A novel specific bioassay for the determination of glucocorticoid bioavailability in human serum.

OBJECTIVE: Some patients develop side-effects even on relatively low doses of topically administered glucocorticoids (GCs), while others appear to be less sensitive to GCs. We have developed and validated a bioassay which can measure glucocorticoid bioavailability directly from small amounts of human serum to help elucidate underlying mechanisms. METHODS: We have stably transfected the human embryonic kidney cell line HEK293 with a plasmid expressing the glucocorticoid receptor, and a plasmid containing the luciferase gene preceded by three concatenated steroid response elements, bringing luciferase expression under control of the liganded glucocorticoid receptor. RESULTS: The assay, with an intra- and interassay coefficient of variance (CV) better than 10%, showed the expected difference in potency between different GCs (fluticasone propionate > budesonide > dexamethasone > hydrocortisone). No cross-reactivity was detected with other steroid hormones such as progesterone, testosterone and oestradiol. The bioassay easily detects the rise and subsequent fall of bioavailable GCs in human serum following ingestion of only 0.5 mg dexamethasone, and clearly reflects the diurnal cortisol rhythm. Moreover, systemic availability following inhalation of 2 x 250 micro g fluticasone propionate using a pressure dose inhaler could be demonstrated. CONCLUSIONS: This assay can be used to determine levels of bioavailable GCs in serum, both endogenous and administered, and thus may help in optimizing treatment regimens. The small amount of serum needed to perform an analysis makes this assay applicable even to infants.

Detection of oestrogenic activity of steroids present during mammalian gestation using oestrogen receptor alpha- and oestrogen receptor beta-specific in vitro assays.

Numerous steroid hormones are present in the foetus but their potential to activate oestrogen receptor (ER) alpha and/or beta is largely unknown. In this study, in vitro assays were developed to rapidly and specifically detect ERalpha or ERbeta activation by these steroid hormones. Our results showed that several oestrogen precursors and androgens are able to activate both ERalpha and ERbeta. Of special interest is that some of these precursors are able to activate ERalpha and ERbeta at concentrations that are present during human gestation. Moreover, some precursors (dehydroepiandrosterone (DHEA) and 17-hydroxylated pregnenolone sulphate) and androgens (5-androsten-3beta,16alpha,17beta-triol and testosterone) showed a more than 100-fold relative preference for ERbeta transactivation over ERalpha transactivation when compared with 17beta-oestradiol. Due to their relatively high levels, the precursor steroids DHEA and pregnenolone may be of particular importance in the regulation of ERbeta activity in vivo. To obtain information about the oestrogenic activity of the total pool of steroid hormones present during mammalian gestation, steroids were extracted from mouse embryos at different prenatal stages and assayed for oestrogenic activity in the established in vitro assays. Oestrogenic activity was detected in steroid extracts from all stages tested. This study has demonstrated that oestrogen receptor agonists are present in the murine embryo and that oestrogen precursors may contribute to the total pool of agonists during foetal life.

Cardiomyocyte differentiation of mouse and human embryonic stem cells.

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. Cardiac ischaemia caused by oxygen deprivation and subsequent oxygen reperfusion initiates irreversible cell damage, eventually leading to widespread cell death and loss of function. Strategies to regenerate damaged cardiac tissue by cardiomyocyte transplantation may prevent or limit post-infarction cardiac failure. We are searching for methods for inducing pluripotent stem cells to differentiate into transplantable cardiomyocytes. We have already shown that an endoderm-like cell line induced the differentiation of embryonal carcinoma cells into immature cardiomyocytes. Preliminary results show that human and mouse embryonic stem cells respond in a similar manner. This study presents initial characterization of these cardiomyocytes and the mouse myocardial infarction model in which we will test their ability to restore cardiac function.

4-hydroxytamoxifen trans-represses nuclear factor-kappa B activity in human osteoblastic U2-OS cells through estrogen receptor (ER)alpha, and not through ER beta.

Estrogens are important mediators of bone homeostasis, and postmenopausal estrogen replacement therapy is extensively used to prevent osteoporosis. The biological effects of estrogen are mediated by receptors belonging to the superfamily of steroid/thyroid nuclear receptors, estrogen receptor (ER)alpha and ER beta. ER alpha, not only trans-activates target genes in a hormone-specific fashion, but it can also neutralize other transcriptional activators, such as nuclear factor (NF)-kappa B, causing repression of their target genes. A major mechanism by which estrogens prevent osteoporosis seems to be repression of transcription of NF-kappa B target genes, such as the osteoclast-activating cytokines interleukin-6 and interleukin-1. To study the capacity of both ERs in repression of NF-kappa B signaling in bone cells, we first carried out transient transfections with ER alpha or ER beta of the human osteoblastic U2-OS cell line, in which endogenous NF-kappa B was stimulated by tumor necrosis factor alpha. Repression by ER alpha was already observed without 17 beta-estradiol, whereas addition of the ligand increased repression to 90%. ER beta, however, was able to repress NF-kappa B activity only in the presence of ligand. Because it is known that some antiestrogens can also display tissue-specific agonistic properties, 4-hydroxytamoxifen was tested for its capacity in repressing NF-kappa B activity and was found to be active (albeit less efficient than 17 beta-estradiol) and, interestingly, only with ER alpha. The pure antagonist ICI 164,384 was incapable of repressing through any ER subtypes. Deletion analysis and the use of receptor ER alpha/ER beta-chimeras showed that the A/B domain, containing activation function-1, is essential for this suppressive action. Next, we developed stable transfectants of the human osteoblastic U2-OS cell line containing ER alpha or ER beta in combination with an NF-kappa B luciferase reporter construct. In these cell lines, repression of NF-kappa B activity was only mediated through ER alpha and not through ER beta. These findings offer new insights into the specific role of both ER subtypes in bone homeostasis and could eventually help in developing more specific medical intervention strategies for osteoporosis.

Retinoic acid hydroxylase (CYP26) is a key enzyme in neuronal differentiation of embryonal carcinoma cells.

Besides nuclear retinoid receptors and cellular retinoid binding proteins also retinoic acid (RA)-synthesizing enzymes (using all-trans-retinal as substrate) and RA-catabolizing enzymes (producing hydroxylated products) may explain the specific effects of retinoids. In the past we have established an active role for 4-hydroxy-RA and 4-oxo-RA, which originally were considered to be inactive retinoids, but in fact are highly active modulators of positional specification in Xenopus development. Here we present evidence for a specific role of hydroxylated RA metabolites in the onset of neuronal differentiation. 4-Hydroxy- and 18-hydroxy-RA are products of the hydroxylation of RA by a novel cytochrome P450 (CYP)-type of enzyme, CYP26, expression of which is rapidly induced by RA. P19 embryonal carcinoma (EC) cell lines stably expressing hCYP26 undergo extensive and rapid neuronal differentiation in monolayer at already low concentrations of RA, while normally P19 cells under these conditions differentiate only in endoderm-like cells. Our results indicate that the effects on growth inhibition and RARbeta transactivation of P19 EC cells are mediated directly by RA, while the onset of neuronal differentiation and the subsequent expression of neuronal markers is mediated by hCYP26 via the conversion of RA to its hydroxylated products.

Embryonal carcinoma cell lines stably transfected with mRARbeta2-lacZ: sensitive system for measuring levels of active retinoids.

Embryonal carcinoma cell lines (F9 EC and P19 EC) were stably transfected with 1.8 kb promoter sequence of RARbeta2 coupled to the lacZ gene as a system for measuring active retinoids. These stable transfectants, designated F9-1.8 and P19-1.8, were used as reporter cell lines to investigate different retinoids for their ability to activate the reporter gene. F9-1.8 cells showed similar EC(50) values for the acidic retinoids all-trans retinoic acid (RA), 4-oxo RA, 9-cis RA, and 13-cis RA, in the range of 1-7 nM, while P19-1.8 cells were less sensitive. Retinal showed decreased activity compared to the RA isomers in both lines. However, P19-1.8 cells hardly showed beta-gal activity after treatment with retinol, while the lacZ reporter in F9-1.8 cells was still inducible by this retinoid. In addition, the reporter system was used to investigate RA metabolism and its inhibition by P450 inhibitors. A combination of RA and liarozole showed a 10 times greater induction of the RARbeta2-lacZ reporter in P19-1.8 cells, but not in F9-1.8 cells. The EC(50) value for 4-oxo RA, however, was not altered, indicating that metabolic conversion of RA to 4-oxo RA is the target for inhibition by liarozole in P19-1.8 cells. HPLC analysis revealed nearly complete inhibition of RA metabolism after liarozole treatment in P19-1.8 cells, resulting in higher levels of RA. Finally, the F9-1.8 cells were used to detect active retinoids during different stages of chick limb bud development, demonstrating that it is the limb bud mesenchyme which generates RA and not the epidermis, with a twofold higher level of RA in the posterior half than in the anterior half.

Development of a stably transfected estrogen receptor-mediated luciferase reporter gene assay in the human T47D breast cancer cell line.

Development of an estrogen receptor-mediated, chemical-activated luciferase reporter gene-expression (ER-CALUX) assay was attempted by stable transfection of luciferase reporter genes in a number of cell lines. Stable transfection of the chimeric Gal4 estrogen receptor and luciferase gene constructs in MCF-7 breast cancer and Hepa.1c1c7 mouse hepatoma cell lines, as well as transfection of a newly constructed luciferase reporter gene pEREtata-Luc in the ECC-1 human endometrial cell line, resulted in constitutive, non-estradiol-inducible clones. Stable transfection of pEREtata-Luc in the T47D breast cancer cell line, however, resulted in an extremely sensitive, highly responsive cell line. Following a 24-h exposure to estradiol (E2), stably transfected T47D.Luc cells demonstrated a detection limit of 0.5 pM, an EC50 of 6 pM, and a maximum induction of 100-fold relative to solvent controls. No clear reduction in responsiveness has been found over extended culture periods (50 passages). Anti-estrogens ICI 182,780, TCDD, and tamoxifen inhibited the estradiol-mediated luciferase induction. Genistein, nonylphenol, and o,p'DDT were the most potent (pseudo-)estrogens tested in this system (EC50 100, 260, and 660 nM, respectively). Determination of interactive effects of the (pseudo-)estrogens nonylphenol, o,p'DDT, chlordane, endosulfan, dieldrin, and methoxychlor revealed that, in combination with 3 pM E2, (pseudo-)estrogens were additive. Slightly more than additive effects (less than 2-fold) were found for combinations of dieldrin and endosulfan tested in the range of 3 to 6 microM. At these concentrations, the combination of endosulfan and chlordane demonstrated additive interaction. The ER-CALUX assay with T47D cells can provide a sensitive, responsive, and rapid in vitro system to detect and measure substances with potential (anti-)estrogenic activity.

Human retinoic acid (RA) 4-hydroxylase (CYP26) is highly specific for all-trans-RA and can be induced through RA receptors in human breast and colon carcinoma cells.

We report on the isolation of a cytochrome P450 (CYP)-like retinoic acid (RA) 4-hydroxylase cDNA from T-47D human breast cancer cells that is identical to the recently cloned hCYP26, which is involved in the metabolic breakdown of RA. Northern analysis showed that this novel human CYP26 is induced within 1 h upon RA treatment in RA-sensitive T-47D breast carcinoma cells but not in RA-resistant MDA-MB-231 breast cancer cells and HCT 116 colon cancer cells. Stable introduction of different RA receptor (RAR) subtypes in HCT 116 cells showed that CYP26 expression is dependent on RARalpha and RARgamma and, to a lesser extent, on RARbeta and closely paralleled RA metabolism, suggesting that it represents the major RA 4-hydroxylase in these human cells. Furthermore, stable introduction of all three RAR subtypes in HCT 116 cells resulted in restored RA sensitivity as assayed by growth inhibition. Interestingly, CYP26 activity was efficiently inhibited by liarozole, an inhibitor of RA metabolism, leading to enhanced growth inhibition by RA. The RA-induced CYP26 was shown to be highly specific for the hydroxylation of all-trans-RA and did not recognize the 13-cis and 9-cis isomers. This substrate specificity is promising for finding retinoids that are not recognized by this enzyme and, therefore, could be more effective in growth inhibition of susceptible cancer cells.

Autoinduction of retinoic acid metabolism to polar derivatives with decreased biological activity in retinoic acid-sensitive, but not in retinoic acid-resistant human breast cancer cells.

Previous studies have shown that all-trans-retinoic acid (RA) inhibits in vitro proliferation of hormone-dependent human breast cancer cells but not the growth of hormone-independent cells. Here we report on RA metabolism in breast cancer cells as examined by high performance liquid chromatography analysis and found a correlation with sensitivity to growth inhibition by RA. RA-sensitive T-47D and MCF-7 cells exhibited high rate metabolism to polar metabolites, whereas RA-resistant MDA-MB-231 and MDA-MB-468 cells metabolized RA to a much lesser extent, and almost no polar metabolites could be detected. The high metabolic rate in RA-sensitive cells appears to be the result of autoinduction of RA metabolism, whereas RA-resistant cells showed no such induction of metabolism. We observed furthermore that transfection with retinoic acid receptor-alpha expression vectors in RA-resistant MDA-MB-231 cells resulted in increased RA metabolism and inhibition of cell proliferation. Metabolism of RA, however, seems not to be required to confer growth inhibition of human breast cancer cells. The biological activity of the polar metabolites formed in RA-sensitive cells was found to be equal or lower than that of RA, indicating that RA itself is the most active retinoid in these cells. Together our data suggest that RA-sensitive cells contain mechanisms to activate strongly the catabolism of RA probably to protect them from the continuous exposure to this active retinoid.

Cell scattering of SK-N-MC neuroepithelioma cells in response to Ret and FGF receptor tyrosine kinase activation is correlated with sustained ERK2 activation.

The c-ret proto-oncogene encodes a receptor tyrosine kinase which plays an important role in kidney and enteric nervous system development. Germline mutations in c-ret are responsible for the dominantly inherited cancer syndromes, multiple endocrine neoplasia types 2A and 2B and familial medullary thyroid carcinoma as well as the developmental disorder Hirschsprung's disease. Using SK-N-MC neuroepithelioma cells stably transfected with an EGFR/Ret chimeric receptor, we have studied cellular consequences and signalling events following activation of exogenous EGFR/Ret and endogenous FGF and PDGF receptor tyrosine kinases in cells of neuroectodermal origin. Here we report that Ret activation led to cell scattering, growth inhibition and loss of anchorage-independent growth. Basic FGF, but not PDGF, evoked similar responses in those cells. Nevertheless, activation of all three receptor tyrosine kinases led to ERK2 activation. Analysis of the kinetics of ERK2 activation and downstream events revealed that Ret and FGF receptor activation led to sustained ERK2 activation and SRE transactivation, while PDGF treatment led to transient ERK2 activation and failed to induce SRE transactivation. Our results suggest that sustained, but not transient ERK2 activation may be involved in cell scattering.

Retinoic acid receptor alpha 1 isoform is induced by estradiol and confers retinoic acid sensitivity in human breast cancer cells.

Retinoic acid (RA) inhibits proliferation of estrogen receptor (ER)-positive human breast cancer cells, but not the growth of ER-negative cells. We have shown previously that ER-positive cells express higher levels of retinoic acid receptor (RAR) alpha, suggesting that RAR alpha gene expression may be regulated in breast cancer cells by estrogens. We here report that estradiol (E2) increases RAR alpha mRNA in a time- and concentration-dependent manner resulting in a marked increase in RAR alpha protein expression, and present evidence that RAR alpha 1 is the only known isoform of RAR alpha regulated by E2 in breast cancer cells. In parallel we demonstrate that ER-positive cells exhibit greater RA sensitivity in the presence of E2, suggesting that E2-induced expression of RAR alpha 1 is involved in growth inhibition by RA. To directly investigate the role of RAR alpha 1 in RA-mediated growth inhibition, we introduced RAR alpha 1 expression vectors into RA-resistant and ER-negative MDA-MB-231 cells. The RAR alpha 1-transfected cells were growth inhibited by RA, while mock- and untransfected cells were unresponsive. Together, our data indicate that adequate levels of RAR alpha 1, either generated by introduction of expression vectors or endogenously induced by estrogens, are required for growth inhibition of breast cancer cells by RA.

Efficacy of all-trans-beta-carotene, canthaxanthin, and all-trans-, 9-cis-, and 4-oxoretinoic acids in inducing differentiation of an F9 embryonal carcinoma RAR beta-lacZ reporter cell line.

A reporter cell line was established from F9 mouse teratocarcinoma cells containing the RAR beta 2 promoter coupled to the lacZ (beta-galactosidase) reporter gene. All-trans-, 9-cis-, and all-trans-4-oxoretinoic acid were equipotent in inducing cell differentiation at 1 microM, determined by induction of collagen IV mRNA expression, of morphological changes, as well as of beta-galactosidase enzyme activity. By the same criteria, beta-carotene at 10 microM also induced differentiation, but less strongly and more slowly than the retinoic acids. In contrast, the oxocarotenoid (or xanthophyll) canthaxanthin, at 10 microM, had little effect on differentiation, unless preincubated in culture medium, from which 4-oxoretinoic acid was recovered and identified as a decomposition product. This indicates that canthaxanthin can act as an effective inducer of differentiation only after breakdown to active metabolites. Likewise, beta-carotene probably also acts subsequent to breakdown to retinoic acid. Throughout these experiments the response of the RAR beta promoter-lacZ reporter gene correlated well with other parameters of differentiation, making this cell line a useful system for examination of inducers of embryonal carcinoma cell differentiation.

The retinoid ligand 4-oxo-retinoic acid is a highly active modulator of positional specification.

Retinoids (vitamin A and its metabolites) are suspected of regulating diverse aspects of growth, differentiation, and patterning during embryogenesis, but many questions remain about the identities and functions of the endogenous active retinoids involved. The pleiotropic effects of retinoids may be explained by the existence of complex signal transduction pathways involving diverse nuclear receptors of the retinoic acid receptor (RAR) and retinoid X receptor (RXR) families, and at least two types of cellular retinoic acid binding proteins (CRABP-I and -II). The different RARs, RXRs, and CRABPs have different expression patterns during vertebrate embryogenesis, suggesting that they each have particular functions. Another level at which fine tuning of retinoid action could occur is the metabolism of vitamin A to active metabolites, which may include all-trans-retinoic acid, all-trans-3,4-didehydroretinoic acid, 9-cis-retinoic acid, and 14-hydroxy-4,14-retroretinol. Formation of the metabolite all-trans-4-oxo-retinoic acid from retinoic acid was considered to be an inactivation pathway during growth and differentiation. We report here that, in contrast, 4-oxo-retinoic acid is a highly active metabolite which can modulate positional specification in early embryos. We also show that this retinoid binds avidly to and activates RAR beta, and that it is available in early embryos. The different activities of 4-oxo-retinoic acid and retinoic acid in modulating positional specification on the one hand, and growth and differentiation on the other, interest us in the possibility that specific retinoid ligands regulate different physiological processes in vivo.

Retinoic acid receptor and retinoid X receptor expression in retinoic acid-resistant human tumor cell lines.

Retinoic acid (RA) has profound effects on cell proliferation and differentiation both in vitro and in vivo. Many human cell lines are known to be sensitive to the growth-inhibitory action of RA. We analyzed established human solid tumor-derived cell lines for their RA sensitivity. Growth inhibition by RA in monolayer was examined by [3H]thymidine incorporation and cell proliferation. Here we report that 11 widely used human cell lines were RA resistant. The majority are carcinoma derived (A-431, BT-20, C-41, ACHN, HCT116, 293, A549, and PA-1); two are sarcoma derived (Saos-2 and A673); and one is a melanoma cell line (A-375). Since nuclear retinoid receptors are implicated in the biological effects of RA, we examined the expression of retinoic acid receptors (RARs) RAR alpha, RAR beta, RAR gamma, and the retinoid X receptors (RXRs) RXR alpha, RXR beta, and RXR gamma in the RA-resistant cell lines by northern blotting and by RNase protection analysis for RAR beta. RAR alpha transcripts were constitutively expressed in all cell lines. By contrast, RAR beta was expressed in only seven RA-resistant cell lines (Saos-2, ACHN, 293, A549, A-375, A673, and PA-1), and its level was enhanced by RA in some cases. In most cell lines, RAR gamma expression was low and was not affected by RA. The RXR genes showed a very distinct expression pattern in the group of selected cell lines. In general, RXR alpha was the most abundantly expressed subtype, RXR beta was expressed at low levels, and RXR gamma could not be detected. In none of the RA-resistant cell lines was RXR expression modulated by RA. The results presented here indicate that the resistance of these human tumor cell lines to RA cannot be simply correlated with expression of RAR or RXR or both.

A cyclic AMP response element is involved in retinoic acid-dependent RAR beta 2 promoter activation.

Activation of the retinoic acid receptor (RAR) beta 2 promoter is known to be mediated by a RA response element located in the proximity of the TATA-box. By deletion studies in P19 embryonal carcinoma cells we have analyzed the RAR beta 2 promoter for the presence of additional regulatory elements. We found that the cyclic AMP response element-related motif, TGATGTCA at position -99 to -92, is able to enhance RA-dependent RAR beta 2 promoter activation. In addition we demonstrate that this element, designated CRE-beta 2, is functionally active as a CRE since it can bind members of the CREB/ATF transcription factor family and, moreover, mediates the stimulatory effect of cAMP on RA-dependent RAR beta 2 promoter activation in human foetal kidney 293 cells.

Embryonic stem cells stably transfected with mRAR beta 2-lacZ exhibit specific expression in chimeric embryos.

Using the embryonic stem (ES) cell/chimera approach, we have studied the activity of the mouse retinoic acid receptor beta 2 (mRAR beta 2) promoter during ES cell differentiation and during embryonic development. Stable ES clones were isolated after introduction of a 1.8 kb mRAR beta 2-lacZ expression cassette. LacZ expression in these stable clones was specifically induced by retinoic acid (RA) in a similar fashion as the endogenous RAR beta 2 gene. Following introduction of three different ES clones into blastocysts, an integration-independent mRAR beta 2-lacZ expression pattern was obtained in chimeric embryos similar to that described by in situ hybridization and transgenic studies. Moreover, mRAR beta 2-lacZ expression was also detected at some additional sites not described before, e.g. body wall, ureter, mesonephric duct and optic stalk. Maternal RA administration at 8.5 days of pregnancy extended lacZ expression to more anterior and posterior regions. Transgenic mice were generated from germ-line transmission of the transfected ES cells; expression pattern and changes in expression upon RA induction in these transgenic embryos were identical to those in chimeric embryos. We conclude that by using the ES/chimera approach, the proximal 1.8 kb of the mRAR beta 2 promoter produces a reliable and reproducible expression pattern of the reporter gene, and that the ES cell/chimera approach is invaluable for the study of gene expression and regulation.

Changes in retinoic acid receptor messenger ribonucleic acid levels in the vitamin A-deficient rat testis after administration of retinoids.

Recently, we have reported that retinoic acid (RA), similarly to retinol acetate, is able to reinitiate spermatogenesis in vitamin A-deficient rats. Here, we investigated the expression of RA receptors RAR alpha, RAR beta, RAR gamma, and retinoid X receptor RXR alpha by Northern blot analysis of poly(A)+ RNA of testes of vitamin A-deficient rats before and after reinitiation of spermatogenesis induced by injection of retinol acetate or RA and testes of 21-day-old and 10-week-old normal rats. In the testis of vitamin A-deficient rats 1.9-, 2.8-, and 3.8-kilobase (kb) transcripts of RAR alpha; 2.8- and 3.3-kb transcripts of RAR beta; 1.8-, 2.8-, and 3.4-kb transcripts of RAR gamma; and two transcripts of RXR alpha of 2.5 and 4.8 kb are expressed. When vitamin A-deficient rats receive RA or retinol acetate, a 3-fold increase in the amount of poly(A)+ RNA per testis can be observed after 8 h, while the amounts of glyceraldehyde-3-phosphate dehydrogenase and sulfated glycoprotein-1 mRNA hardly change. Also, the expression of several transcripts of each RAR type is significantly increased from 1.8- up to 3.6-fold. Moreover, additional transcripts of RAR beta and RXR alpha (1.8 and 1.0 kb, respectively) can be detected. In the testes of 21-day-old rats, three transcripts of each RAR type and two RXR alpha transcripts are expressed. In contrast, in the normal adult rat testis the expression of all RARs, if present, is lower than that in the 21-day-old rat testis or the adult vitamin A-deficient rat testis. The expression of all transcripts of each RAR in the testis of 21-day-old rats shows great similarity with the expression in the testis of the vitamin A-deficient rat after replacement of retinol acetate or RA. These changes in expression indicate that RARs and RXR alpha may play a role in the process of proliferation and differentiation of A spermatogonia, which is induced in vitamin A-deficient rats shortly after replacement of RA or retinol acetate.

Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RAR alpha.

P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.

Commitment to differentiation induced by retinoic acid in P19 embryonal carcinoma cells is cell cycle dependent.

The rate at which P19 embryonal carcinoma cells in monolayer culture become anchorage dependent during differentiation induced by retinoic acid (RA) was investigated. In both nonsynchronized cultures and cultures synchronized by mitotic selection, the ability to grow in semisolid medium, characteristic of the malignant stem cell, decreased after a lag period of about 12 hr in the continuous presence of RA, prior to an increase in cell generation time. However, striking differences between synchronized and nonsynchronized cultures were observed in their commitment to differentiation following RA removal. After only 2 hr of exposure to RA, synchronized cells continued a program of differentiation in which they became anchorage dependent, while at least 24 hr of exposure was required for exponentially growing cells to become similarly committed. Induction of anchorage dependence by RA was also strikingly cell cycle dependent; 2 or 4 hr of exposure of synchronized cells to RA in G1 phase, when the intrinsic capacity for soft agar growth is low, was sufficient to commit cells to anchorage dependence, but a similar exposure in S phase was not. Together, these results suggested that interactions between cells in different cell cycle phases in asynchronous cultures influenced commitment since exposure to RA for more than one cycle (13 hr) was required for all cells to become anchorage dependent. Increased plasminogen activator secretion and epidermal growth factor binding, markers of certain differentiated cell types, increased only 3 and 5 days after RA addition, respectively, and were not induced by pulsed exposure to RA of less than 24 hr, even in synchronized cells.