Osteoconductive and bioresorbable composites based on poly(L,L-lactide) and pseudowollastonite: from synthesis and interfacial compatibilization to in vitro bioactivity and in vivo osseointegration studies.

This contribution reports on the elaboration of novel bioresorbable composites consisting of pseudowollastonite (psW) (a silicate-based polycrystalline ceramic (α-CaSiO(3))) and poly(L,L-lactide) as a valuable polymeric candidate in bone-guided regeneration. These composites were prepared by direct melt-blending to avoid the use of organic solvents harmful for biomedical applications. Amphiphilic poly(ethylene oxide-b-L,L-lactide) diblock copolymers synthesized by ring-opening polymerization were added to psW-based composites to modulate the bioactivity of the composites. The bioactivity of the composites was first evaluated by monitoring the release of bioactive Ca(2+) and (SiO(4))(4-) ions as well as the concomitant formation of hydroxyapatite on the material surface after soaking them in physiological fluid. Subsequently, the composites were studied in vitro to evaluate their cytotoxicity in the presence of SaOS-2 osteoblastic cells and in vivo to assess their osteoconductivity in an orthotopic rat tibia model. This study provides a first insight into the use of direct melt-blended psW-poly(L,L-lactide) composites for bone-regeneration applications.

[Cascade of gene activation in Landouzy Dejerine muscular dystrophy]. / Une activation de gènes en cascade dans la dystrophie musculaire facioscapulohumérale.

Our laboratory studies the Landouzy Dejerine muscular dystrophy or FSHD, a genetic disease which affects 7 in 100,000 individuals. The genetic defect is a deletion on chromosome 4 that decreases the copy number of a repeated DNA element, disturbs chromatin structure and activates the expression of neighbouring genes. The originality of our team has been to identify a gene within the repeated element itself and to show its activation in FSHD muscle cells. This gene expresses DUX4, a transcription factor that targets tens of genes, some of which express other transcription factors which target other genes, leading to a general deregulation. This DUX4-mediated cascade recapitulates by itself the major pathological features of FSHD: muscle atrophy, differentiation defect, oxidative stress... The homologous DUX4c gene located 42 kb from the repeat array expresses a protein that triggers myoblast proliferation. Its high expression level in severe cases of FSHD most probably contributes to the pathology by interfering with myoblast fusion with the muscle fibers at the last steps of muscle regeneration. We are performing global analyses of proteins and metabolites in healthy and FSHD myotubes (collaboration R Wattiez and JM Colet, UMONS) to identify abnormalities and their links with DUX4 or DUX4C.

The helicase-like transcription factor and its implication in cancer progression.

The helicase-like transcription factor (HLTF) belongs to the SWI/SNF family of chromatin-remodeling factors. Several SWI/SNF genes are disrupted in cancer, suggesting their possible role as tumor suppressors. Similarly, the HLTF gene was found to be inactivated by hypermethylation in a significant number of colon, gastric and uterine tumors, indicating that HLTF silencing may confer a growth advantage and that HLTF could be considered as a tumor suppressor gene. However, 20-fold HLTF overexpression was detected in various transformed cell lines, suggesting that HLTF could be associated with neoplastic transformation and act more like an oncogene. Moreover, HLTF activation was recently linked to the initial steps of carcinogenesis in an experimental model of estrogen-induced kidney tumors. Those apparently contradictory observations suggest that HLTF might play various roles in cancer. In this review, we will try to reconcile all these data in order to specify the role of HLTF in cancer progression.

[Functional study of a gene candidate for Landouzy-Dejerine muscular dystrophy]. / Etude fonctionnelle d'un gène candidat pour la dystrophie musculaire de Landouzy-Dejerine.

Facioscapulohumeral muscular dystrophy (FSHD) or Landouzy Dejerine muscular dystrophy affects one in 20,000 individuals. It is characterized by a progressive degeneration of skeletal muscles that progresses asymmetrically from the upper to the lower parts of the body. Although the genetic defect has been known for twelve years, the causative gene has not been identified. Patients exhibit a partial deletion in a series of repeated elements mapping close to the end of the chromosome 4 long arm. Investigators believe this deletion disturbs chromatin structure over a long distance, affecting the expression of several genes. Our laboratory has identified a gene within the repeated element itself. This gene is functional and encodes a protein, DUX4, located at the periphery of the cell nucleus. DUX4 is detected in small amounts in primary myoblasts cultures of patient but not of non-affected individuals. Its forced expression in control cells causes cell death within 48 hours. Patient primary myoblasts present oxidative stress: this phenomenon is also induced by DUX4 forced expression. In conclusion, we propose the DUX4 gene as a candidate for FSHD.

Active genes in junk DNA? Characterization of DUX genes embedded within 3.3 kb repeated elements.

The human genome contains hundreds of repeats of the 3.3 kb family in regions associated with heterochromatin. We have previously isolated a 3.3 kb-like cDNA encoding a double homeodomain protein (DUX1). Demonstration that the protein was expressed in human rhabdomyosarcoma TE671 cells, and characterization of a homologous promoter suggested that functional DUX genes might be present in 3.3 kb elements. In the present study, we describe two nearly identical 3.3 kb/DUX genes derived from PAC 137F16 (DUX3), and TE671 genomic DNA (DUX5), both mapping to all the acrocentric chromosomes. Their promoters harbor a GC and a TATAA box, and the open reading frame of the intronless structural part encodes two DUX proteins differing by alternative translation initiation. The shorter protein of the DUX5 gene is identical to DUX1. Using a protein truncation test, we could show that these two proteins are encoded by total RNA, but not by poly (A)(+) RNA, from different human tissues and cell lines. Our results indicate that active genes of unusual structure are present in chromosome regions characterized by large amounts of heterochromatic repetitive DNA.

Far upstream sequences regulate the human prolactin promoter transcription.

The human prolactin gene is mainly expressed in pituitary lactotrope cells, but transcription from an alternative, far upstream promoter was detected in lymphoid, placental and mammary cells. We describe the transcriptional activity in rat pituitary cells of the complete region separating the two promoters, using transient transfection experiments. A far upstream activating region was only functional in combination with the prolactin promoter. DNaseI protection experiments revealed, in addition to binding sites for the pituitary-specific factor Pit-1, sites (e.g. SD1) for several ubiquitous factors and one lymphoid-specific factor (SD4). A single copy of the ubiquitous site SD1 or the lymphoid-specific site SD4 was unable to activate transcription of a heterologous promoter in pituitary cells. However, SD1 activated transcription in nonpituitary cells and SD4 was functional specifically in lymphoid cells. Five copies of a distal site (D8) activated transcription in each cell type tested. Gel retardation experiments show that this site binds the specific factor C/EBP in liver and a distinct factor in other cell types. Our results suggest that different elements within this large region direct specific expression from each promoter via a complex interplay between cell-specific and ubiquitous transcription factors.

1,25-Dihydroxyvitamin D(3) induction of the tissue-type plasminogen activator gene is mediated through its multihormone-responsive enhancer.

Tissue-type plasminogen activator (t-PA) is a positive modulator of the plasminogen-plasmin system, which is involved in bone remodeling. In the present study, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] was found to stimulate t-PA gene expression in ROS17/2.8 osteosarcoma cells. Transient transfection analysis and in vitro DNA binding studies identified two vitamin D-responsive elements (VDRE) in the human t-PA enhancer. The first VDRE (bp -7175 to -7146) comprised an inverted palindrome separated by 9 bp (IP9) overlapping a palindrome separated by 3 bp. The second VDRE (bp -7315 to -7302) is an IP2 element overlapping the previously identified retinoic acid-responsive element. 1,25(OH)(2)D(3) treatment of primary osteoblasts derived from t-PAlacZ transgenic mice containing 9 kb of 5' sequence of the human t-PA gene increased the number of lacZ-positive cells, fitting with the probability model of enhancer function.

Involvement of Sp1 in basal and retinoic acid induced transcription of the human tissue-type plasminogen activator gene.

Transcription of the human tissue-type plasminogen activator (t-PA) gene is regulated by a multi-hormonal responsive enhancer at -7 kb. Transient co-transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t-PA reporter constructs showed that Sp1 and Sp3 activate the t-PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.

Pit-1 mediates cell-specific and cAMP-induced transcription of the tilapia GH gene.

Expression of the tilapia growth hormone (tiGH) gene is pituitary-specific and controlled by intracellular cAMP levels. DNaseI protection experiments allowed us to identify four Pit-1 binding sites in the tiGH - 465/ + 19 region. Deletion and mutagenesis analysis revealed that the - 131/+ 19 region, containing two Pit-1 sites, or four copies of the most proximal site tiGHF1 fused to the heterologous Tk promoter, confer high level expression in rat pituitary cells and direct transcription in non-pituitary cells only after expression of rat Pit-1. We show that a tilapia pituitary factor specifically binds to site tiGHF1 and obtained a partial cDNA sequence coding for tilapia Pit-1. The cAMP stimulation is mediated by the proximal (- 131/- 31) promoter region. It is Pit-1-dependent and requires the tiGHF1 site. In addition, four copies of this site confer cAMP inducibility to the Tk promoter in GC cells.

Nucleotide sequence of the partially deleted D4Z4 locus in a patient with FSHD identifies a putative gene within each 3.3 kb element.

Facioscapulohumeral muscular dystrophy (FSHD) is linked to the polymorphic D4Z4 locus on chromosome 4q35. In non-affected individuals, this locus comprises 10-100 tandem copies of members of the 3.3kb dispersed repeat family. Deletions leaving 1-8 such repeats have been associated with FSHD, for which no candidate gene has been identified. We have determined the complete nucleotide sequence of a 13.5kb EcoRI genomic fragment comprising the only two 3.3kb elements left in the affected D4Z4 locus of a patient with FSHD. Sequence analyses demonstrated that the two 3.3kb repeats were identical. They contain a putative promoter that was not previously detected, with a TACAA instead of a TATAA box, and a GC box. Transient expression of a luciferase reporter gene fused to 191bp of this promoter, demonstrated strong activity in transfected human rhabdomyosarcoma TE671 cells that was affected by mutations in the TACAA or GC box. In addition, these 3.3kb repeats include an open reading frame (ORF) starting 149bp downstream from the TACAA box and encoding a 391 residue protein with two homeodomains (DUX4). In-vitro transcription/translation of the ORF in a rabbit reticulocyte lysate yielded two (35)S Cys/ (35)S Met labeled products with apparent molecular weights of 38 and 75kDa on SDS-PAGE, corresponding to the DUX4 monomer and dimer, respectively. In conclusion, we propose that each of the 3.3kb elements in the partially deleted D4Z4 locus could include a DUX4 gene encoding a double homeodomain protein.

Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene.

Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families.

Structure and functional analysis of a tilapia (Oreochromis mossambicus) growth hormone gene: activation and repression by pituitary transcription factor Pit-1.

A gene encoding the Tilapia mossambica (Oreochromis mossambicus) growth hormone (tiGH) was isolated and sequenced. The gene spans 5.6 kb, including 3.7 kb of 5' and 0.2 kb of 3' flanking sequences and a 1.7-kb transcription unit comprised of six exons and five introns. The gene and the 5' flanking region contain several potential binding sites for Pit-1, a key transcription activator of mammalian GH genes. One of these (-57/-42) is highly conserved in fish GH genes. It activates transcription in pituitary cells and binds Pit-1. Transfection of luciferase reporter plasmids containing either the -3602/+19 tiGH sequence or one of its 5' deletion mutants (-2863/, -1292/, and -463/+19) resulted in strong activity in Pit-1-producing rat pituitary GC cells. A dose-dependent activation of the tiGH promoter was achieved in nonpituitary fish EPC and monkey COS cells cotransfected with a rat Pit-1 expression vector, demonstrating the crucial role played by Pit-1 as an activator of the tiGH gene. Fusion of the tiGH promoter with the beta-galactosidase gene led to transient expression specifically in the nervous system of microinjected zebrafish embryos. The activity of the tiGH promoter in GC and EPC cells was strongly repressed by extending its 3' end from +19 to +40, a sequence in which a Pit-1-binding site was identified using gel retardation assays. Point mutations of the site that suppressed Pit-1 binding in vitro restored full tiGH promoter activity. Thus, a Pit-1-binding site located in the 5' untranslated region mediates Pit-1-dependent repression of the tiGH gene.

Characterization of a double homeodomain protein (DUX1) encoded by a cDNA homologous to 3.3 kb dispersed repeated elements.

Target genes for the helicase-like transcription factor (HLTF), a member of the SNF/SWI family, were immunoprecipitated from HeLa chromatin fragments with an anti-HLTF antibody. A 182 bp fragment ( HEFT1 ) presented 87% sequence identity with 3.3 kb dispersed repeats from the 4q35 D4Z4 locus linked to facioscapulohumeral muscular dystrophy (FSHD). The HEFT1 loci were, however, not genetically linked to FSHD. Transfection and in vitro binding studies identified within HEFT1 a promoter whose basal activity required a GC box activated by Sp1 or Sp3. A 4.4 kb homologous transcript was found mostly in human skeletal muscle and heart. A 1.2 kb cDNA fragment was cloned that encoded a 170 amino acid protein (DUX1) with two paired-type homeodomains. In vitro translated DUX1 specifically interacted in electrophoretic mobility shift assay (EMSA) with a P5 oligonucleotide (5'-GATCTGAGTCTAATTGAGAATTACTGTAC-3'). DUX1 co-expression activated up to 5-fold transient expression in insect cells of a minimal promoter-luciferase construct fused to P5. The presence of 20 kDa DUX1 in vivo in rhabdomyosarcoma TE671 cell extracts was shown by western blotting with a rabbit antiserum raised against a DUX1 peptide. This antiserum suppressed a TE671 protein-P5 complex in EMSA with identical migration as the in vitro translated DUX1-P5 complex. Genomic PCR experiments could not identify a gene fragment linking the HEFT1 and DUX1 sequences, which present one mismatch in their overlapping region. However, a similar gene was found in another 3.3 kb element comprising the HEFT1 promoter and a DUX1 -like open reading frame. In addition, homologous gene sequences were identified in 3.3 kb elements of the D4Z4/FSHD locus, considered until now 'junk' DNA.

Characterization and targeting of the murine alpha2-antiplasmin gene.

Alpha2-Antiplasmin (alpha2-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of alpha2-AP deficient mice, the murine alpha2-AP gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the alpha2-AP gene, but failed to identify the 5' boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical size and intron-exon boundaries obeying the GT/AG rule. The 5' boundary of exon 10 is identical in both genes while the 3' non-coding region is 64 bp longer in the human gene. Introns 2, 3, 6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5' flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse alpha2-AP gene encodes a 491-amino-acid protein comprising the experimentally determined NH2-terminus of the mature protein Val-Asp-Leu-Pro-Gly-. A targeting vector, pPNT.alpha2-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.alpha2-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding the mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.alpha2-AP yielded three targeted clones with correct homologous recombination at the 5'- and 3'-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate alpha2-AP deficient mice.

Nonisotopic quantitative analysis of protein-DNA interactions at equilibrium.

Two versions of an enzyme-linked immunosorbent assay-type method to quantify protein-DNA interactions at equilibrium were developed. The first variant comprised immobilization of DNA-binding protein on microtiter plates, incubation with biotinylated DNA, and tagging of bound DNA with streptavidin- and biotin-substituted horseradish peroxidase. In the second version, biotinylated DNA was immobilized on streptavidin-substituted microtiter plates, incubated with DNA-binding protein, and bound protein was quantified with specific antibodies. To illustrate the method, the interaction of a fusion protein between glutathione-S-transferase and the DNA-binding domain of the helicase-like transcription factor with its cis-element (the B box of the plasminogen activator inhibitor-1 promoter) was determined with both versions: a 1:1 stoichiometric interaction with an equilibrium dissociation constant (Kd) of 1 nM was found, which is similar to the value determined by electrophoretic mobility shift assay, demonstrating the validity of the assays.

Identification of a multihormone responsive enhancer far upstream from the human tissue-type plasminogen activator gene.

A 2.4-kilobase (kb) DNA fragment, located 7.1 kb upstream from the human tissue-type plasminogen activator (t-PA) gene (t-PA2.4), acts as an enhancer which is activated by glucocorticoids, progesterone, androgens, and mineralocorticoids. Transient expression of t-PA-chloramphenicol acetyltransferase reporter constructs in HT1080 human fibrosarcoma cells identified a glucocorticoid responsive unit with four functional binding sites for the glucocorticoid receptor, located between bp -7,501 and -7,974. The region from bp -7,145 to -9,578 (t-PA2.4) was found to confer a cooperative induction by dexamethasone and all-trans-retinoic acid (RA) to its homologous and a heterologous promoter, irrespective of its orientation. The minimal enhancer, defined by progressive deletion analysis, comprised the region from -7.1 to -8.0 kb (t-PA0.9) and encompassed the glucocorticoid responsive unit and the previously identified RA-responsive element located at -7.3 kb (Bulens, F., Ibañez-Tallon, I., Van Acker, P., De Vriese, A., Nelles, L., Belayew, A., and Collen, D. (1995) J. Biol. Chem. 270, 7167-7175). The amplitude of the synergistic response to dexamethasone and RA increased by reducing the distance between the enhancer and the proximal t-PA promoter. The synergistic interaction was also observed between the aldosterone and the RA receptors. It is postulated that the t-PA0.9 enhancer might play a role in the hormonal regulation of the expression of human t-PA in vivo.

The enhancers of the human placental lactogen B, A, and L genes: progressive activation during in vitro trophoblast differentiation and importance of the DF-3 element in determining their respective activities.

The hCS-A and hCS-B genes encoding human chorionic somatomammotropin and the related hCS-L gene are very similar in their coding and flanking sequences. For each of these genes, downstream enhancers, varying in strength, have been identified with the help of cytotrophoblast-derived JEG-3 cells, which do not express the hCS genes. Here we study the activity of the hCS enhancers in human syncytiotrophoblast in primary culture, which naturally expresses the hCS genes. We show that the activity of the hCS-B gene enhancer is mediated by two elements, DF-3 and DF-4, whereas the hCS-L and hCS-A gene enhancers display weaker activity due to mutations in their respective DF-3 sites. Replacement of the hCS-B DF-3 site with the homologous hCS-A sequence causes hCS-B enhancer activity to decrease. Primary cytotrophoblasts differentiate in culture to form the syncytiotrophoblast. We show that during this process the production of hCS progressively increases and that concomitantly all three hCS enhancers are progressively activated. A targeted mutation in the 3' part of the DF-4 element abolishes the binding of a protein present only in syncytiotrophoblast extracts and inactivates the DF-4 element. Thus, a direct correlation exists between the appearance of this syncytiotrophoblast-specific protein and hCS enhancer activity. This primary culture model proves useful in studying the regulation of the hCS genes.

The tilapia prolactin I gene: evolutionary conservation of the regulatory elements directing pituitary-specific expression.

To study the elements involved in the pituitary specific transcriptional regulation of the tilapia prolactin I gene (tiPRL I), we have cloned and entirely sequenced a 3.4-kb genomic fragment immediately upstream from the first exon. In footprinting experiments, three tilapia sequences are protected from DNase I digestion by rat pituitary extracts (base pair coordinates -643 to -593, -160 to -111, and -73 to -46). Computer analysis of the nucleotide sequence reveals significant homology to mammalian binding sites for Pit-1, a transcription factor that is known to mediate pituitary-specific expression of the PRL genes in mammals. The tiPRL I 5'-flanking sequences can direct transient expression of a linked luciferase reporter gene in transfected rat pituitary cell lines and tilapia pituitary primary cell cultures. Transient expression experiments with 5'-deletion mutants reveal three regulatory regions. Two have a stimulatory effect on transcription and one an inhibitory effect. Electrophoretic mobility-shift assays (EMSA) demonstrate that the rat Pit-1 factor specifically binds to tilapia DNA sequences. Several such tilapia Pit-1 binding sites mediate activation of a linked heterologous promoter in transfected rat and tilapia pituitary cells. As evidenced by EMSA, a Pit-1-like protein is present in tilapia pituitary extracts. All these data point to a high conservation of the molecular mechanisms involved in pituitary-specific expression of the PRL genes in vertebrates.

Characterization of a helicase-like transcription factor involved in the expression of the human plasminogen activator inhibitor-1 gene.

A 5.4-kb cDNA encoding the protein that binds to the B Box of the plasminogen activator inhibitor-1 (PAI-1) gene was isolated and sequenced. The protein, named helicase-like transcription factor (HLTF), contains a DNA-binding domain, a RING finger domain, and seven helicase domains and is homologous to SWI/SNF proteins. Two HLTF mRNAs of 5.5 and 4.5 kb were detected in most human tissues, a single gene was located on chromosome 3q24-25, and the protein was located in the nucleoplasm. Two HLTF proteins differing in translation start site (Met-1 or Met-123) were obtained by in vitro translation in reticulocyte lysate or by immunoprecipitation from HeLa cell nuclear extracts. In vitro transcription from the PAI-1 promoter in HeLa cell extracts was inhibited by HLTF antibodies and by the HLTF DNA binding domain. Over-expression of HLTF or HLTFMet123 produced a three-fold induction of PAI-1-LUC transient expression in HeLa cells. Mutation of the PAI-1 B Box led to an eight-fold reduction of basal PAI-1-LUC expression in these cell lines, but did not affect the four- to six-fold induction by phorbol esters.

Cyclosporin A, rapamycin and FK506 decrease prolactin release from rat pituitary cells in primary culture.

It is at present well established that prolactin exerts a non-specific immunoactivating function. In this work we tested whether the immunosuppressant drugs cyclosporin A, FK506 and rapamycin influence prolactin release from rat pituitary cells in primary culture. The tested drugs had no effect on the prolactin release measured during a 2h incubation period, indicating that they do not influence the secretion of prolactin from intracellular stores into the culture medium. During longer incubation times (48h), however, prolactin release was diminished to 56% +/- 18 (10 microM cyclosporin A), 64% +/- 14 (1 microM rapamycin) or 64% +/- 7 (1 microM FK506), suggesting an effect on prolactin production. At these drug concentrations no toxic effects were observed. The data indicate that inhibition of pituitary prolactin synthesis might contribute to the immunosuppressant action of cyclosporin A, rapamycin and FK506.