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C Jean

Publications and source records attributed to C Jean.

At least 19 recordsLinked to original sources
PubMed2005

TNFalpha stimulates NKG2D-mediated lytic activity of acute myeloid leukemic cells.

The mechanism by which leukemic cells interfere with normal hematopoiesis remains unclear. We show here that, whereas the leukemic KG1a cells are naturally devoid from cellular cytotoxicity, once activated by TNFalpha, they display cytolytic activity toward various cellular targets including CFU-GM. This mechanism is dependent on stimulation of the granzyme B/perforin system. In addition, KG1a cells expressed the NKG2D receptor and its signal-transducing adaptator DAP 10, which were functional as confirmed by redirected lysis experiments. Interestingly, flow cytometry analysis of 20 samples of patients with acute myeloid leukemia (AML) (FAB M0-M5) revealed the expression of NKG2D (40%) and other natural cytotoxicity receptors (40% for NKp30, 74% for NKp44, 39% for NKp46) by a pool >15% of leukemic cells. Furthermore, CD34+ hematopoietic progenitors undergoing granulomonocytic differentiation expressed NKG2D ligands. Altogether, we propose a model in which, upon stimulation by TNFalpha, leukemic cells may exert cytotoxicity against myeloid progenitors. This finding may have important clinical implications in the context of diseases characterized by TNFalpha accumulation, such as AML or myelodisplasic syndromes.

Acute Disease↗
PubMed2003

A carbohydrate diet rich in sucrose increased insulin and WAT in macronutrient self-selecting rats.

In order to evaluate the influence of a carbohydrate (CHO) diet rich in sucrose (37%) on food choice and body composition, Wistar rats received a food selection diet (protein, CHO, fat) from the time of weaning to 13 weeks of age. Three groups of animals were examined: the first received a CHO diet containing 37% sucrose; the second, a diet containing only 10% sucrose; and the third, control group, received a complete standard diet (14% protein, 72% CHO including 10% sucrose). Food intakes and body weight (BW) were recorded four times a week. No differences in total food intake were observed between the two self-selecting groups and the control group, and no differences were observed between the two self-selecting groups in terms of their protein intake (about 45% of the total calorie intake). The latter groups modified their selections during the 10-week period, but the variations were similar. BW gain in the 37% group was lower but the white adipose tissue (WAT)/total BW ratio was significantly higher than those seen in the control and 10% groups. Insulinemia was higher in 37% and control groups. In conclusion, the high preferences for protein and fat were identical, whatever the CHO diet composition. The sucrose level in the diet was an essential factor for the development of hyperinsulinemia, leptin resistance and thus a higher prevalence of obesity. These results confirm the importance of the quality of CHO sources in the diet.

Adipose Tissue↗
PubMed2003

Hormonal and developmental regulation of the mouse aldose reductase-like gene akr1b7 expression in Leydig cells.

The akr1b7 gene encodes an aldose reductase-like protein that is responsible for detoxifying isocaproaldehyde generated by the conversion of cholesterol to pregnenolone. The regulation of gene expression by human chorionic gonadotropin (hCG) was first investigated in the MA-10 Leydig tumor cell line. The akr1b7 gene was constitutively expressed and accumulation of its mRNA was increased in a dose- and time-dependent manner by treatment with hCG. akr1b7 mRNA accumulation was sharply increased in the presence of 0.25 nM hCG and it reached a fivefold increase within 2 h. AKR1B7 protein accumulation was delayed compared with that of the corresponding mRNA. In agreement, hCG significantly increased the levels of mRNA and protein of akr1b7 in primary cultures of adult mouse Leydig cells, thus suggesting that LH potentially regulates akr1b7 gene expression in vivo. Expression of akr1b7 was developmentally regulated in the testis. Unexpectedly, levels of akr1b7 mRNA increased from embryonic day 15 to the day of birth and declined until adulthood while AKR1B7 protein levels followed an inverse pattern, suggesting an important role for translational mechanisms.

Aldehyde Reductase↗
PubMed2001

Role of protein kinase C zeta isoform in Fas resistance of immature myeloid KG1a leukemic cells.

Leukemic CD34(+) immature acute myeloid leukemia (AML) cells express Fas receptor but are frequently resistant to Fas agonistic reagents. Fas plays an important role in T-cell-mediated cytotoxicity, and recently it has been suggested that altered Fas signaling may contribute to drug resistance. Therefore, Fas resistance could be one of the mechanisms by which AML progenitors escape chemotherapy or T-cell-based immune intervention. However, the molecular mechanism of Fas resistance in AML cells has not been identified. Fas signaling can be interrupted at 3 mains levels: Fas clustering, alteration of death-inducing-signaling-complex (DISC) formation, and effector caspase inhibition of downstream caspase-8. This study shows that in the Fas-resistant CD34(+)CD38(-) KG1a cells, Fas agonists resulted in Fas aggregation but not in caspase-8 activation, related to a defect in DISC formation. However, pretreatment with chelerythrin, but not with calphostin C, resulted in the restoration of Fas-induced caspase-8 activation and cytotoxicity, suggesting that some atypical protein kinase C (PKC) isoforms contributed to the lack of DISC formation. Indeed, treatment with antisense oligonucleotides directed against PKC zeta and enforced expression of Par-4, a negative regulator of PKC zeta activity, restored Fas-induced caspase-8 activity and apoptosis. Moreover, it was found that PKC zeta interacts with FADD and that PKC zeta immunoextracts prepared from KG1a cells are able to phosphorylate FADD in vitro, whereas this phosphorylation is dramatically reduced in Par-4 transfectant cells. In conclusion, it is suggested that in AML cells, PKC zeta plays an important role in Fas resistance by inhibiting DISC formation, possibly by phosphorylating FADD.

Alkaloids↗
PubMed2001

Physiological functions and hormonal regulation of mouse vas deferens protein (AKR1B7) in steroidogenic tissues.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) highly expressed in vas deferens epithelium and zona fasciculata of the adrenal cortex. Recombinant MVDP showed kinetic properties distinct from those of aldose reductase, including its spectrum of substrates, cofactor preference and sensitivity to inhibitors. We demonstrate that in adrenocortical cells, MVDP, rather than aldose reductase, is the principal reductase for isocaproaldehyde (a product of side-chain cleavage of cholesterol) and 4-hydroxynonenal (a lipid peroxidation product). In steroidogenic tissues MVDP expression is regulated by pituitary trophic hormones, namely ACTH in adrenals, FSH in ovaries, and LH in testicular Leydig cells.

Adrenal Cortex↗
PubMed2001

Metabolic evidence for adaptation to a high protein diet in rats.

This study was designed to assess the effects of long-term adaptation to a high protein diet on energy intake, body weight gain, body composition and splanchnic metabolic indicators in rats. For this purpose, adult male Wistar rats were fed either a 50 g/100 g dry matter (DM) protein diet (P50 group) or a 14 g/100 g DM protein diet (P14 group) for 21 d. These two groups were compared with a P14 pair-fed (P14-pf) group that consumed the same daily energy as the P50 group. The energy intake of the P50 group was 16 +/- 1% less than that of the P14 group (P < 0.05), and the P50 group had significantly lower body weight. The P50 group had significantly less adipose tissue compared with both P14 and P14-pf rats. The activities of the brush border membrane enzymes, neutral aminopeptidase and gamma-glutamyl transferase, were significantly higher in the P50 group than in the P14 rats. Similarly, the activities of alanine aminotransferase, arginase and serine dehydratase were significantly higher in the liver of P50 rats compared with P14 rats. Both amino acid transporter system A and X(A,G-) activities, measured in freshly isolated hepatocytes, were significantly higher in the P50 group (8- and 1.5-fold, P < 0.05, respectively) compared with the P14 group. The 1.5-fold increase in the steady-state activity of X(A,G-) was accompanied by a doubling of EAAT2 mRNA, involved in the system X(A,G-). This study provides confirmation that specific biochemical and molecular adaptive processes of the splanchnic area are involved in the response to variations in the protein content of the diet.

Adaptation, Physiological↗
PubMed2001

SF-1 (steroidogenic factor-1), C/EBPbeta (CCAAT/enhancer binding protein), and ubiquitous transcription factors NF1 (nuclear factor 1) and Sp1 (selective promoter factor 1) are required for regulation of the mouse aldose reductase-like gene (AKR1B7) expression in adrenocortical cells.

The MVDP (mouse vas deferens protein) gene encodes an aldose reductase-like protein (AKR1B7) that is responsible for detoxifying isocaproaldehyde generated by steroidogenesis. In adrenocortical cell cultures, hormonal regulation of MVDP gene occurs through the cAMP pathway. We show that in adrenals, the pituitary hormone ACTH regulates MVDP gene expression in a coordinate fashion with steroidogenic genes. Cell transfection and DNA-binding studies were used to investigate the molecular mechanisms underlying MVDP gene regulation in Y1 adrenocortical cells. Progressive deletions of upstream regulatory regions identified a -121/+41 fragment that was sufficient for basal and cAMP-mediated transcriptional activities. Gel shift assays showed that CTF1/nuclear factor 1 (NF1), CCAAT enhancer binding protein-ss (C/EBPss), and selective promoter factor 1 (Sp1) factors bound to cis-acting elements at positions -76, -61, and -52, respectively. We report that the cell-specific steroidogenic factor-1 (SF-1) interacts specifically with a novel regulatory element located in the downstream half-site of the proximal androgen response element (AREp) at position -102. Functional analysis of SF-1 and NF1 sites in the -121/+41 promoter showed that mutation of one of them decreases both constitutive and forskolin-stimulated promoter activity without affecting the fold induction (forskolin stimulated/basal). Individual mutations of C/EBP and Sp1 sites resulted in a loss of more than 50% of the cAMP-dependent induction. When both sites were mutated simultaneously, cAMP responsiveness was nearly abolished. Thus, in adrenocortical cells, both SF-1 and NF1 are required for high expression of the MVDP promoter while Sp1 and C/EBPss functionally interact in an additive manner to mediate cAMP-dependent regulation. Furthermore, we report that MVDP gene regulation is impaired in stably transfected Y1 clones expressing DAX-1. Taken together, our findings suggest that detoxifying enzymes of the aldose reductase family may constitute new potential targets for regulators of adrenal and gonadal differentiation and function, e.g. SF-1 and DAX-1.

Adrenal Cortex↗
PubMed2000

Adult Colorado potato beetles, Leptinotarsa decemlineata compensate for nutritional stress on oryzacystatin I-transgenic potato plants by hypertrophic behavior and over-production of insensitive proteases.

Protease inhibitors have been proposed as potential control molecules that could be engineered into potato plants for developing crops resistant to the Colorado potato beetle, Leptinotarsa decemlineata, a major pest of potato and other Solanaceae. In this study, we examined the effects of feeding young female beetles with foliage from a cultivar of the "Kennebec" potato line (K52) transformed with a gene encoding oryzacystatin I (OCI), a specific cysteine proteinase inhibitor with proven activity against cathepsin H-like enzymes of larvae and adults of the potato beetle. To evaluate the insect's performance, we collected data over a 16-d postemergence period on survival, diapause incidence, foliage consumption, weight gain, and oviposition of females. Tested individuals were fed untransformed (control) and OCI-transformed foliage at two stages of potato leaf differentiation, corresponding to "low" and "high" levels of OCI expression in leaves of K52. The OCI-expressing foliage did not affect female survival (close to 100%), incidence of diapause (15-30%), relative growth rate (RGR) during postemergence growth (5-9% d(-1)) or maximum weight reached (140-160 mg). Neither did it affect female reproductive fitness as measured by preoviposition time (8-9 d), 16-d fecundity (220-290 eggs), or egg eclosion incidence (86-91%). However, nutritional stress to females feeding on OCI foliage was evident, as reflected in their lower efficiency of conversion of ingested foliage (ECI) during postemergence growth, increased foliage consumed per egg laid (up to 119% more), and adaptation of their digestive proteolytic system to the inhibitory effect of OCI. Interestingly, beetles fed foliage expressing the highest level of OCI reacted rapidly to the presence of OCI by producing OCI-insensitive proteases, and exhibiting strong hypertrophic behavior by ingestion of 2.4-2.5 times more OCI rich foliage apparently as a compensatory response for nutritional stress due to the protease inhibitor in their diet.

Animals↗
PubMed2000

Down-regulation of AP1 activities after polarization of vas deferens epithelial cells correlates with androgen-induced gene expression.

Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i. e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.

Aldehyde Reductase↗
PubMed2000

Adrenal tumorigenesis targeted by the corticotropin-regulated promoter of the aldo-keto reductase AKR1B7 gene in transgenic mice.

Studies of ACTH functions in adrenal steroidogenesis have been facilitated by the availability of immortalized mouse adrenocortical Y1 cells. In order to obtain alternative cell lines with a more differentiated zona fasciculata (ZF) phenotype we used targeted tumorigenesis strategy. We have generated transgenic mice expressing the SV40 T antigen under the control of the ACTH-dependent promoter for the AKR1B7/MVDP gene (aldo-keto reductase 1B7/mouse vas deferens protein), which encodes an enzyme responsible for detoxifying isocaproaldehyde, the product of side-chain cleavage of cholesterol generated by steroidogenesis. Our previous data indicated that in the mouse adrenal, AKR1B7 expression was restricted to the ZF and that a 0.5-kb promoter region was able to target specific adrenal expression in transgenic mice. In situ hybridization analyses indicate that AKR1B7 expression during fetal and post-natal periods paralleled the onset of glucocorticoid synthesis and the development of ZF. In transgenic mice, ACTH control and developmental programming of the CAT gene driven by the 0.5-kb promoter followed endogenous gene regulation. Then transgenic mice harboring the 0.5-kb/SV40 T antigen construct were generated and two founders out of three developed adrenal tumors. Cells derived from the tumor of founder 1 (ATC1) were grown in presence of forskolin to maintain ACTH receptor expression and were tested for ACTH responsiveness by immunocychemistry and northern blot analyses. Even after several passages, the ACTH induced AKR1B7 and P450c11beta mRNAs accumulations were similar to that observed in mouse primary adrenocortical cell cultures. Our findings suggest that ATC1 cells have conserved essential features of ZF cells. In order to achieve complete characterization of these cells further analyses are currently performed to investigate their steroidogenic activity.

Adrenal Gland Neoplasms↗
PubMed1999

Product of side-chain cleavage of cholesterol, isocaproaldehyde, is an endogenous specific substrate of mouse vas deferens protein, an aldose reductase-like protein in adrenocortical cells.

Mouse vas deferens protein (MVDP) is an aldose reductase-like protein that is highly expressed in the vas deferens and adrenal glands and whose physiological functions were unknown. We hereby describe the enzymatic characteristics of MVDP and its role in murine adrenocortical Y1 cells. The murine aldose reductase (AR) and MVDP cDNAs were expressed in bacteria to obtain recombinant proteins and to compare their enzymatic activities. Recombinant MVDP was functional and displayed kinetic properties distinct from those of murine AR toward various substrates, a preference for NADH, and insensitivity to AR inhibitors. For MVDP, isocaproaldehyde, a product of side-chain cleavage of cholesterol generated during steroidogenesis, is the best natural substrate identified so far. In Y1 cells, we found that NADH-linked isocaproaldehyde reductase (ICR) activity was much higher than NADPH-linked ICR activity and was not abolished by AR inhibitors. We demonstrate that in Y1 cells, forskolin-induced MVDP expression enhanced NADH-linked ICR activity by 5-6-fold, whereas no variation in ICR-linked NADPH activity was observed in the same experiment. In cells stably transfected with MVDP antisense cDNA, NADH-linked ICR activity was abolished even in the presence of forskolin, and the isocaproaldehyde toxicity was increased compared with that of intact Y1 cells, as measured by isocaproaldehyde LD(50). In Y1 cells transfected with MVDP antisense cDNA, forskolin-induced toxicity was abolished by aminoglutethimide. These results indicate that in adrenocortical cells, MVDP is responsible for detoxifying isocaproaldehyde generated by steroidogenesis.

Adrenal Cortex↗
PubMed1999

The mammalian interphase centrosome: two independent units maintained together by the dynamics of the microtubule cytoskeleton.

In mammalian cells the centrosome or diplosome is defined by the two parental centrioles observed in electron microscopy and by the pericentriolar material immunostained with several antibodies directed against various centrosomal proteins (gamma-tubulin, pericentrin, centrin and centractin). Partial destabilization of the microtubule cytoskeleton by microtubule-disassembling substances induced a splitting and a slow migration of the two diplosome units to opposite nuclear sides during most of the interphase in several mammalian cell lines. These units relocated close together following drug removal, while microtubule stabilization by nM taxol concentrations inhibited this process. Cytochalasin slowed down diplosome splitting but did not affect its relocation after colcemid washing. These results account for the apparently opposite effects induced by microtubule poisons on centriole separation. Moreover, they provide new information concerning the centrosome cycle and stability. First, the centrosome is formed by two units, distinguished only by the number of attached stable microtubules, but not by pericentrin, gamma-tubulin, centrin and centractin and their potency to nucleate microtubules. Second, the centrosomal units are independent during most of the interphase. Third, according to the cell type, these centrosomal units are localized in close proximity because they are either linked or maintained close together by the normal dynamics of the microtubule cytoskeleton. Finally, the relocalization of the centrosomal units with their centrioles in cells possessing one or two centrosomes suggests that their relative position results from the overall tensional forces involving at least partially the microtubule arrays nucleated by each of these entities.

Animals↗
PubMed1999

Entry-into-human study with the novel immunosuppressant SDZ RAD in stable renal transplant recipients.

AIMS: To evaluate the tolerability of single oral SDZ RAD doses in stable renal transplant recipients and the pharmacokinetics of ascending SDZ RAD doses when coadministered with steady-state cyclosporin A microemulsion (Neoral). METHODS: This randomized, double-blind, placebo-controlled, sequential study involved 54 patients in six treatment groups; a different SDZ RAD dose (0.25, 0. 75, 2.5, 7.5, 15, 25 mg) was assessed in each group. Patients received a single oral dose of SDZ RAD (n=6) or placebo (n=3) with their usual Neoral dose. SDZ RAD and cyclosporin A pharmacokinetic parameters were determined. RESULTS: All SDZ RAD doses were well tolerated, with no discontinuations due to adverse events, serious adverse events, or deaths. Similar proportions of patients receiving SDZ RAD and placebo had at least one adverse event (44% and 50%, respectively). Mean changes in laboratory variables (baseline to endpoint) showed no clinically meaningful differences between SDZ RAD and placebo groups. SDZ RAD was absorbed rapidly and showed dose-proportional pharmacokinetics (dose: 2.5-25 mg), based on systemic exposure. Multiple postabsorptive phases in the pharmacokinetic profile indicate tissue distribution. The elimination half-life ranged from 24 to 35 h across the five highest dose groups. Pharmacokinetics were similar in men and women. Co-administration of escalating single oral SDZ RAD doses did not affect steady-state cyclosporin A pharmacokinetics. CONCLUSIONS: SDZ RAD was well tolerated; safety profiles of SDZ RAD and placebo were similar. SDZ RAD pharmacokinetics were dose-proportional across the range 2.5-25 mg in conjunction with cyclosporin A-based therapy, according to systemic exposure. Cyclosporin A pharmacokinetics were not affected by coadministration of single oral doses of 0.25-25 mg SDZ RAD.

Adolescent↗
PubMed1999

5'-flanking and intragenic sequences confer androgenic and developmental regulation of mouse aldose reductase-like gene in vas deferens and adrenal in transgenic mice.

The MVDP (mouse vas deferens protein) gene, which encodes an aldose reductase-like enzyme, is mainly expressed in vas deferens epithelium and adrenal cortex. Vas deferens MVDP gene transcription was known to be under androgenic control, we now have evidence for androgen and probable ACTH responsiveness of the MVDP gene in the adrenal. To analyze the role of potential regulatory regions in hormonal, developmental, and tissue-specific aspects of MVDP regulation, we generated transgenic mice harboring MVDP-CAT fusion genes. The constructs carried either -1.8 or -0.5 kb 5'-flanking sequence attached to the chloramphenicol acetyltransferase gene in presence or absence of a 3.5-kb intragenic fragment in a downstream position. We show that at least two regions ensure proper gene regulation in vivo. The first, located within the 1.8-kb promoter fragment, directs tissue specificity; positive elements necessary for vas deferens and adrenal expression lay within positions -1804 to -510 and -510 to +41, respectively. The second, located within the 3.5-kb intragenic fragment spanning intron 1 to intron 2, increases percentage of expressing lines and behaves as a vas deferens-specific enhancer. Hormonal and developmental control of transgenes closely parallel endogenous gene regulation. Androgen and ACTH responsiveness in adrenals is conferred by 0.5-kb promoter, whereas in vas deferens, full androgenic response of the 1.8-kb promoter required the 3.5-kb intragenic fragment. Thus, vas deferens and adrenals use distinct cis-acting elements to direct and regulate the expression of the MVDP gene.

Adrenal Glands↗
PubMed1999

Structural and functional analysis of the promoter of a mouse gene encoding an androgen-regulated protein (MSVSP99).

MSVSP99 (mouse seminal vesicle secretory protein of 99 amino acids) is a member of the rat and mouse seminal vesicle secretory protein family. The gene encoding MSVSP99 is under androgenic control and we demonstrate here that this regulation involves a complex interplay of positive and negative regions. First, we show that the promoter region (-387/+16) sufficient to mediate a full androgen induction is a complex enhancer organized in two regulatory regions. These two regions are inactive individually and must act together to confer a 40-fold androgen induction to the MSVSP99 gene and androgen responsiveness is not only dependent on the presence of functional androgen response element (ARE) sequences but results from complex cooperations between ARE and non-ARE sequences forming an androgen response unit. Secondly, we characterized a new regulatory region (-824/-632) that decreases androgen-dependent transcriptional activity of the MSVSP99 promoter. This region, also able to repress the transcriptional activity of the heterologous thymidine kinase promoter, contains a functional promoter on the inverted strand (-826 to -387) and we identified a transcription initiation site located at position -639 with respect to the cap site of the MSVSP99 promoter. Sequence analysis of the flanking DNA also revealed that the MSVSP99 gene is surrounded by long interspersed repeated sequences called LINEs.

Androgens↗
PubMed1999

Differential processing of neuropeptides influences Drosophila heart rate.

Peptides that play critical physiological roles are often encoded in precursors that contain several structurally-related gene products. Differential processing of a precursor by cell-specific processing enzymes can yield multiple messengers with diverse distributions and activities. We have reported the isolation of SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide from adult Drosophila melanogaster. The peptides are encoded in the FMRFamide gene and have a common C-terminal FMRFamide but different N-terminal extensions. In order to investigate the processing of the FMRFamide polypeptide protein precursor, we generated antisera to distinguish among the structurally-related neuropeptides. Utilizing a triple-label immunofluorescent protocol, we mapped the distribution of the peptides. Each peptide has a unique, non-overlapping cellular expression pattern in neural tissue suggesting that the precursor is differentially processed. In order to identify a biological activity of the peptides, we established an in vivo heart rate assay. SDNFMRFamide decreases heart rate but DPKQDFMRFamide and TPAEDFMRFamide do not, indicating that the N-terminal residues are critical for this activity. SDNFMRFamide immunoreactivity is present in the aorta, implying that SDNFMRFamide acts locally to affect heart rate; DPKQDFMRFamide and TPAEDFMRFamide antisera do not stain cardiac tissue. Our data support the conclusion that Drosophila contains cell-specific proteolytic enzymes to differentially process a polypeptide protein precursor resulting in unique expression patterns of structurally-related, yet functionally distinct neuropeptides.

Analysis of Variance↗
PubMed1998

Phorbol ester causes ligand-independent activation of the androgen receptor.

We have examined the human androgen receptor (hAR) for its ability to activate AR-dependent transcription of a transgene in a ligand-independent manner. The transcriptional activity was determined by analysis of chloramphenicol acetyltransferase (CAT) activity in T47D cells cotransfected with a plasmid expressing the hAR and a natural AR-regulated promoter (the MVDP androgen-dependent enhancer) ligated to the reporter CAT gene. In this study, the effects of the protein kinase C (PKC) activator 12-O-tetradecanoyphorbol-13 acetate (TPA) on AR activity were tested. We demonstrated that in the absence of androgen, TPA enhanced AR-mediated transactivation by 10-12-fold. This effect was specific of the PKC pathway since stimulation to the PKA pathway did not activate the unliganded AR. This ligand-independent pathway can function through another androgen-regulated promoter as shown by the use of the mouse mammary tumor virus MMTV-CAT reporter. The human glucocorticoid receptor (hGR) and the rabbit progesterone receptor (rPR) could not be activated by TPA, indicating that the effects are not universal for steroid receptors. A reporter plasmid containing the MVDP androgen response element (ARE) in front of the thymidine kinase promoter ligated to the CAT gene was activated by DHT but not by TPA, indicating that the context of the natural promoter is critical for ligand-independent activation of the AR. Exogenous c-jun enhanced transcriptional activation by the AR in a ligand-dependent manner, but had no effect in the absence of DHT. Base pair substitutions in both AR-binding (5'-TGTTCT-3' to 5'-TTTTTT-3') and NF1-binding (5'-GTGGCTG-3' to 5'-GTTTTTG-3') sites resulted in a loss of TPA responsiveness. Our results suggest that ligand-independent activation of the AR by TPA results from interaction of unliganded AR with other proteins in the transcription machinery.

Animals↗