Retinoids cell preparation

Benzofurotriazoles 353 were prepared and their activity in the inhibition of CYP26A1 evaluated in a MCF-7 cell assay. These compounds are analogues of the advanced pharmaceutical candidate liarozole, 354 <2006MIP308-86>. CYP26A1 catalyzes the metabolism of all-/ra j retenoic acid (ATRA) and is believed to be principally responsible for controlling the levels of this retenoid. Elevated levels of ATRA are used to treat hormone refractory prostate cancer and psoriasis. It is hoped that inhibition of CYP26A1 will limit the development of resistance to these retinoid treatments. 

Cyclodextrins were important components of the remaining systems evaluated, in which mainly retinoids and sex hormones were studied. Retinoids, compounds related to vitamin A, are effective in the treatment of several skin disorders (e.g., acne) and are possibly chemopre-ventive agents of some forms of cancers. The therapeutic usefulness of retinoids is impeded by their high toxicity and thus, their present use is rather limited. Biochemical data collected on retinoids suggested to us that toxic effects of retinoids occur when the concentration of these very lipophilic compounds is in excess of the concentration of their specific carrier protein, i.e., when there are free retinoids in contact with cells. Consequently, we attempted to create synthetic carriers for retinoids. The most successful system we prepared (4) was composed of retinal, bound chemically to semicarbazide groupings which were... 

For further work, serum has been replaced by insulin and transferrin, and a routine assay for retinoids in serum-free medium is performed as follows (Breitman et al., 1980a) HL-60 cells are seeded at a density of 2 x 10 cells per ml in defined medium, which is a 1 1 mixture of Dulbecco s modified Eagle s minimum essential medium and Ham s F-12 medium supplemented with 3 x 10 M selenium dioxide, insulin (5 xg per ml), and transferrin (5 ig per ml). Retinoids are added to the HL-60 cells at the start of the assay, using ethanol as the vehicle final concentration of ethanol does not exceed 0.1%. The cells are incubated for 4 or 5 days, and differentiation is then measured by NBT reduction (Collins et al., 1979) of cytospin slide preparations. Results are expressed as percentage of NBT positive cells. Approximately 4-8% of the cells will differentiate spontaneously in the absence of added retinoid. As with the F9 system, dose-response curves have been determined for a large number of retinoids, and some of the more significant results are shown in Table III. 

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... 

Preparation of Cell Lysates and Labeling with Retinoid... 

Efficiency and specificity of the prepared probe (e.g., ADAM-3) for the binding to RAR are crucial. These fractions can be evaluated by ligand competition assay using radiolabeled retinoids and RAR samples. As RAR samples, RARs extracted from various cells or tissues, including cells (mammalian, E. coli, and so on) transfected with an RAR-expression vector, can be used. (1-3,5,7,9-11) (see Note 3). 

Duplicate tissue or cell samples should be prepared that can be treated with the retinoid of interest and the vehicle used to deliver the retinoid. The duration of treatment may vary depending on the specific conditions of interest, however shorter exposure times may be helpful in limiting the number of genes which are not directly regulated by RA, but are involved further downstream in retinoid-signalmg pathways. Samples should be kept in the dark during treatment to avoid breakdown of retinoids. 

Bexarotene is an RXR-selective retinoid marketed by Ligand Pharmaceuticals Inc., for treatment of refractory advanced-stage cutaneous T-cell lynq>homa. Bexarotene is orally administered, safe and side effects are reversible.(Jtl) The synthesis of Bexarotene starts with tetrahydronaphthalene 11, prepared in 91% yield by Friedel-Crafts alkylation of toluene with 2,5-dichloro-2,S-dime1hylhexane 10 (Scheme 5).(31-33) Friedel-Crafts acylation of 11 with chloromethylterphthalate 12 afforded ester 13 in 72% yield. 

A comparison of the results above suggests that subtle differences in experimental conditions could have pronounced effects on the apparent requirements for T cell activation. Lessard and Dupuis [12] reported that the effects of retinol and RA on T cell blastogenesis induced by Concanavalin A differed markedly depending on the preparation of the retinoid-containing medium added to cells. Blastogenesis was reduced when retinol was first diluted in RPMI medium and then added to serum-containing medium, but in contrast was increased when retinol was first diluted in fetal calf serum. The association of retinoids with serum proteins may be critical, and/or the effective concentrations or stability of retinoids may differ importantly depending on preparation. In any case, these disparate results illuminate the importance of mul-... 

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