 600 micrograms/g purified diet containing no other retinoid or carotenoid) were assessed over a period of 42 weeks. Carcinoma yield was reduced by approximately 50% in the 600 micrograms/g diet group (mean 0.22 carcinomas/mouse) compared with the 6 micrograms/g diet group (mean 0.44 carcinomas/mouse, p = 0.003). The 60 micrograms/g diet group showed a pattern of inhibition similar to the 600 micrograms/g diet group. A protective effect (25% reduction) of beta-carotene (in the 600 micrograms/g diet group) on papilloma formation was also found. However, the intermediate 60 micrograms/g diet group showed the same incidence as the low 6 micrograms/g diet group. This points to a lack of correlation between papilloma and carcinoma incidence, as we also found in previous work on dietary retinoids and carotenoids. The purpose of the second study was to assess whether topical beta-carotene (2 micrograms) has tumor-promoting or chemopreventive activity in the two-stage protocol. In the absence of TPA, beta-carotene had no significant tumor-promoting activity. Instead, papilloma yield induced by TPA was decreased by topical beta-carotene from an average of 20 to approximately 10 papillomas/mouse (p = 2.5 x 10(-7)). The effect of topical beta-carotene persisted beyond the treatment period (Week 24) until the termination of the study at Week 42. Western blot analysis of mouse skin extracts showed that topical beta-carotene upregulated retinoic acid receptor-alpha and -gamma expression in the dorsal skin. This finding suggests that beta-carotene may work as a chemopreventive agent by upregulating the expression of retinoid receptors in mouse skin. In conclusion, our data show that beta-carotene prevents skin carcinoma formation, induces retinoic acid receptor expression, and fails to act as a tumor promoter in the two-stage model of skin tumorigenesis.
A biomic approach by integrating three independent methods, DNA microarray, proteomics and bioinformatics, is used to study the differentiation of human myeloid leukemia cell line HL-60 into macrophages when induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Analysis of gene expression changes at the RNA level using cDNA against an array of 6033 human genes showed that 5950 (98.6%) of the genes were expressed in the HL-60 cells. A total of 624 genes (10.5%) were found to be regulated during HL-60 cell differentiation. Most of these genes have not been previously associated with HL-60 cells and include genes encoded for secreted proteins as well as genes involved in cell adhesion, signaling transduction, and metabolism. Protein analysis using two-dimensional gel electrophoresis showed a total of 682 distinct protein spots; 136 spots (19.9%) exhibited quantitative changes between HL-60 control and macrophages. These differentially expressed proteins were identified by mass spectrometry. We developed a bioinformatics program, the Bulk Gene Search System (BGSS, http://www.sinica.edu.tw:8900/perl/genequery.pl) to search for the functions of genes and proteins identified by cDNA microarrays and proteomics. The identified regulated proteins and genes were classified into seven groups according to subcellular locations and functions. This powerful holistic biomic approach using cDNA microarray, proteomics coupled to bioinformatics can provide in-depth information on the impact and importance of the regulated genes and proteins for HL-60 differentiation.
Psoriasis responds favourably to treatment with retinoids but the cellular pathways mediating these effects are poorly understood. Retinoids regulate keratinocyte proliferation and maturation via binding to nuclear retinoic acid receptors (mainly RARalpha and RARgamma) which form heterodimers with the 9-cis-RA receptor, RXRalpha. We have previously shown that mRNA expression of RARalpha and RXRalpha is down-regulated in psoriatic lesions as compared with non-lesional human skin. In the present study, we investigated the protein expression of RARalpha, RARgamma and RXRalpha in normal and psoriatic skin using indirect immunofluorescence analysis. Epidermal keratinocytes of normal and non-lesional psoriatic skin displayed similar nuclear localization of all three receptors; RARalpha was detected with decreasing intensity from basal to suprabasal layers, RARgamma showed the opposite trend, whereas RXRalpha was evenly expressed throughout the epidermis. In lesional psoriatic skin, however, all three receptor proteins showed a much higher staining intensity in the lower half of the epidermis; in particular, RARalpha immunoreactivity was low or even absent in the upper layers of epidermis. The results support the idea that psoriasis is associated with abnormal retinoid signalling in lesional epidermis.
Cldn, Claudin; TJ, tight junction; TPA, 12-O-tetradecanoyl-phorbol-13-acetate
We describe a technique for transferring electrophoretically separated bands of double-stranded DNA from agarose gels to diazobenzyloxymethyl-paper. Controlled cleavage of the DNA in situ by sequential treatment with dilute acid, which causes partial depurination, and dilute alkali, which causes cleavage and separation of the strands, allows the DNA to leave the gel rapidly and completely, with an efficiency independent of its size. Covalent attachment of DNA to paper prevents losses during subsequent hybridization and washing steps and allows a single paper to be reused many times. Ten percent dextran sulfate, originally found to accelerate DNA hybridization in solution by about 10-fold [J.G. Wetmur (1975) Biopolymers 14, 2517-2524], accelerates the rate of hybridization of randomly cleaved double-stranded DNA probes to immobilized nucleic acids by as much as 100-fold, without increasing the background significantly.
A dot hybridization method is presented for rapidly determining the relative concentrations of nucleic acids in a mixture,
as well as the extent of sequence homology between related RNA or DNA species.
We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression.
A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described. The method provides a pure preparation of undegraded RNA in high yield and can be completed within 4 h. It is particularly useful for processing large numbers of samples and for isolation of RNA from minute quantities of cells or tissue samples.
Retinoic acid receptors (RARs) are retinoic acid (RA)-inducible enhancer factors belonging to the superfamily of steroid/thyroid nuclear receptors. We have previously characterized two human RAR (hRAR-alpha and hRAR-beta) cDNAs and have recently cloned their murine cognates (mRAR-alpha and mRAR-beta) together with a third RAR (mRAR-gamma) whose RNA was detected predominantly in skin, a well-known target for RA. mRAR-gamma cDNA was used here to clone its human counterpart (hRAR-gamma) from a T47D breast cancer cell cDNA library. Using a transient transfection assay in HeLa cells and a reporter gene harboring a synthetic RA responsive element, we demonstrate that hRAR-gamma cDNA indeed encodes a RA-inducible transcriptional trans-activator. Interestingly, comparisons of the amino acid sequences of all six human and mouse RARs indicate that the interspecies conservation of a given member of the RAR subfamily (either alpha, beta, or gamma) is much higher than the conservation of all three receptors within a given species. These observations indicate that RAR-alpha, -beta, and -gamma may perform specific functions. We show also that hRAR-gamma RNA is the predominant RAR RNA species in human skin, which suggests that hRAR-gamma mediates some of the retinoid effects in this tissue.
Specific [3H]retinoic acid (RA)-binding sites in nuclear and cytosolic extracts prepared from human myeloblastic leukemia HL-60 cells have been detected by sucrose density gradient sedimentation and size-exclusion high-performance liquid chromatography (HPLC) analyses. This RA-binding activity migrated as a single peak with an apparent molecular weight of 50,000 and greater than 95% of the total binding activity was associated with the nuclear extract. Nuclear extracts prepared from COS-1 cells transfected with an expression vector for the nuclear RA receptors RAR alpha or RAR beta were enriched (20- to 100-fold) with a RA-binding activity that coeluted by size-exclusion HPLC with the putative RAR from HL-60 cells. The HL-60 nuclear receptor exhibited high-affinity binding of RA and its benzoic acid analogs Ch55, Ch30, Ro 13-7410, and SRI 6409-40 and low-affinity binding of retinol, Ro 8-8717, and SRI 5442-60, correlating well with the biological activity of these compounds in HL-60 cells. Saturation binding and Scatchard plot analyses of the binding of RA to the nuclear HL-60 receptor yielded an apparent dissociation constant of approximately 0.46 nM and 1400 +/- 100 receptor sites per cell. Northern blot analyses of poly(A)+ RNA with cDNA probes specific for RAR alpha and RAR beta indicated that HL-60 cells contain predominantly transcripts encoded by the RAR alpha gene. Our results suggest that the observed nuclear RA-binding activity in HL-60 cells might mediate the action of RA in these cells.
Incubation of turkey erythrocytes with the phorbol ester phorbol 12-myristate 13-acetate (PMA) results in a dose- and time-dependent desensitization of isoproterenol-stimulated adenylate cyclase activity. Compared to controls, membranes from PMA-treated cells have an isoproterenol-stimulated adenylate cyclase activity that is decreased 20%-40%, with little effect on forskolin or fluoride activation of adenylate cyclase. No change in beta-adrenergic receptor number is observed after PMA treatment, indicating that the major effect of PMA is to uncouple receptor interactions with Ns, the stimulatory guanine nucleotide regulatory protein of adenylate cyclase. Purification of beta-adrenergic receptors from 32Pi-labeled turkey erythrocytes, incubated in the presence or absence of PMA, indicates that the phorbol ester is capable of inducing a 3-fold increase in phosphorylation of the beta-adrenergic receptor. The PMA effect is similar to the phosphorylation of the beta-adrenergic receptor during isoproterenol- and dibutyryl cAMP-induced desensitization of adenylate cyclase in turkey erythrocytes. The findings indicate that decreased receptor-Ns coupling is correlated with receptor phosphorylation and that phorbol esters can influence the responsiveness of hormone-sensitive adenylate cyclase in certain cell types.
The effects of dose and duration of treatment with the potent tumor-promoting agent 12-O-tetra-decanoylphorbol-13-acetate (TPA) on the formation of skin tumors in Charles River CD-1 mice was studied. Mice were initiated with a single application of 0.2 μmol of 7, 12-dimethylbenz[a]anthracene (DMBA) in 0.2 ml acetone. Beginning two weeks after initiation, mice were treated twice weekly with various doses (0.01 – 20 nmol) of TPA in 0.2ml acetone. Application of either 0.01 or 0.1 nmol of TPA did not elcity tumors during the 50 weeks duration of treatment. A dose-dependent increase in the number of papillomas was observed through the range of 1 to 10 nmol of TPA. Twice weekly applications of 20nmol of TPA did not further enhance the papilloma incidence. A good correlation was observed between the induction of ornithine decarboxylase (ODC) activity and the formation of skin tumors by various doses of TPA.
To determine the effect of promotion duration on the incidence of papillomas and carcinomas, mice were treated with 10 nmol of TPA for various durations (6,12,18,24,30, or 36 weeks) beginning 2 weeks after initiation with 0.2 μmol of DMBA. Mice promoted for only 6 weeks developed papilllomas and carcinomas after promotion had been discontinued. There was an intermediate incidence of tumors in the group treated for 12 weeks. Promotion for 18, 24, 30, or 36 weeks elicited virtually identical yields of papillomas. The incidence of carcinomas was proportional to promotion duration times of 6, 12, and 18 weeks, but carcinoma incidence was less than maximal in mice promoted for 24 weeks or longer.
The results indicate that a) the incidence of papillomas serves as a rapid (18 weeks) index for subsequent appearance of carcinomas, b) twice weekly applications of 10 nmol of TPA for 18 weeks following initiation of female CD-1 mice with 0.2 μmnol of DMBA is an appropriate protocol for maximum tumor yield in initiation-promotion experiments, and c) ODC induction may be an important component of the mechanism of skin tumor promotion by TPA.
Thyroid hormones and retinoic acid function through nuclear receptors that belong to the steroid/thyroid-hormone receptor superfamily. Thyroid hormone receptors (TRs) and retinoic acid receptors (RARs) require auxiliary nuclear proteins for efficient DNA binding. Here we report that retinoid X receptors RXR alpha is one of these nuclear proteins. RXR alpha interacts both with TRs and with RARs, forming heterodimers in solution that strongly interact with a variety of T3/retinoic acid response elements. Transfection experiments show that RXR alpha can greatly enhance the transcriptional activity of TR and RAR at low retinoic acid concentrations that do not significantly activate RXR alpha itself. Thus, RXR alpha enhances the transcriptional activity of other receptors and its own ligand sensitivity by heterodimer formation. Our studies reveal a new subclass of receptors and a regulatory pathway controlling nuclear receptor activities by heterodimer formation.
We have purified and cloned a HeLa cell nuclear protein that strongly stimulates binding of retinoic acid and thyroid hormone receptors (RARs and TRs) to response elements. The purified protein is a human retinoid X receptor beta (hRXR beta). Three murine members of the RXR family (mRXR alpha, beta, and gamma) have also been cloned, and their interactions with RARs and TRs have been investigated. Under conditions where RAR, RXR, and TR bound poorly as homodimers to various response elements, strongly cooperative RAR-RXR and TR-RXR binding was observed. The binding efficiency was dependent on the sequence, relative orientation, and spacing of the repeated motifs of response elements. We show also that unstable RAR-RXR heterodimers were formed in solution, and that C-terminal sequences and the DNA-binding domains of both receptors were required for efficient formation of stable heterodimers on response elements. These findings suggest a convergence of the signaling pathways of some members of the nuclear receptor superfamily.
Hydrolysis of inositol phospholipids by phospholipase C is initiated by either receptor stimulation or opening of Ca2+ channels.
This was once thought to be the sole mechanism to produce the diacylglycerol that links extracellular signals to intracellular
events through activation of protein kinase C. It is becoming clear that agonist-induced hydrolysis of other membrane phospholipids,
particularly choline phospholipids, by phospholipase D and phospholipase A2 may also take part in cell signaling. The products
of hydrolysis of these phospholipids may enhance and prolong the activation of protein kinase C. Such prolonged activation
of protein kinase C is essential for long-term cellular responses such as cell proliferation and differentiation.
The induction of cancer on mouse skin by initiation-promotion protocols occurs through stages in which a benign squamous papilloma is an obligate precursor of squamous cell carcinoma. Activation of the Ha-ras gene is sufficient to produce the papilloma phenotype, while additional genetic changes are required for malignant conversion. The introduction of Ha-ras into normal keratinocytes suppresses the expression of differentiation markers, keratin K1 and K10, and loricrin (a cornified envelope precursor) and, to a lesser extent, filaggrin, at the level of transcription. However, cells initiated by Ha-ras express a nonepidermal keratin, K8. The transcription of K8 in these cells is sensitive to the level of medium Ca2+, being abundant in 0.5 mM Ca2+ and not detected in 0.05 mM Ca2+. Epidermal differentiation is regulated by signalling, which involves changes in phosphatidylinositol turnover and intracellular Ca2+. Cells initiated by Ha-ras do not differ from normal keratinocytes in their intracellular Ca2+ response patterns, at least in response to changes in extracellular Ca2+ and serum factors. However, c-Ha-ra keratinocytes have a high basal level of phosphatidylinositol (PI) turnover, which is additive with several other inducers of this pathway, including Ca2+ and aluminum fluoride. Additional studies suggest that high turnover of the PI pathway is incompatible with differentiation-specific gene expression in keratinocytes. We suggest this negative relationship is mediated through elevated diacylglycerol production and chronic down-modulation of protein kinase C. Protein kinase C is known to be essential for expression of differentiation-related genes in keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Retinoic acid, the first morphogen described so far in vertebrates, is a vitamin A derivative which exerts striking effects on development and differentiation. The identification of three retinoic acid receptors as members of the nuclear receptor super-family provides an explantation for the molecular action of morphogens on gene expression. Functional analysis of the receptors requires the identification of target genes and of their cis-acting retinoic acid-responsive elements. We have previously shown that the retinoic acid receptor beta gene is transcriptionally up-regulated by retinoic acid and now report the characterization of a functional retinoic acid responsive element in the beta gene that mediates trans-activation by retinoic acid. Using deletion mapping, we have identified a 27-base pair fragment, located 59 base pairs upstream of the transcriptional start, which confers retinoic acid responsiveness on the herpes virus thymidine kinase promoter. This sequence contains a perfect direct repeat of the motif GTTCAC, which is reminiscent of the 5' half-palindrome of the thyroid and oestrogen hormone responsive elements. Specific binding of the beta protein to the retinoic acid responsive element is demonstrated and is independent of the presence of retinoic acid. Both alpha and beta receptors enhance retinoic acid response in CV1 cells, indicating that they can both act through the same DNA sequence.
We present evidence that the glucocorticoid receptor (GR) and transcription factor Jun/AP-1 can reciprocally repress one another's transcriptional activation by a novel mechanism that is independent of DNA binding. Overexpression of c-Jun prevents the glucocorticoid-induced activation of genes carrying a functional glucocorticoid response element (GRE). Conversely, GR is able to repress AP-1-mediated transcriptional activation. Mutant analysis reveals that the ligand binding and DNA binding domains of GR and the region including the leucine zipper of c-Jun are required for repression. Gel retardation analysis demonstrates that bacterially expressed c-Jun disrupts GR-GRE complexes. These data indicate that members of two distinct classes of transcription factors can oppose one another's activity through a mechanism likely involving protein-protein interactions.
Glucocorticoids are potent inhibitors of collagenase induction by phorbol esters and inflammatory mediators. The target for this negative effect is the AP-1 site within the collagenase promoter, which also mediates its induction. Negative regulation is due to repression of AP-1 activity by the glucocorticoid receptor (GCR). While the GCR is a potent inhibitor of AP-1 activity (Jun/Fos), both c-Jun and c-Fos are potent repressors of GCR activity. In vitro experiments using purified GCR and c-Jun proteins suggest that mutual repression is due to direct interaction between the two. Direct interaction between GCR and either c-Jun or c-Fos is demonstrated by cross-linking and coimmunoprecipitation. These findings reveal a cross talk between two major signal transduction systems used to control gene transcription in response to extracellular stimuli, and a novel mechanism for transcriptional repression.
Okadaic acid is a polyether derivative of 38-carbon fatty acid, and is implicated as the causative agent of diarrhetic shellfish poisoning. It is a potent tumour promoter that is not an activator of protein kinase C, but is a powerful inhibitor of protein phosphatases-1 and -2A (PP1 and PP2A) in vitro. We report here that okadaic acid rapidly stimulates protein phosphorylation in intact cells, and behaves like a specific protein phosphatase inhibitor in a variety of metabolic processes. Our results indicate that PP1 and PP2A are the dominant protein phosphatases acting on a wide range of phosphoproteins in vivo. We also find that okadaic acid mimics the effect of insulin on glucose transport in adipocytes, which suggests that this process is stimulated by a serine/threonine phosphorylation event.
A cDNA encoding a protein that binds retinoic acid with high affinity has been cloned. The protein is homologous to the receptors for steroid hormones, thyroid hormones and vitamin D3, and appears to be a retinoic acid-inducible trans-acting enhancer factor, suggesting that the molecular mechanisms of the effect of retinoids (vitamin A) on embryonic development, differentiation and tumour cell growth are similar to those described for other members of this nuclear receptor family.
We have previously described a human complementary DNA that encodes a novel protein which is homologous to members of the steroid/thyroid nuclear receptor multigene family. This novel protein (hap for hepatoma) exhibits strong homology with the human retinoic acid receptor (RAR) which has been recently characterized. To test the possibility that the hap protein might also be a retinoid receptor, a chimaeric receptor was created by replacing the putative DNA binding domain of hap with that of the human oestrogen receptor (ER). The resulting hap-ER chimaera was then tested for its ability to trans-activate an oestrogen-responsive reporter gene (vit-tk-CAT) in the presence of possible receptor ligands. Here we show that retinoic acid (RA) at physiological concentrations is effective in inducing the expression of this reporter gene by the hap-ER chimaeric receptor. This demonstrates the existence of two human retinoic acid receptors designated RAR-alpha and RAR-beta.
Processes as diverse as growth, vision and reproduction depend on the presence of vitamin A and its metabolites (retinoids), but the molecular mechanisms which govern these diverse actions remain unclear (for reviews see refs 1,2). A crucial advance recently was the isolation of a specific nuclear receptor for retinoic acid, one of the physiologically active vitamin A derivatives. This nuclear receptor is a member of the steroid/thyroid hormone receptor family. Our analysis of an uncharacterized member of this class of intracellular receptors, encoded by a complementary DNA clone from a human placental library, has led us to discover a second retinoic acid receptor. This new receptor is expressed at high levels in a number of epithelial-type tissues. The gene for the receptor was first identified in a hepatocellular carcinoma where it surrounds a site of integration of hepatitis B virus. Activation by this virus may play a role in tumour development in liver cells, where it is normally not expressed.
Studies of steroid receptors have led to the identification of a superfamily of ligand-inducible regulatory proteins that includes receptors for thyroid hormones and retinoic acid. This family of receptors regulates gene expression through binding to short cis-acting sequences referred to as hormone-response elements. Identification of a functional retinoic acid responsive element is crucial to our understanding of the mechanisms by which retinoic acid receptors activate gene expression and regulate cell differentiation. One impediment to such a study is the absence of any identified gene whose transcription is directly dependent on the receptor-hormone complex. Because the DNA-binding domains of the retinoic acid and thyroid hormone receptors are highly related (62% identical in their amino acid sequences), we have investigated the possibility that the retinoic acid receptor could activate gene expression through a thyroid hormone response element. We now report that a human retinoic acid receptor expressed from cloned complementary DNA or the endogenous retinoic acid receptor present in F9 teratocarcinoma cells can activate gene expression from promoters fused to a natural or synthetic thyroid hormone response element. The product translated in vitro from the human retinoic acid receptor cDNA can bind to a thyroid hormone response element with high affinity. The unexpected implication of these findings is that retinoic acid and thyroid hormones, acting through their respective receptors, could control overlapping gene networks involved in the regulation of vertebrate morphogenesis and homeostasis.
Okadaic acid is a polyether compound of a C38 fatty acid, isolated from a black sponge, Halichondria okadai. Previous studies showed that okadaic acid is a skin irritant and induces ornithine decarboxylase (OrnDCase; 3-hydroxyl-L-glutamate 1-carboxy-lyase, EC 4.1.1.17) in mouse skin 4 hr after its application to the skin. This induction was strongly inhibited by pretreatment of the skin with 13-cis-retinoic acid. A two-stage carcinogenesis experiment in mouse skin initiated by a single application of 100 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) and followed by application of 10 micrograms of okadaic acid twice a week revealed that okadaic acid is a potent additional tumor promoter: tumors developed in 93% of the mice treated with DMBA and okadaic acid by week 16. In contrast, tumors were found in only one mouse each in the groups treated with DMBA alone or okadaic acid alone. An average of 2.6 tumors per mouse was found in week 30 in the group treated with DMBA and okadaic acid. Unlike phorbol 12-tetradecanoate 13-acetate (TPA), teleocidin, and aplysiatoxin, okadaic acid did not inhibit the specific binding of [3H]TPA to a mouse skin particulate fraction when added up to 100 microM or activate calcium-activated, phospholipid-dependent protein kinase (protein kinase C) in vitro when added up to 1.2 microM. Therefore, the actions of okadaic acid and phorbol ester may be mediated in different ways. These results show that okadaic acid is a non-TPA-type tumor promoter in mouse skin carcinogenesis.
Alterations in NMRI mouse skin induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate in "stage I of tumor promotion" are slowly reversible, and this reversibility has a half-time of 10 to 12 weeks. The tumor response observed in the course of an initiation-promotion experiment in vivo is independent of whether stage I of promotion occurs before or after initiation. Since the time interval between treatment with the promoter, and subsequent initiation can be extended up to at least 6 weeks, an enhancement of initiation because of promoter-induced cellular DNA synthesis seems to be unlikely. This result may be inconsistent with the two-stage model of tumor promotion because it indicates that in skin the existence of initiated cells is not required for the induction of cellular alterations that are essential for the stage of skin tumorigenesis called stage I of promotion.
The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the non-promoter mezerein both induce ornithine decarboxylase activity in mouse epidermis by a route which can be blocked by indomethacin. In two-stage tumor promotion experiments in mice with mezerein as the stage II promoter, TPA was effective as the stage I promoter whether it was applied before or after an initiating dose of 7,12-dimethylbenz[a]anthracene (DMBA). There appear to be at least 4 events in promotion, only 3 of which are caused by second stage promoters.
A simple and rapid method for transferring RNA from agarose gels to nitrocellulose paper for blot hybridization has been developed. Poly(A)+ and ribosomal RNAs transfer efficiently to nitrocellulose paper in high salt (3 M NaCl/0.3 M trisodium citrate) after denaturation with glyoxal and 50% (vol/vol) dimethyl sulfoxide. RNA also binds to nitrocellulose after treatment with methylmercuric hydroxide. The method is sensitive: about 50 pg of specific mRNA per band is readily detectable after hybridization with high specific activity probes (10(8) cpm/microgram). The RNA is stably bound to the nitrocellulose paper by this procedure, allowing removal of the hybridized probes and rehybridization of the RNA blots without loss of sensitivity. The use of nitrocellulose paper for the analysis of RNA by blot hybridization has several advantages over the use of activated paper (diazobenzyloxymethyl-paper). The method is simple, inexpensive, reproducible, and sensitive. In addition, denaturation of DNA with glyoxal and dimethyl sulfoxide promotes transfer and retention of small DNAs (100 nucleotides and larger) to nitrocellulose paper. A related method is also described for dotting RNA and DNA directly onto nitrocellulose paper treated with a high concentration of salt; under these conditions denatured DNA of less than 200 nucleotides is retained and hybridizes efficiently.
A rapid, microanalytical procedure for the reproducible isolation of RNA from small cultured cell samples and application to dot-blot hybridization is described. The procedure employs guanidine hydrochloride solubilization of whole cells, disruption by syringing, and selective precipitation of RNA with ethanol. The method can be performed in a single tissue culture tube and obviates the need for removal of nuclei or for organic solvent extractions. Recovery of RNA from small cell samples (10(6) cells) is 51%, while 97% of the DNA and 99% of the protein are eliminated by the procedure. Detection of specific RNA by dot-blot hybridization using a labeled probe demonstrates high reproducibility of recovered RNA and lack of "masking" with up to a 10-fold excess of starting cell material. Applicability of the procedure to detection of virus-specific RNA in cells persistently infected with mouse hepatitis virus is described.
It has been suggested that the phorbol ester tumor promoters act via the receptor-effector system for epidermal growth factor (EGF), since they interact with the EGF receptor system and mimic many of the effects of EGF in cultured cells. We have studied the interaction of phorbol esters with the EGF-responsive MCF-7 human breast cancer cell line. Similar to other systems, phorbol esters inhibit EGF binding in MCF-7 cells in a manner paralleling their potency as tumor promoters in mice. The effect is specific for EGF since the membrane binding of insulin is unaffected. Like EGF, the potent phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA) stimulates protein synthesis as indicated by a twofold increase in [(3)H]leucine incorporation into protein after 24 h in TPA. Cell morphology, however, is significantly different with TPA treatment. After 24-48 h in TPA, cells become markedly enlarged with increased cytoplasmic vacuolization and increased membrane microvilli. This is reflected in a fourfold increase in the protein/DNA ratio (control 13.1; TPA 55.9). Furthermore, TPA inhibits cell division in media with or without serum, and prevents growth stimulation by EGF. Low TPA concentrations (1.0 ng/ml) are active, and 10 ng/ml results in maximal inhibition of cell replication. Other phorbol esters inhibit MCF-7 cells relative to their tumor promoting activity in vivo and their ability to inhibit EGF binding in these cells. After 24 h in TPA, incorporation of [(3)H]thymidine into DNA is markedly reduced and the thymidine labeling index falls (33% to 2%) indicating very few S-phase cells. Growth inhibition is reversible by removing TPA from the medium. Similar inhibitory effects are seen with the two other human breast cancer cell lines studied, ZR75-1 and MDA-MB-231. In conclusion, phorbol esters may interact with the EGF receptor domain in MCF-7 human breast cancer cells, but they have distinct effects on cell morphology and growth suggesting alternative pathways of action. The antineoplastic activity of these compounds needs further investigation.
The effects of fluocinolone acetonide (FA), retinoic acid (RA), and tosylphenylalanine chloromethyl ketone (TPCK) on two-stage promotion after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female Sencar mice were investigated. The two-stage promotion protocol was achieved by twice weekly applications of 2 microgram of 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks (stage I) followed by twice weekly applications of mezerein for 18 weeks (stage II). Separately stage I and II do not cause any tumors to develop after DMBA initiation. FA was found to be a potent inhibitor of stages I and II but to a greater degree for stage I than for stage II. RA was ineffective in stage I but was a potent inhibitor of stage II; TPCK specifically inhibited stage I but not stage II. FA and TPCK effectively counteract the appearance of the dark basal keratinocytes, whereas RA has no effect. These results provide additional evidence for the importance of dark basal keratinocytes in stage I of promotion and indicate that most of the other biochemical and morphological responses normally associated with promotion (such as polyamines) are actually associated with stage II of promotion.
The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4-O-methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 mug of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 mug of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 mug of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 mug of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 mug of 4-O-methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4-O-methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen.