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PubMed2003

Ras signaling and transcriptional synergy at a flexible Ets-1/Pit-1 composite DNA element is defined by the assembly of selective activation domains.

Pit-1 and Ets-1 binding to a composite element synergistically activates and targets Ras-mitogen-activated protein kinase signaling to the rat prolactin promoter. These transcriptional responses appear to depend on three molecular features: organization of the Ets-1/Pit-1 composite element, physical interaction of these two factors via the Pit-1 homeodomain (amino acids 199-291) and the Ets-1 regulatory III domain (amino acids 190-257), and assembly of their transcriptional activation domains (TADs). Here we show that the organization of the Ets-1/Pit-1 composite element tolerates significant flexibility with regard to Ras stimulation and synergy. Specifically, the putative monomeric Pit-1 binding site can be substituted with bona fide binding sites for either a Pit-1 monomer or dimer, and these sites tolerated a separation of 28 bp. Additionally, we show that the physical interaction of Ets-1 and Pit-1 is not required for Ras responsiveness or synergy because block mutations of the Pit-1 interaction surface in Ets-1, which reduced Ets-1/Pit-1 binding in vitro, did not significantly affect Ets-1 stimulation of Ras responsiveness or synergy. We also show differential use of distinct TAD subtypes and Pit-1 TAD subregions to mediate either synergy or Ras responsiveness. Specifically, TADs from Gal4, VP16, or Ets-2 regulatory III domain linked to Ets-1 DNA binding domain constructs restored synergy to these TAD/Ets-1 DNA binding domain fusions. Conversely, deletion of the defined Pit-1 TAD (amino acids 2-80) retained synergy, but not Ras responsiveness. Consequently, we further defined the Pit-1 amino-terminal TAD into region 1 (R1, amino acids 2-45) and region 2 (R2, amino acids 46-80). R1 appears to regulate basal and synergistic responses, whereas the Ras response was mapped to R2. In summary, Ras responsiveness and Pit-1/Ets-1 synergy are mediated through the assembly of distinct TADs at a flexible composite element, indicating that different mechanisms underlie these two transcriptional responses and that the Pit-1 R2 subregion represents a novel, tissue-specific Ras-responsive TAD.

Amino Acid Sequence↗

PubMed2000

Nonspecific adherence by Actinobacillus actinomycetemcomitans requires genes widespread in bacteria and archaea.

The gram-negative coccobacillus, Actinobacillus actinomycetemcomitans, is the putative agent for localized juvenile periodontitis, a particularly destructive form of periodontal disease in adolescents. This bacterium has also been isolated from a variety of other infections, notably endocarditis. Fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms, a property likely to be critical for colonization of teeth and other surfaces. Here we report the identification of a locus of seven genes required for nonspecific adherence of A. actinomycetemcomitans to surfaces. The recently developed transposon IS903phikan was used to isolate mutants of the rough clinical isolate CU1000 that are defective in tight adherence to surfaces (Tad(-)). Unlike wild-type cells, Tad(-) mutant cells adhere poorly to surfaces, fail to form large autoaggregates, and lack long, bundled fibrils. Nucleotide sequencing and genetic complementation analysis revealed a 6.7-kb region of the genome with seven adjacent genes (tadABCDEFG) required for tight adherence. The predicted TadA polypeptide is similar to VirB11, an ATPase involved in macromolecular transport. The predicted amino acid sequences of the other Tad polypeptides indicate membrane localization but no obvious functions. We suggest that the tad genes are involved in secretion of factors required for tight adherence of A. actinomycetemcomitans. Remarkably, complete and highly conserved tad gene clusters are present in the genomes of the bubonic plague bacillus Yersinia pestis and the human and animal pathogen Pasteurella multocida. Partial tad loci also occur in strikingly diverse Bacteria and Archaea. Our results show that the tad genes are required for tight adherence of A. actinomycetemcomitans to surfaces and are therefore likely to be essential for colonization and pathogenesis. The occurrence of similar genes in a wide array of microorganisms indicates that they have important functions. We propose that tad-like genes have a significant role in microbial colonization.

Aggregatibacter actinomycetemcomitans↗

PubMed2006

Differential utilization of transcription activation subdomains by distinct coactivators regulates Pit-1 basal and Ras responsiveness.

The POU-homeodomain transcription factor Pit-1 governs ontogeny and cell-specific gene expression of pituitary lactotropes, somatotropes, and thyrotropes. The splice isoform, Pit-1beta, inserts a 26-amino acid (AA) repressor at AA48 in the Pit-1 transcription activation domain (TAD). The Pit-1 TAD contains a basal regulatory subregion, R1 (AA1-45), and a basal and Ras-responsive region, R2 (AA46-80). To precisely map these activities, we generated GAL4-Pit-1/Pit-1betaTAD fusions and, in full-length HA-Pit-1, a series of substitution mutants of R2. Analysis in GH4 cells identified an activation domain at AA50-70, followed by an overlapping, dual-function, Ras-responsive-inhibitory domain, located from AA60-80. In contrast, GAL4-Pit-1betaTAD repressed both basal and Ras-mediated TAD activity. To determine the functional interplay between TAD subregions and the beta-domain, we inserted the beta-domain every 10 AA across the 80-AA Pit-1 TAD. Like wild-type Pit-1beta, each construct retained transcriptional activity in HeLa cells and repressed the Ras response in GH4 cells. However, beta-domain insertion at AA61 and 71 resulted in greater repression of Ras responsiveness, defining a critical R2 TAD spanning AA61-71 of Pit-1. Furthermore, Ras activation is augmented by steroid receptor coactivator 1, whereas cAMP response element binding protein-binding protein is not a Ras mediator in this system. In summary, the Pit-1/Pit-1beta TADs are composed of multiple, modular, and transferable subdomains, including a regulatory R1 domain, a basal activation region, a selective inhibitory-Ras-responsive segment, and a beta-specific repressor domain. These data provide novel insights into the mechanisms by which the Pit-1 TAD integrates DNA binding, protein partner interactions, and distinct signaling pathways to fine-tune Pit-1 activity.

Animals↗

PubMed1999

Double induction strategy for acute myeloid leukemia: the effect of high-dose cytarabine with mitoxantrone instead of standard-dose cytarabine with daunorubicin and 6-thioguanine: a randomized trial by the German AML Cooperative Group.

Early intensification of chemotherapy with high-dose cytarabine either in the postremission or remission induction phase has recently been shown to improve long-term relapse-free survival (RFS) in patients with acute myeloid leukemia (AML). Comparable results have been produced with the double induction strategy. The present trial evaluated the contribution of high-dose versus standard-dose cytarabine to this strategy. Between March 1985 and November 1992, 725 eligible patients 16 to 60 years of age with newly diagnosed primary AML entered the trial. Before treatment started, patients were randomized between two versions of double induction: 2 courses of standard-dose cytarabine (ara-C) with daunorubicin and 6-thioguanine (TAD) were compared with 1 course of TAD followed by high-dose cytarabine (3 g/m2 every 12 hours for 6 times) with mitoxantrone (HAM). Second courses started on day 21 before remission criteria were reached, regardless of the presence or absence of blast cells in the bone marrow. Patients in remission received consolidation by TAD and monthly maintenance with reduced TAD courses for 3 years. The complete remission (CR) rate in the TAD-TAD compared with the TAD-HAM arm was 65% versus 71% (not significant [NS]), and the early and hypoplastic death rate was 18% versus 14% (NS). The corresponding RFS after 5 years was 29% versus 35% (NS). An explorative analysis identified a subgroup of 286 patients with a poor prognosis representing 39% of the entire population; they included patients with more than 40% residual blasts in the day-16 bone marrow, patients with unfavorable karyotype, and those with high levels of serum lactate dehydrogenase. Their CR rate was 65% versus 49% (p =.004) in favor of TAD-HAM and was associated with a superior event-free survival (median, 7 v 3 months; 5 years, 17% v 12%; P =.012) and overall survival (median, 13 v 8 months; 5 years, 24% v 18%; P =.009). This suggests that the incorporation of high-dose cytarabine with mitoxantrone may contribute a specific benefit to poor-risk patients that, however, requires further substantiation. Double induction, followed by consolidation and maintenance, proved a safe and effective strategy and a new way of delivering early intensification treatment for AML.

Adolescent↗

PubMed1999

Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinase C-mediated modulation of the AhR pathway.

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the "PMA effect." A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR x ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR x ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRDeltaCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRDeltaCVP x ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR. ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.

Animals↗

PubMed1977

Metabolism of 5-hydroxytryptamine and levels of tricyclic antidepressant drugs in rat brain after acute and chronic treatment.

Because tricyclic antidepressants (TAD) are usually given chronically to patients, both their acute and their chronic effects on 5-hydroxytryptamine (5-HT) metabolism were studied. The probenecid method was used and, in addition to 5-hydroxy-indoleacetic acid (5-HIAA), some other indole compounds in brain were measured. Simultaneously, TAD levels in brain and plasma were determined. Dimethylated as well as monomethylated TADs were administered, both at 10 and 25 mg/kg i.p. Treatment with either 10 mg/kg during 14 days or 25 mg/kg given acutely resulted in a similar brain level of TAD, so any differences found could be attributed to differences in administration schedule. Drug levels in brain and plasma differed considerably after chronic and acute treatments but no major differences in the effect on 5-HIAA level in the brain were found, although accumulation of 5-HIAA following probenecid treatment was mostly lowered after treatment with dimethylated TAD. The TAD level in rat brain was not decisive for the effect on central 5-HT turnover. The monomethylated TAD affected the 5-HT turnover very little, not only acutely but also chronically.

Animals↗

PubMed1984

Treatment of disseminated breast cancer with tamoxifen, aminoglutethimide, hydrocortisone, and danazol, used in combination or sequentially.

222 patients with disseminated breast cancer have been randomised to receive either a combination of hormone therapies using tamoxifen, aminoglutethimide with hydrocortisone, and danazol (TAD), or tamoxifen alone. The response to the combination was significantly better (43%) than that to tamoxifen alone (31%). Patients who relapsed after response or failed to respond to tamoxifen were subsequently treated with aminoglutethimide and then, if possible, with danazol. Some patients who received TAD were subsequently treated with alternative endocrine therapy, which was usually medroxyprogesterone acetate. Of the 111 patients who initially received tamoxifen, 43 responded to the tamoxifen and/or subsequent endocrine therapy. Of the 111 patients who initially received TAD, 50 responded. Although the duration of response to TAD was the same as for tamoxifen, the TAD patients achieved remission more quickly. The total time in endocrine remission for patients receiving TAD is 303 months to date, compared with 264 months for patients receiving tamoxifen. Survival for patients randomised to receive TAD or tamoxifen is the same.

Aminoglutethimide↗

PubMed1997

Resorbable and non-resorbable augmentation devices for tenorrhaphy of xenografts in extensor tendon deficits: 12 week study.

Resorbable (poly-L-lactide) and non-resorbable (polyethylene terephathalate) tendon augmentation devices (TAD) in conjunction with a pericardial adhesion barrier, were designed to strengthen tenorrhaphies and were evaluated in an ovine extensor tendon deficit model in a short term study. Fifteen centimetres of tendon were resected and replaced with kangaroo tail tendon xenografts that had been cross-linked with 0.075% glutaraldehyde (GA) at 4 degrees C for one or seven days. Compared with tenorrhaphies performed with Kessler sutures alone, both types of TAD were more effective at preventing tenorrhaphy dehiscence, and thus maintaining tendon function. Furthermore, tensile strength of TAD tenorrhaphies increased significantly between zero and twelve weeks. For xenografts cross-linked in GA for one day, the tensile strength of tenorrhaphies with the resorbable TAD rose from 38 +/- 9 N at time zero, to 116 +/- 46 N at twelve weeks, while non-resorbable TAD tenorrhaphy strength at time zero was 42 +/- 16 N and 99 +/- 27 N at twelve weeks. For xenografts cross-linked with GA for seven days, similar increases in tensile strength of tenorrhaphies, with the two types of TAD were found. As there was no significant difference in mechanical performance or tissue response between the two TAD types in the first 12 weeks, use of the resorbable poly-L-lactide device may be advantageous clinically. Tensile strengths of midsections of the tendon xenograft cross-linked for 7 days was not significantly diminished 12 weeks after implantation and these xenografts were partially remodelled around the periphery. However, the tensile strength of xenografts cross-linked for one day declined significantly between time zero (319 +/- 80 N) and twelve weeks (239 +/- 92 N), suggesting that this degree of cross-linking was inadequate for maintenance of mechanical strength. Evaluation of the performance of tenorrhaphy augmentation devices with xenografts, over a longer implantation period, is required to further understand their usefulness for reconstruction of traumatic tendon injuries.

Animals↗

PubMed2005

Posterior cortical atrophy: clinical presentation and cognitive deficits compared to Alzheimer's disease.

BACKGROUND: Posterior cortical atrophy (PCA) is an uncommon dementia syndrome with initial manifestations of visual dysfunction and preservation of memory and language until late in the disease. Since prognosis and management differ from typical Alzheimer's disease (tAD), clinical tests to distinguish PCA from tAD are needed. METHODS: Fifteen PCA cases and 15 tAD cases, defined by clinical and MRI criteria, were compared by present symptoms and scores on four cognitive tests. RESULTS: Symptoms of visual disturbance and dyslexia were more commonly reported in PCA cases (p = 0.0001 and p = 0.006, respectively), and memory loss was more commonly reported in tAD (p = 0.006). Patients with PCA were less accurate on the Cortical Vision Screening Test (t = 6.0; p < 0.001) and in copying the Rey-Osterreith Complex Figure (t = 6.0; p < 0.001), in comparison to the tAD group. Memory, evaluated by the Rey Auditory Verbal Learning Test, was impaired in both groups; however, delayed recall was more impaired in the tAD group (t = 2.5; p = 0.03). CONCLUSION: Compared to patients with tAD, patients with PCA are more likely to present to their providers with symptoms of visual dysfunction. Performance on simple tests of visual perception and copying can be used to distinguish the two disorders even a few years after initial symptoms.

Age of Onset↗

PubMed2006

The oxygen sensor factor-inhibiting hypoxia-inducible factor-1 controls expression of distinct genes through the bifunctional transcriptional character of hypoxia-inducible factor-1alpha.

The function of the hypoxia-inducible factor-1 (HIF-1), the key transcription factor involved in cellular adaptation to hypoxia, is restricted to low oxygen tension (pO(2)). As such, this transcription factor is central in modulating the tumor microenvironment, sensing nutrient availability, and controlling anaerobic glycolysis, intracellular pH, and cell survival. Degradation and inhibition of the limiting HIF-1alpha subunit are intimately connected in normoxia. Hydroxylation of two proline residues by prolyl hydroxylase domain (PHD) 2 protein earmarks the protein for degradation, whereas hydroxylation of an asparagine residue by factor-inhibiting HIF-1 (FIH-1 or FIH) reduces its transcriptional activity. Indeed, silencing of either PHD2 or FIH in normoxia partially induced hypoxic genes, whereas combined PHD2/FIH silencing generated a full hypoxic gene response. Given the fact that HIF-1alpha possesses two transcriptional activation domains [TAD; NH(2)-terminal (N-TAD) and COOH-terminal (C-TAD)], we hypothesized on a possible bifunctional activity of HIF-1alpha that could be discriminated by FIH, an inhibitor of the C-TAD. In human cell lines engineered to overexpress or silence FIH in response to tetracycline, we show by quantitative reverse transcription-PCR that a set of hypoxic genes (ca9, phd3, pgk1, and bnip3) respond differently toward FIH expression. This finding, extended to 26 hypoxia-induced genes, indicates differential gene expression by the N-TAD and C-TAD in response to the hypoxic gradient. We propose that the oxygen-sensitive attenuator FIH, together with two distinct TADs, is central in setting the gene expression repertoire dictated by the cell pO(2).

Adenocarcinoma↗

PubMed1985

Biochemical mechanisms of resistance to tiazofurin.

The purpose of this investigation was to examine factors which regulate the reprogramming of gene expression in tumors responsible for resistance to tiazofurin. To study the resistance phenomenon drug-induced tumor lines were selected and examined for the mechanism of resistance. A comparison of the biochemical expression of resistance to tiazofurin in drug-induced resistant lines of hepatoma 3924A, leukemias L1210 and P388 revealed that the 3 lines expressed similar genetic alterations related to reduced TAD content, decreased NAD pyrophosphorylase activity and increased synthesis of guanylates from salvaging preformed guanine indicating that these 3 factors play an important role in the resistance to tiazofurin. Resistance was stable in the leukemia lines and did not require drug to maintain resistance. Hepatoma 3924A resistant line reverted to sensitive state in the absence of drug selection pressure. NAD pyrophosphorylase activity was substantially deleted in the tiazofurin resistant leukemia lines, but was only significantly decreased in the hepatoma resistant line. Extensive biochemical alterations including enhanced activity of IMP dehydrogenase, increased inosinate and guanylate pools, and reduced uptake of tiazofurin were found in the hepatoma line resistant to tiazofurin. To examine the applicability of these results to naturally sensitive and spontaneously resistant tumors, murine tumors were examined. In murine tumors, TAD accumulation, ratios of enzyme activities responsible for the synthesis and degradation of TAD, and the ratios of perturbation of inosinate and guanylate pools following tiazofurin challenge demonstrated significant correlation with the sensitive or resistant nature of the tumors. To extrapolate these observations to human tumor systems, cytotoxicity of tiazofurin and its metabolic effects were compared in 6 human lung cancer cell lines derived from cancer patients with small cell lung cancer (4 lines) and lung adenocarcinoma (2 lines). Cell lines exhibiting greater sensitivity to tiazofurin accumulated significantly larger amounts of TAD and showed significant reduction of guanylate pools following tiazofurin incubation. The activity of the enzyme responsible for the formation of TAD, NAD pyrophosphorylase, did not correlate with responsiveness to tiazofurin but the enzyme which hydrolyzes TAD, TADase, correlated positively with the status of resistance.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenine Nucleotides↗

PubMed1992

Inhibition by tiazofurin of inosine 5'-phosphate dehydrogenase (IMP DH) activity in extracts of ovarian carcinomas.

Cancer cells have an increased ability to synthesize GTP (guanosine triphosphate) because of increased activity of IMP DH (inosine 5'-phosphate dehydrogenase, EC 1.1.1.205). Because IMP DH activity is rate limiting for de novo biosynthesis of GTP, this enzyme was suggested as a sensitive target for chemotherapy. Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) is converted in the cells into the active metabolite, TAD, (thiazole-4-carboxamide adenine dinucleotide) which potently inhibits IMP DH activity. By adding TAD to tissue extracts one can determine the extent of inhibition of IMP DH. We applied the IMP DH assay method to extracts of normal ovaries (N = 11) and epithelial ovarian carcinomas (N = 10). The IMP DH activity (mean +/- SE) in ovarian carcinoma was 21.1 +/- 5.8 which was markedly higher than that observed in normal ovaries (2.9 +/- 0.7 nmol/hr/mg protein) (P < 0.05%). The inhibition by TAD of IMP DH activity in ovarian carcinomas (N = 4) was 81%. The results indicate that IMP DH activity is elevated sevenfold in ovarian carcinomas as compared to normal ovary and can be inhibited by exposure to tiazofurin (TAD). Similar high IMP DH activity and inhibition of the activity by TAD was observed in patients with chronic granulocytic leukemia in blast crisis among whom 70 to 80% remissions were reported. Since there is increased IMP DH activity in human ovarian carcinomas and in OVCAR-5 cells and tiazofurin and TAD inhibit IMP DH activity of these cells and the proliferation of human ovarian carcinoma xenografts in the mouse, tiazofurin may merit serious consideration for a Phase II trial for patients with recurrent/refractory epithelial ovarian carcinoma.

Adenocarcinoma↗

PubMed2004

Regulation of HSF1-responsive gene expression by N-terminal truncated form of p73alpha.

DNp73 is a transactivation domain (TAD)-truncated form of p73. The ability of DNp73alpha to regulate gene expression was examined using reporter assays with luciferase gene constructs. Among various promoter-regulated reporter genes tested, heat shock factor (HSF)-responsive gene expression was selectively activated by DNp73alpha, but not by other p73-isoforms with TAD and DNp73beta. Deletion of TAD endowed p73alpha with the ability to activate HSF-responsive gene expression, but deletion of N-terminal proline-rich domain (PRD) rendered both DNp73alpha and the TAD-deleted p73alpha inactive. Considering the inability of DNp73beta, which is the C-terminus-truncated form of DNp73alpha, to function, these results indicate that both the PRD and C-terminus are necessary for DNp73alpha to be able to activate the HSF-dependent gene expression. In addition to the reporter gene expression, both DNp73alpha and TAD-deleted p73alpha activated the expression of an endogenous gene, hsp70, corresponding with an increase in the active form of HSF1. Taken together, these results demonstrate that TAD-truncated p73alpha can activate HSF-dependent gene expression via induction of active HSF1.

Base Sequence↗

PubMed2005

PSA bouncing after short term androgen deprivation and 3D-conformal radiotherapy for localized prostate adenocarcinoma and the relationship with the kinetics of testosterone.

OBJECTIVES: To assess the factors effecting PSA bounce and to identify any possible relationship with biochemical control after 3-D conformal radiotherapy (3D-CRT) and total androgen deprivation (TAD) for prostate cancer by evaluating four previously described PSA bounce definitions. METHODS: Between January 1998 and January 2001, 83 consecutive patients with clinically localized prostate cancer were treated by 3D-CRT with neoadjuvant 3 months and/or 6 months adjuvant TAD. All patients had a pretreatment PSA level, at least eight post-external beam radiotherapy (EBRT) PSA and testosterone levels and minimum two years of follow-up. Total radiotherapy dose was 73.6 Gy at ICRU reference point. Four previous definitions of PSA bounce were used: Critz definition (>or=0.1 ng/mL), Cavanagh definition (>or=0.2 ng/mL), Hanlon definition (>or=0.4 ng/mL) and Rosser definition (>or=0.5 ng/mL) according to original methodology performed to report PSA bounce. Biochemical failure was defined in accordance with the ASTRO consensus guidelines. RESULTS: The median follow-up time was 40 months. PSA bounce was recorded as follows: Critz definition, 33 patients (40%); Cavanagh definition, 21 patients (25%); Hanlon definition, 11 patients (13%); and Rosser definition, 7 patients (8%). In multivariate analysis, pre-EBRT PSA level and the duration of TAD for Critz definition; age, pre-EBRT PSA and the duration of TAD for Cavanagh definition; age and duration of TAD for Hanlon definition; age and pre-biopsy PSA for Rosser definition were significant independent prognostic factors determining PSA bounce. A significant increase of mean testosterone level in bouncers was detected at the 6th-9th and 18th-21st months. PSA bounce did not predict for PSA failure in multivariate analysis. CONCLUSIONS: We observed no correlation between biochemical failure and PSA bounce. The longer duration of TAD and older age were found to be inversely proportional with PSA bouncing in this cohort. Notably, recovery of testosterone might cause PSA bouncing.

Adenocarcinoma↗

PubMed1999

The effects of hyperoxic and hypercarbic gases on tumour blood flow.

Carbogen (95% O2 and 5% CO2) has been used in preference to 100% oxygen (O2) as a radiosensitizer, because it is believed that CO2 blocks O2-induced vasoconstriction. However, recent work suggests that both normal and tumour arterioles of dorsal flap window chambers exhibit the opposite: no vasoconstriction vs constriction for O2 vs carbogen breathing respectively. We hypothesized that CO2 content might cause vasoconstriction and investigated the effects of three O2-CO2 breathing mixtures on tumour arteriolar diameter (TAD) and blood flow (TBF). Fischer 344 rats with R3230Ac tumours transplanted into window chambers breathed either 1%, 5%, or 10% CO2 + O2. Intravital microscopy and laser Doppler flowmetry were used to measure TAD and TBF respectively. Animals breathing 1% CO2 had increased mean arterial pressure (MAP), no change in heart rate (HR), transient reduction in TAD and no change in TBF. Rats breathing 5% CO2 (carbogen) had transiently increased MAP, decreased HR, reduced TAD and a sustained 25% TBF decrease. Animals exposed to 10% CO2 experienced a transient decrease in MAP, no HR change, reduced TAD and a 30-40% transient TBF decrease. The effects on MAP, HR, TAD and TBF were not CO2 dose-dependent, suggesting that complex physiologic mechanisms are involved. Nevertheless, when > or = 5% CO2 was breathed, there was clear vasoconstriction and TBF reduction in this model. This suggests that the effects of hypercarbic gases on TBF are site-dependent and that use of carbogen as a radiosensitizer may be counterproductive in certain situations.

Adenocarcinoma↗

PubMed1996

CREB-binding protein activates transcription through multiple domains.

CREB-binding protein (CBP) functions as a coactivator molecule for a number of transcription factors including CREB, c-Fos, c-Jun, c-Myb, and several nuclear receptors. Although binding sites for these factors within CBP have been identified, the regions of CBP responsible for transcriptional activation are unknown. In this report, we show that the N-terminal half of CBP is sufficient for activation of CREB-mediated transcription and that this region contains a strong transcriptional activation domain (TAD). Both deletion of this TAD or sequestering of factors that the TAD binds using a squelching assay were found to greatly decrease the ability of CBP to activate CREB-mediated transcription. In vivo studies by others have shown that p300/CBP associates with TBP; using an in vitro approach, we show the N-terminal TAD binds TBP. We also examined the ability of the C terminus of CBP to activate transcription using GAL-CBP chimeras. With this approach, we identified two C-terminal TADs located adjacent to the c-Fos binding site. In previous studies, cAMP-dependent protein kinase A (PKA) increased the transcriptional activity of a GAL full-length CBP chimera in F9 cells, and of the C terminus in PC-12 cells. Here, we demonstrate that PKA also increased the ability of the N-terminal TADs of CBP to activate transcription in PC-12 but not F9 or COS-7 cells, suggesting that this PKA-responsiveness is cell type-specific.

Animals↗

PubMed2002

Identification of residues critical for regulation of protein stability and the transactivation function of the hypoxia-inducible factor-1alpha by the von Hippel-Lindau tumor suppressor gene product.

Under normoxic conditions the hypoxia-inducible factor-1alpha (HIF-1alpha) protein is targeted for degradation by the von Hippel-Lindau (pVHL) tumor suppressor protein acting as an E3 ubiquitin ligase. Binding of pVHL to HIF-1alpha is dependent on hydroxylation of specific proline residues by O(2)-dependent prolyl 4-hydroxylases. Upon exposure to hypoxia the hydroxylase activity is inhibited, resulting in stabilization of HIF-1alpha protein levels and activation of transcription of target genes. One of the two critical proline residues, Pro(563) in mouse HIF-1alpha, is located within a bifunctional domain, the N-terminal transactivation domain (N-TAD), which mediates both pVHL-dependent degradation at normoxia and transcriptional activation at hypoxia. Here we have identified two N-TAD residues, Tyr(564) and Ile(565), which, in addition to Pro(563), were critical for pVHL-mediated degradation at normoxia. We have also identified D568A/D569A/D570A, F571A, and L573A as mutations of the N-TAD that abrogated binding to pVHL both in vitro and in vivo, and constitutively stabilized N-TAD against degradation. Moreover, the mutations Y564G, L556A/L558A, and F571A/L573A drastically reduced the transactivation function of either the isolated N-TAD or full-length HIF-1alpha in hypoxic cells. Interestingly, the P563A mutant exhibited a constitutively active and potent transactivation function that was enhanced by functional interaction with the transcriptional coactivator protein CREB-binding protein. In conclusion, we have identified by mutation analysis several residues that are critical for either one or both of the interdigitated and conditionally regulated degradation and transactivation functions of the N-TAD of HIF-1alpha.

Amino Acid Motifs↗

PubMed2003

Lack of effect of long-term octreotide therapy in severe thyroid-associated dermopathy.

Severe thyroid associated dermopathy (TAD), a rare complication of Graves' disease, currently lacks effective treatment. Mediators of growth and inflammation, including insulin-like growth factor-1 (IGF-1) and somatostatin (SST) have been implicated in its pathogenesis. Octreotide, a potent SST analogue, has been used in TAD with anecdotal success. Therefore, we evaluated the efficacy of long-term octreotide therapy in moderate to severe TAD. Three obese women with TAD were studied. Baseline treatment included levothyroxine, methimazole, fluocinonide ointment under occlusive dressing, and physiotherapy for lymphedema. After establishing baseline clinical status, octreotide was started (300 microg subcutaneously daily) for 10 to 28 months. Studies obtained at baseline and every 3 months included: leg circumference, skin examination, clinical photography, self-reported clinical status, body mass index (BMI), serum thyrotropin (TSH) and free thyroxine, titers of antithyroidal and anti-TSH receptor antibodies. Minimal changes in the edema, erythema, skin texture and size of nodules were observed. The minor changes seen followed significant decreases in the patients' BMI, and hence, cannot be specifically attributed to octreotide administration. No clinically significant benefit from long-term octreotide therapy was demonstrated in three patients with moderate to severe TAD. Weight loss and measures such as compression bandaging and topical corticosteroid application still remain the most cost effective treatment modalities for TAD.

Adult↗