Synergetic bitherapy in mice with xenografts of human prostate cancer using a methional mimic (METLICO) and an aldehyde dehydrogenase 3 inhibitor (MATE): systemic intraperitoneal (IP) and targeted intra-tumoral (IT) administration.

An intraperitoneal (IP) monotherapy in nu/nu mice with subcutaneous xenografts of a human prostate epithelial cancer cell line:DU145 was undertaken with an aldehyde dehydrogenase 3 inhibitor MATE, that is a potent apoptogen on (DU145) in culture but not on their human prostate epithelial normal counterparts [13] . Tumour growth was slowed down but treatment had to be done 5days/week. To try to potentiate the action of MATE in vivo, a bitherapy was undertaken based on the synergetic apoptotic effect that had been observed previously in culture on DU145 treated with a methional mimic METLICO and DIMATE, an inhibitor of ALDH1 and ALDH3 [19]. The bitherapy with METLICO/MATE administered IP was as effective as the monotherapy with MATE alone by IP, but at a 2-fold lower dose of MATE and at a dose of METLICO that had no growth-inhibitory effect as a monotherapy . Hence there was definite synergism with bitherapy. To try to increase the efficacy of bitherapy, it was administered by the intra-tumoral (IT) route using the recently developed 20-bars-pressurized microinjection system from CERMA [16, 17]. IT administration of the bitherapy was indeed more effective than that by IP as regards tumour volumes are concerned. Histopathological analysis of IT-treated tumours confirmed that there were many necrotized zones but intact cells were still present. Approaches for treating a wider zone of tumour tissue by IT-bitherapy are discussed.

Comparison of two strategies for systemic antibiotic treatment of dairy cows with retained fetal membranes: preventive vs. selective treatment.

The objective of this study was to evaluate the efficacy of a blanket systemic preventive treatment (PT) of cows having retained fetal membranes (RFM) with 1 mg/kg of ceftiofur administered the first day after calving regardless of their body temperature. This strategy was compared with a selective treatment (ST) strategy in which only cows having RFM and a rectal temperature > or = 39.5 degrees C within 10 d postpartum received ceftiofur. Cows that retained their fetal membranes for at least 24 h after calving were allocated to 2 groups. Rectal temperature was measured daily for 10 d postpartum. Sixty PT cows having RFM received a daily ceftiofur (1 mg/kg of body weight) treatment, administered subcutaneously during the first 3 d after diagnosis of RFM. If rectal temperature was > or = 39.5 degrees C after 3 daily treatments, cows received ceftiofur for 2 more days. Therapy in 53 ST cows was based on selective administration of ceftiofur to cows having fever during the first 10 d postpartum. Treatment was conducted for 3 to 5 consecutive days as described for PT cows, beginning on the first day of fever. In both groups, manual removal of the placenta was not attempted and antibiotic drugs were not administered into the uterus. For every cow having RFM enrolled in PT or ST, 1 cow without RFM that had calved on the same day was enrolled in a healthy control group (n = 113). All cows received two 25-mg doses of PGF(2alpha): 1 dose between 18 and 24 d and 1 dose between 32 and 38 d postpartum. The PT did not reduce the proportion of cows experiencing fever during 10 d postpartum compared with ST cows (71.7 vs. 69.8%). Results were compared using logistic regression models and survival analyses. The artificial insemination submission rate between 42 and 62 d postpartum was greater in PT (41.2 vs. 20.8 vs. 24.5%), but total conception rate was less in ST and control cows, respectively (25.0 vs. 38.9 vs. 36.2%). In this trial, a preventive systemic antibiotic treatment of all cows having RFM was not superior to a selective antibiotic treatment of cows only in case of fever.

Strategies to improve the therapy of retained fetal membranes in dairy cows.

In this field trial, a protocol for the treatment of retained fetal membranes (RFM) without any intrauterine therapy was compared with 3 protocols based on the intrauterine use of antibiotic pills (AP), the manual removal (MR) of the fetal membranes, or the combination of both (PR). The study was conducted on 5 commercial dairy farms in Germany. Cows with RFM for at least 24 h after calving were assigned to 1 of 4 treatment groups. Cows of all groups with a rectal temperature >or= 39.5 degrees C received a systemic antibiotic treatment with ceftiofur (1 mg/kg per d) for 3 to 5 consecutive days. In case of continued fever after 5 treatments, cows received a different antibiotic as an escape therapy. In the reference group (REF; n = 131), cows did not receive any additional treatment. All cows in group AP (n = 119) received intrauterine treatment with antibiotic pills consisting of 1,000 mg of ampicillin and 1,000 mg of cloxacillin for 3 consecutive days. In group MR (n = 121), an attempt was made to remove the fetal membranes manually, but uterine pills were not administered. In group PR (n = 130), an attempt was made to remove the fetal membranes manually and all cows received a local antibiotic treatment as in group AP. All cows received 2 doses of 25 mg of PGF(2alpha): one dose between 18 and 24 d and another between 32 and 38 d postpartum. Statistical analyses were performed using binary logistic regression models and survival analyses with group REF as reference. Of all animals, 79.8% had a body temperature of >or= 39.5 degrees C at least once within 10 d postpartum and were treated with ceftiofur. Occurrence of fever within 10 d postpartum was significantly lower in groups AP and PR compared with reference group REF, but was not different between groups MR and REF. Risk of receiving an escape therapy in case of fever after 5 treatments with ceftiofur did not differ among groups. Reproductive performance measures did not differ significantly between group REF and any of the comparison groups. Compared with a treatment protocol based only on systemic treatment with antibiotics for cows with a fever, neither intrauterine antibiotics nor manual removal of fetal membranes alone or in combination reduced proportions of cows needing an escape therapy nor did those treatments improve reproductive measures in the current lactation. Systemic treatment alone based on elevated rectal temperature was effective and reduced use of antibiotics compared with therapies that included intrauterine antibiotics.

Effects of retinoic acid receptor-selective agonists on human nasal epithelial cell differentiation.

Retinoids play a critical role in the maintenance of the mucociliary phenotype of epithelial cells in the upper respiratory tract. To determine the role of retinoic acid receptors (RARs) in the regulation of epithelial differentiation, we tested the effect of the synthetic retinoids CD336, CD2019, and CD666, selective agonists for RARalpha, RARbeta, and RARgamma, respectively, during differentiation of human nasal epithelial (HNE) cells in vitro. Using glutamylated tubulin and transglutaminase I (Tg I) as markers of ciliated cell and squamous cell differentiation, respectively, we showed that retinoic acid (RA) stimulated mucociliary differentiation and, in parallel, inhibited squamous cell differentiation. The agonists of the three RARs independently induced ciliogenesis and inhibited squamous cell differentiation by downregulating Tg I expression in a dose- and time-dependent manner. Antagonists specific for the three RARs abolished the effects of the corresponding agonists, demonstrating an RAR-specific mediated effect. Moreover, treatment of retinoid-deficient cultures with RAR agonists induced conversion of the squamous-like phenotype into a ciliated phenotype. In conclusion, all three RARs are potentially involved in the differentiating effects of RA in respiratory epithelial cells.

Limited cooperation between peroxisome proliferator-activated receptors and retinoid X receptor agonists in sebocyte growth and development.

We have found that sebaceous epithelial cell (sebocyte) differentiation is induced by cognate ligand-agonists of either peroxisome proliferator-activated receptors (PPARs) or retinoid X receptors (RXRs). In this study, we tested the hypothesis that PPAR-RXR cooperation is used in sebocytes as was reported to occur in gene transfection systems and liposarcoma cells through PPAR-RXR heterodimerization. PPAR agonists at maximally effective concentrations were tested in combination with a specific RXR agonist (the rexinoid CD2809) at doses ranging from submaximal to maximal in a primary rat preputial cell monolayer culture system. We evaluated ligand-agonists of PPARalpha (WY-14643 = WY), PPARgamma (troglitazone = TRO), and PPARdelta,alpha (carbaprostacyclin = cPGI2). Cell differentiation was determined by analysis of lipid staining and proliferation by cell counting. The RXR agonist induced a more diffuse and granular pattern of lipid staining throughout colonies than did PPAR agonists. The PPAR ligands WY, TRO, and cPGI2 induced 37, 35, and 59% lipid-forming colonies (LFCs), respectively (P < 0.05 vs controls, which averaged 19%). Low-dose rexinoid (10(-8) M) alone exerted no significant effect but amplified the effect of cPGI2 (P < 0.05). Middose rexinoid (10(-7) M), which alone induced about 40% LFCs, had an additive effect on differentiation with WY, TRO, and cPGI2 (71, 48, and 83% LFCs respectively, P < 0.05 vs each agonist alone). Proliferation was enhanced significantly by either rexinoid or cPGI2, but there was no change in growth when the two were added together. The greater effectiveness of cPGI2 than the other PPAR agonists may be explained by the predominance of PPARdelta gene expression in cultured sebocytes, as demonstrated by RNase protection assay. These studies demonstrate that a submaximal dose of RXR agonist augmented the stimulation of sebocyte differentiation by PPAR agonists, as expected from PPAR-RXR heterodimerization. However, the evidence for PPAR-RXR cooperativity is limited. The pattern of lipid staining is compatible with an independent effect of rexinoid on sebocyte differentiation. Furthermore, since there is no enhancement of the growth-promoting effects of RXR agonist and cPGI2 when they are combined, this effect also does not appear to be mediated by PPAR-RXR interaction.

Isolation of a recombinant formate dehydrogenase by pseudo-affinity expanded bed adsorption.

Formate dehydrogenase (FDH) is an enzyme of industrial interest, which is recombinantly expressed as an intracellular protein in Escherichia coli. In order to establish an efficient and reliable purification protocol, an expanded bed adsorption (EBA) process was developed, starting from the crude bacterial homogenate. EBA process design was performed with the goal of finding operating conditions which, on one hand, allow efficient adsorption of the target protein and which, on the other hand, support the formation of a perfectly classified fluidised bed (expanded bed) in the crude feed solution. A pseudo-affinity ligand (Procion Red HE3B) was used to bind the FDH with high selectivity and reasonable capacity (maximum equilibrium capacity of 30 U/ml). Additionally, a simplified modelling approach, involving small packed beds for generation of process parameters, was employed for defining the operating conditions during sample application. In combination with extended elution studies, a process was set up, which could be scaled up to 7.5 l of adsorbent volume yielding a total amount of 100,000 U of 94% pure FDH per run. On this scale, 19 l of a benzonase-treated E. coli homogenate of 15% wet-weight (pH 7.5, 9 mS/cm conductivity) were loaded to the pseudo-affinity adsorbent (0.25 m sed. bed height, 5 x 10(-4) m/s fluid velocity). After a series of two wash steps, a particle-free eluate pool was obtained with 85% yield of FDH. This excellently demonstrates the suitability of expanded bed adsorption for efficient isolation of proteins by combining solid-liquid separation with adsorptive purification in a single unit operation.

Activation-induced apoptosis and cell surface expression of Fas (CD95) ligand are reciprocally regulated by retinoic acid receptor alpha and gamma and involve nur77 in T cells.

It has been previously shown that CD4+ T cells enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. In Jurkat cells TCR stimulation regulates the de novo synthesis of FasL, while in the influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) the cell surface appearance of a preformed FasL is initiated. Both processes are dependent on new mRNA and protein synthesis, involve up-regulation of nur77, and can be inhibited by retinoic acids (RA). Two groups of nuclear receptors for RA have been identified: retinoic acid receptors (RAR) and retinoid X receptors (RXR). In this study various synthetic retinoids were used to define which receptors regulate TCR-mediated apoptosis. It is demonstrated that the inhibition is mediated via RARalpha, while RARgamma enhances TCR-mediated apoptosis, and when both receptors are stimulated, the costimulation by RXR will promote the effect of RARalpha. Evidence is presented that these receptors affect the transcriptional activity of nur77 and consequently the expression of FasL. Our data suggest a complex interaction between the various isoforms of retinoid receptors in regulating T cell death and demonstrate that the target through which retinoids regulate TCR-mediated apoptosis is nur77.

Influence of imidazole replacement in different structural classes of histamine H(3)-receptor antagonists.

The reference compounds for histamine H(3)-receptor antagonists carry as a common feature an imidazole moiety substituted in the 4-position. Very recently novel ligands lacking an imidazole ring have been described possessing a N-containing non-aromatic heterocycle instead. In this study we investigated whether imidazole replacement, favourably by a piperidine moiety, is generally applicable to different structural classes of reference compounds, e.g., thioperamide, carboperamide, clobenpropit, FUB 181, ciproxifan, etc. While replacement led to a loss of affinity for many of the compounds, it was successfully applied to some ether derivatives. The piperidine analogues of FUB 181 and ciproxifan, 3-(4-chlorophenyl)propyl 3-piperidinopropyl ether hydrogen oxalate (6) and cyclopropyl 4-(3-piperidinopropyloxy)phenyl methanone hydrogen maleate (7), almost maintained in vitro affinities, pK(i) values of 7.8 and 8.4, respectively, and showed high potency in vivo after p.o. administration (ED(50) values of 1.6 and 0.18 mg/kg, respectively).

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death.

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.

Cell death in HIV pathogenesis and its modulation by retinoids.

Patients infected with the human immunodeficiency virus exhibit a progressive decline in the CD4 T-cell number, resulting in immunodeficiency and increased susceptibility to opportunistic infections and malignancies. Although CD4 T cell production is impaired in patients infected with HIV, there is now increasing evidence that the primary basis of T cell depletion is accelerated apoptosis of CD4 and CD8 T cells. The rate of lymphocyte apoptosis in HIV infection correlates inversely with the progression of the disease: it is low in long-term progressors and in patients undergoing highly active antiretroviral therapy. Interestingly, only a minor fraction of apoptotic lymphocytes are infected by HIV, indicating that the enhanced apoptosis does not necessarily always serve to remove the HIV+ cells and results from mechanisms other than direct infection. Thus, understanding and influencing the mechanisms of HIV-associated lymphocyte apoptosis may lead to new therapies for HIV disease. In this paper the potential effects of retinoids on CD4 T cell apoptosis is discussed.

13-cis retinoic acid exerts its specific activity on human sebocytes through selective intracellular isomerization to all-trans retinoic acid and binding to retinoid acid receptors.

Despite its potent biologic effect on human sebocytes, 13-cis retinoic acid exhibits low binding affinity for cellular retinoic acid binding proteins and nuclear retinoid receptors. Hence, 13-cis retinoic acid may represent a pro-drug possibly acting through all-trans isomerization. In this study, marked isomerization of 13-cis retinoic acid has been confirmed in cultured SZ95 sebocytes showing 2- to 15-fold higher levels of all-trans retinoic acid at 12-72 h, as measured by high performance liquid chromatography. In contrast, only low amounts of all-trans retinoic acid were converted intracellularly to its 13-cis isoform. 9-cis retinoic acid was not detected after either 13-cis retinoic acid or all-trans retinoic acid treatment. The rapid isomerization of 13-cis retinoic acid to high levels of all-trans retinoic acid was a sebocyte-specific event, as no significant isomerization of 13-cis retinoic acid to all-trans retinoic acid occurred in HaCaT keratinocytes. De novo mRNA expression of cytochrome P450 1A1, a major xenobiotic metabolizing enzyme, in SZ95 sebocytes was induced by all-trans retinoic acid, but not by 13-cis retinoic acid. In addition, mRNA levels of cellular retinoic acid-binding protein II, which is supposed to regulate the concentration of intracellular all-trans retinoic acid, rapidly increased under all-trans retinoic acid treatment (30 min-6 h), whereas the 13-cis retinoic acid effect was markedly weaker and delayed. Both 13-cis retinoic acid and all-trans retinoic acid suppressed mRNA expression of cytochrome P450 1A2. In parallel experiments, 13-cis retinoic acid significantly reduced SZ95 sebocyte proliferation at 10-7 M, show- ing 30-40% inhibition after 9 d. All-trans retinoic acid and 9-cis retinoic acid exhibited similar anti-proliferative effects. AGN 193109, a pan-antagonist of the retinoic acid receptors, antagonized the anti-proliferative activity of all retinoic acid isomers tested in a concentration-dependent manner with complete abolishment at ratios of 1:10 13-cis retinoic acid and 1:1 all-trans retinoic acid. Coincubation of SZ95 sebocytes with 13-cis retinoic acid and AGN 193109 did not alter the intracellular concentration of 13-cis retinoic acid and its isomerization profile. In contrast, the retinoid X receptor antagonist CD 3507 did not affect the inhibition of SZ95 sebocyte proliferation induced by retinoic acids. Our findings indicate: (i) a selective 13-cis retinoic acid isomerization to all-trans retinoic acid in the intracellular compartment of SZ95 sebocytes; (ii) a reduced all-trans retinoic acid inactivation process after 13-cis retinoic acid treatment as compared with treatment with all-trans retinoic acid; and (iii) a retinoic acid receptor-mediated inhibition of SZ95 sebocyte proliferation. These data explain the sebocyte-specific activity of 13-cis retinoic acid and support a pro-drug/drug relation between 13-cis retinoic acid and all-trans retinoic acid.

Identification of a unique binding protein specific for a novel retinoid inducing cellular apoptosis.

The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalenecarboxylic acid (AHPN, CD437) induces apoptosis in a variety of cell types, many of which are cancer cells that resist the antiproliferative and/or differentiating effects of retinoids. While the retinoids exert their effects by binding to the retinoic acid nuclear receptors (RARs) or retinoid X receptors (RXRs), AHPN (CD437) binds to another protein with different ligand specificity. In nuclear extracts from HL-60R cells the binding of AHPN (CD437) was only minimally competed by either retinoic acid (tRA)or 9-cis-retinoic acid (9-cis-RA), the natural ligands for the RARs and RXRs, respectively. Moreover, AHPN (CD437) was unable to compete with either tRA or 9-cis-RA for binding to endogenous retinoid receptors in nuclear extracts from the MDA-MB-468 breast carcinoma cell line. Size exclusion chromatography revealed AHPN binding to a 95 kDa protein(s) which is neither an RAR or RXR. Our results suggest that apoptosis induction by AHPN (CD437) may occur through interaction with another protein and is independent of the RAR/RXR-signaling pathways.

Retinoid-dependent growth inhibition, differentiation and apoptosis in acute promyelocytic leukemia cells. Expression and activation of caspases.

In the NB4 model of acute promyelocytic leukemia (APL), ATRA, 9-cis retinoic acid (9-cis RA), the pan-RAR and RARalpha-selective agonists, TTNPB and AM580, induce growth inhibition, granulocytic differentiation and apoptosis. By contrast, two RXR agonists, a RARbeta agonist and an anti-AP1 retinoid have very limited activity, ATRA- and AM580-dependent effects are completely inhibited by RAR antagonistic blockade, while 9-cis RA-induced cell-growth-inhibition and apoptosis are equally inhibited by RAR and RXR antagonists. ATRA, 9-cis RA and AM580 cause upregulation of the mRNAs coding for pro-caspase-1, -7, -8, and -9, which, however, results in increased synthesis of only pro-caspase-1 and -7 proteins. These phenomena are associated with activation of pro-caspase-6, -7 and -8, cytochrome c release from the mitochondria, inversion of Bcl-2/Bax ratio and degradation of PML-RARalpha. Caspase activation is fundamental for retinoid-induced apoptosis, which is suppressed by the caspase-inhibitor z-VAD.

Differential effects of several retinoid receptor-selective ligands on squamous differentiation and apoptosis in airway epithelial cells.

The roles of the different retinoid receptors on the differentiation of rabbit tracheal epithelial (RbTE) cells in primary culture were analysed using selective agonists for the retinoid acid receptor subtypes RARalpha (CD336), RARbeta (CD2019), RARgamma (CD437), an RAR panagonist (CD367), a retinoid X receptor RXR panagonist (CD2624) and an antagonist for RARbeta/gamma (CD2665). Squamous differentiation was assessed via expression of cytokeratins CK13/CK4 and transglutaminase I (TGI), specific markers of metaplasia. Treatment with RARalpha and beta agonists or RAR panagonist, but not the RARgamma agonist or RXR agonist, is required for the inhibition of squamous metaplasia, evidenced by inhibition of CK13/CK4 and TGI expression. The expression of CK10 cytokeratin of keratinizing epithelia, CK14/CK5 basal cell cytokeratins, and CK6 marker of cell proliferation decreases upon exposure of the RARaalpha/beta and RXR agonists. The RARgamma agonist CD437, inactive in the decrease in CK13/CK4, CK10 and CK14, reduces CK5/CK6 amounts. CD437 is responsible for a dose-dependent apoptotic response. Nuclear labelling with propidium iodide (PI) and electron microscopy revealed chromatin condensation and nuclear fragmentation. DNA cleavage and cell fragmentation were confirmed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The RARbetagamma antagonist was also slightly active. The results indicate that CD437 causes growth arrest in the early S-phase of the cell cycle and prevents the transition G1-S-phase. CD437 was demonstrated to induce apoptosis in the S-phase cells identified by bromodeoxyuridine (BrdU) incorporation. In conclusion, RARalpha/beta ligands are effective inhibitors of squamous differentiation. On the contrary, RARgamma ligand appears to be inefficient in metaplasia inhibition, but the selective RARgamma agonist CD437 induces growth arrest and apoptosis of basal proliferative cells.

S-phase arrest and apoptosis induced in normal mammary epithelial cells by a novel retinoid.

The addition of all-trans-retinoic acid has been found to mediate a G1 cell cycle phase arrest but not apoptosis in normal mammary epithelial cells. We have now found that addition of the novel retinoid 6-[3-(1-adamantyl)]-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), which appears to function through a pathway independent of retinoic acid nuclear receptors, results in an S-phase arrest that is preceded by a 4-fold elevation in the levels of the cyclin-cyclin dependent kinase (cdk) inhibitor p21WAF1/CIP1. Failure to inhibit E2F-1 activation of genes through its phosphorylation by the cyclin cdk2 kinase has been shown to result in S-phase arrest and apoptosis in a number of cell types. Although exposure of the normal mammary cells to CD437 does not result in modulation of cyclin A or cdk2 levels, an increase in E2F-1 levels and a marked inhibition of cyclin A/cdk2 kinase activity are observed. Exposure to CD437 results in enhanced E2F-1 binding to its DNA consensus sequences and transcriptional activity during S phase. We hypothesize that this enhanced E2F-1 transcriptional activity results in S-phase arrest and subsequent apoptosis that has been observed in other systems.

Selective agonists of retinoic acid receptors: comparative toxicokinetics and embryonic exposure.

Three biologically active synthetic retinoids were investigated that bind selectively to retinoic acid receptors RARs (alpha, beta and gamma). The retinoids were previously demonstrated to have different teratogenic effects in the mouse in terms of potency and regioselectivity. The teratogenic potency rank order (alpha >beta >gamma) was found to be more or less compatible with the receptor binding affinities and transactivation potencies of the retinoid ligands to their respective receptors. The RARalpha agonist (Am580; CD336) induced a wide spectrum of malformations; CD2019 (RARbeta agonist) and especially CD437 (RARgamma agonist) produced more restricted defects. In the current study we tried to address whether the differences in teratogenic effects are solely related to binding affinity and transactivation differences or also due to differences in embryonic exposure. Therefore, transplacental kinetics of the ligands were assessed following administration of a single oral dose of 15 mg/kg of either retinoid given to NMRI mice on day 11 of gestation. Am580 was rapidly transferred to the embryo resulting in the highest embryonic exposure [embryo to maternal plasma area under the time vs concentration curve (AUC)(0-24 h )ratio (E/M) was 1.7], in accordance with its highest teratogenic potency. The low placental transfer of CD2019 (E/M of 0.3) was compatible with its lower teratogenic potential. Of major interest was the finding that the CD437, though being least teratogenic, exhibited considerable embryonic exposure (E/M of 0.6). These findings suggest that both the embryonic exposure and receptor binding transactivation selectivity are crucial determinants of the teratogenicity of these retinoid ligands.

The role of specific retinoid receptors in sebocyte growth and differentiation in culture.

Retinoic acid derivatives (retinoids) exert their pleiotropic effects on cell development through specific nuclear receptors, the retinoic acid receptors and retinoid X receptors. Despite recent progress in understanding the cellular and molecular mechanisms of retinoid activity, it is unknown which of the retinoid receptor pathways are involved in the specific processes of sebocyte growth and development. In this study, we investigated the roles of specific retinoid receptors in sebocyte growth and differentiation, by testing the effects of selective retinoic acid receptor and retinoid X receptor ligands at concentrations between 10-10 M and 10-6 M in a primary rat preputial cell monolayer culture system. Cell growth was determined by number of cells and colonies, and cell differentiation by analysis of lipid-forming colonies. All-trans retinoic acid and selective retinoic acid receptor agonists (CD271 = adapalene, an RAR-beta,gamma agonist; CD2043 = retinoic acid receptor pan-agonist; and CD336 = Am580, an RAR-alpha agonist) caused significant decreases in numbers of cells, colonies, and lipid-forming colonies, but with an exception at high doses of all-trans retinoic acid (10-6 M), with which only a small number of colonies grew but they became twice as differentiated as controls (42.2 +/- 4.0% vs 22.6 +/- 2.7%, mean +/- SEM, lipid-forming colonies, p < 0.01). Furthermore, the RAR-beta,gamma antagonist CD2665 antagonized the suppressive effects of all-trans retinoic acid, adapalene, and CD2043 on both cell growth and differentiation. In contrast, the retinoid X receptor agonist CD2809 increased cell growth slightly and lipid-forming colonies dramatically in a clear dose-related manner to a maximum of 73.7% +/- 6.7% at 10-6 M (p < 0. 001). Our data suggest that retinoic acid receptors and retinoid X receptors differ in their roles in sebocyte growth and differentiation: (i) retinoic acid receptors, especially the beta and/or gamma subtypes, mediate both the antiproliferative and antidifferentiative effects of retinoids; (ii) retinoid X receptors mediate prominent differentiative and weak proliferative effects; (iii) the antiproliferative and antidifferentiative effects of all-trans retinoic acid are probably mediated by retinoic acid receptors, whereas its differentiative effect at high dose may be mediated by retinoid X receptors via all-trans retinoic acid metabolism to 9-cis retinoic acid, the natural ligand of retinoid X receptors.

Isolation and characterization of an acute promyelocytic leukemia cell line selectively resistant to the novel antileukemic and apoptogenic retinoid 6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid.

6-[3-adamantyl-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) is a novel compound that represents the prototype of a new class of synthetic retinoids with apoptogenic properties in acute promyelocytic leukemia (APL) and other types of leukemia. In this article, using SCID mice xenografted with APL-derived NB4 cells, we demonstrate that CD437 has significant antileukemic activity in vivo. In addition, we report on the isolation and characterization of an APL cell line (NB4.437r) resistant to CD437. The cell line retains expression of PML-RARalpha and is approximately 33-fold more resistant than the parental counterpart to the apoptogenic effects of the retinoid. Resistance is relatively specific to CD437 and structural congeners because the NB4.437r cell line is still sensitive to various types of apoptogenic compounds. The CD437-resistant cell line maintains sensitivity to the antiproliferative and apoptotic action of all-trans-retinoic acid, AM580, and fenretinide, though it shows partial resistance to the cytodifferentiating effects of the first 2 compounds. Resistance to CD437 lays upstream of the CD437-induced release of cytochrome c from the mitochondria and the activation of caspase-3, -7, -8, and -9. Furthermore, NB4.437r cells are deficient in the CD437-dependent activation of nuclear NFkb and AP1-binding activities and in the phosphorylation of the protein kinase Akt. In the case of AP1, deficient assembly of the complex is not caused by the lack of activation of the Jun N-terminal kinase (JNK) family of kinases. The novel cell line will be useful in the elucidation of the molecular mechanisms underlying the apoptogenic action of CD437 and structurally related retinoids. (Blood. 2000;95:2672-2682)