Retinoids reporter cells

This retinoid reporter cell assay has been successfully used to determine the distribution of retinoids in embryonic tissues (7,15,16). Retinoid reporter cell lines similar to those described here have been constructed by other laboratories (17,18). 

The F9-lacZ reporter assay provides a qualitative indication of retinoid release from tissue explants maintained m short term coculture with reporter cells. There are two important requirements that must be kept in mind in order to successfully use the assay, particularly when tissue samples are taken from developing embryos. The first requirement is that the tissue being studied must be viable under the culture conditions required for maintaining the reporter cell monolayer (see Note 7). The second requirement is that the development of the embryos must be timed in accordance with the plating of the reporter cells to ensure that tissue from the desired developmental stage is plated onto near-confluent reporter cell monolayers. 

Visualization of retinoid-mduced reporter gene expression is dependent on the type of reporter gene. For this assay using lacZ as reporter gene, a his-tochemical procedure using X-gal as substrate allows visualization of lacZ gene expression in the responding reporter cells. The procedure used is essentially that of Lim and Chae (23). 

In certain instances, it may be critical to know the relative amount of retinoid released from individual tissues or dissected regions of the embryo. To obtain a quantitative measure of retinoids released from tissues, we developed a reporter cell line using luciferase as the reporter molecule. When luciferase oxidizes its substrate luciferin, a photon is emitted that can be detected and quantified by use of a luminometer or scintillation counter. By relating known concentrations of retinoids to the amount of luciferase activity that they induce, the concentration of retinoids released by tissue samples can be determined. For quantifying retinoid release, a standard curve relating known concentrations of released retinoids to induced luciferase activity must be established. Known concentrations of released retinoids can be achieved by the use of ion-exchange beads that are loaded with retinoic acid and act as a point source of retinoid released onto reporter cell monolayers. 

Assay of Retinoids in Tissue or Ceii Homogenates Using lacZ and Luciferase Reporter Cells... 

Whereas the reporter assays described thus far measure retinoids released from tissue explants, these assays can also be adapted to measure retinoid levels within tissue samples. McCaffery et al. have modified the F9 reporter cell assay to give a measure of tissue retinoid content (16)... 

The N3 supplement is used as a serum substitute to avoid retinoids m serum triggering the response of reporter cells N3, however, can be arduous and expensive to prepare (see Table 1). Given the short-term nature of this assay, it may be advisable first to try the assay in serum-contaimng medium or in medium using serum that has been stripped of retinoids by treatment with charcoal followed by sterile filtration Alternatively, serum-free medium supplement containing insulin, transferrin, and selenite can be obtained from Boehnnger Mannheim (Indianapolis, IN) Whichever medium or serum treatment is chosen, it is important that the viability of both the tissue explant and reporter cell monolayer is ensured over the duration of the assay... 

Serum contains inhibitors of trypsin that will terminate trypsimzation of the cells It IS important for the tissue explants to adhere to the monolayer of reporter cells. This IS best achieved by using minimal amounts of medium to avoid turbulence that might otherwise dislodge tissue explants before they have had a chance to adhere. Adherence to a single spot on the monolayer is required for the reporter cells in the immediumte area of the tissue explants to detect locally released retinoids. 

Before removing explants, check each culture well under a dissecting microscope or low-magnification microscope Assay only those wells in which tissue explants successfully adhered to the reporter cell monolayer Direct contact between tissue explant and cell monolayer appears crucial to the detection of released retinoids... 

The luciferase reporter cell line may also be used to measure retinoids in tissue homogenates. The luciferase reporter cells can detect as little as 0.1 fmol of RA, and their response is linear up to 10 fmol of RA (7). 

It is difficult to generalize from the data available on the reported effects of retinoids on these two related enzyme activities. Results obtained in different cell culture systems are at variance, especially because in certain systems retinoids promote proliferation whereas in others they inhibit it. Moreover, different retinoids have been employed in many of the studies, and the time points chosen for analysis vary from a few hours to several days after the onset of retinoid treatment. For example, Scott and Russell (1982), show a temporal relationship between the peaks of G,-specific ODC and TG activity in synchronized cultures of both CHO cells and murine Cloudman S91 melanoma cells. Retinoid treatment (10 A/ retinol in the case of the CHO cells 10 A/ retinoic acid for the... 

Identifying candidates for mechanistic studies based on the ability of retinoids to activate the retinoid receptors should be approached knowledgeably because of differences in cotransfection assays (see Tab. 3). For example, RE, receptor type, reporter, cell type, and method of expressing activation have been varied. 

Systemic treatment of 13-cis retinoic acid frequently leads to cheilitis and eye irritations (e.g., unspecific cornea inflammation). Also other symptoms such as headache, pruritus, alopecia, pains of joints and bone, and exostosis formation have been reported. Notably, an increase of very low density lipoproteins and triglycerides accompanied by a decrease of the high density lipoproteins has been reported in 10-20% of treated patients. Transiently, liver function markers can increase during oral retinoid therapy. Etretinate causes the side effects of 13-cis retinoid acid at lower doses. In addition to this, generalized edema and centrilobulary toxic liver cell necrosis have been observed. 

It has been shown that the expression of connexin 43 (Cx43) is upregulated by cancer-preventive retinoids and carotenoids, which correlate with the suppression of carcinogen-induced transformation in 10T1/2 cells. Recently, it has been reported that Cx43 induction by astaxanthin, but not by a RAR-specific retinoid, was inhibited by GW9662, a PPARy antagonist (Bertram et al., 2005). 

Some isomers of trans-RA have been reported to exert very potent biological effects. Most noteworthy of these is 9-cis-RA (see Fig. 7,2), which was originally demonstrated to be a retinoid that activates both RARs and RXRs, whereas trans-RA activates only RAR family members (32-34). Levin and colleagues originally isolated [ H]9-cis-RA using RXRs to "trap it in cells fed [ Hl nzres-RA, implying... 

Many oleane and ursane triterpenoids are reported to have interesting biological, pharmacological, and medicinal activities similar to those of retinoids and steroids. These include antiinflammatory activity, suppression of tumor promotion, suppression of immunoglobulin synthesis, protection of the liver against toxic injury, induction of collagen synthesis, and induction of differentiation in leukemia or teratocarcinoma cells [197]. 

Promotion of cellular differentiation is a promising postulated mechanism for chemoprevention. Several compounds have been reported to induce cell differentiation, including retinoids, vitamin D3, and genistein. " Resveratrol is capable of inducting differentiation in some hematological malignant cell lines, colon cancers, and neuroblastoma. Jang et al. reported that incubation of the promyelocytic leukemia HL-60 cells with resveratrol induces cell differentiation and reduction of DNA synthesis. 

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