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PubMed1985

Regeneration and the mechanism of epidermal tumor promotion.

Chemically induced epidermal carcinogenesis is often divided into two stages: initiation, which involves the conversion of some epidermal cells into latent neoplastic cells, and promotion, which allows the evolution of this neoplastic change into the formation of a neoplasm. The hallmark of epidermal tumor promotion is the transformation of the normal epidermis into a hyperplastic epidermis. A major unanswered question about epidermal tumor promotion is whether the epidermal hyperplasia that characterizes promoted skin is a regenerative epidermal hyperplasia resulting from damage produced by the promoter. The opinion currently held is that the epidermal hyperplasia produced by tumor promoters is not simply a regenerative epidermal hyperplasia and possesses characteristics which a regenerative hyperplasia does not have, enabling it to evolve into an epidermal neoplasm. The purpose of this review is to present recent evidence which strongly suggests that promoter-induced epidermal hyperplasia is a regenerative hyperplasia. Three principal lines of evidence are reviewed. The first demonstrates that an epidermal regenerative hyperplasia repeatedly produced by wounding or abrasion can promote epidermal carcinogenesis in the initiated skin of mice. The second line of evidence demonstrates that the epidermal hyperplasia produced by the application of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the most powerful and widely used promoter of skin carcinogenesis, is preceded by damage to the epidermis. This strongly suggests that the epidermal hyperplasia which ensues is a regenerative hyperplasia. Thirdly, evidence is presented which demonstrates that hyperplasia-producing agents which do not promote, produce an epidermal hyperplasia which is different from that produced by tumor promoters. Finally, the review discusses the evidence which suggests that the production of a hyperplasia may be the mechanism for tumor promotion in other organs, such as the liver, bladder, and intestine.

Acetates↗
PubMed1999

[Relationships among health-promoting activities, going out and perceived transportation problems of elderly people living in a small town far from the nearest train station].

To estimate the change in health-promoting activities among elderly people affected by community organizing environments, we examined the relationships among health-promoting activity, going out and perceived transportation problems. A questionnaire was sent to 567 men and women aged 60 years old and over living in a small town in Kanagawa prefecture between July 27 and August 12 in 1995. The questionnaire consisted of 42 items concerning health, social ability of daily living (including the desire to participate in social activities), attitude toward health-promoting activities, and perceived transportation problems. A total of 397 people responded and the answers from 368 people were analyzed after excluding responses from those unable to go out by themselves and those who seldom went out. Single regression analysis and multiregression analysis were used with the sum of responses for each question representing factors related to health-promoting behavior. A probability level of 5 percent was considered significant. The reliability of the data was examined with Cronbach's coefficient alpha. Coefficients of determination for health promoting behavior were 42% in men and 48% in women. In both men and women, age, social ability of daily living and attitudes toward health-promoting behavior were related to health-promoting activity. In women, more actively going out was related to more active health-promoting activity. Higher perception of transportation problems had a negative effect on going out. In men, neither of these factors had any relationship with health-promoting activity. In men, poorer health conditions were related to more active health-promoting activity, but in women, there was no relationship between those factors. These results show that there are gender differences in the relationships among the factors related to health-promoting activities in elderly people. In women, a higher perception of transportation problems restrained actively going out and health-promoting activity.

Aged↗
PubMed1975

Selective effects of MgCl2 and temperature on the initiation of transcription at lac, gal, and lambda promoters.

We have studied the effect of Mg2+ on the formation of transcription preinitiation complexes (open complexes) at two adenosine 3':5'-monophosphate (cyclic AMP)-cyclic AMP receptor protein (CRP)-dependent promoters (lac and gal) and two phage lambda promoters, PL and PR. Mg2+ strongly interferes with open complex formation at the lac and gal promoters, partially inhibits the lambda PR promoter, and is without effect on the lambda PL promoter. Mutations in the lac and gal promoters can affect the response of the promoter to Mg2+. Cyclic AMP and CRP specifically reduce the inhibition of the lac and gal promoters by Mg2+. These factors also affect open complexes at lac and gal by (a) increasing the rate at which they are formed and (b) lowering the midpoint of the temperature transition curve for their formation by about 10 degrees. Open complexes at the lac promoter are more unstable to cooling, even in the presence of CRP and cyclic AMP, than open complexes at lambda promoters. Our studies suggest that the DNA of the lac and gal promoter regions is more resistant to denaturation than the DNA of phage promoter regions. Cyclic AMP and CRP act to decrease this stability, stimulating open complex formation under conditions unfavorable for DNA melting, e.g. low temperature and high Mg2+ concentrations.

Binding Sites↗
PubMed1991

Comparative bladder tumor promoting activity of sodium saccharin, sodium ascorbate, related acids, and calcium salts in rats.

Sodium saccharin and sodium ascorbate are known to promote urinary bladder carcinogenesis in rats following initiation with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N-butyl-N-(4-hydroxybutyl) nitrosamine. Sodium salts of other organic acids have also been shown to be bladder tumor promoters. In addition, these substances increase urothelial proliferation in short term assays in rats when fed at high doses. When they have been tested, the acid forms of these salts are without either promoting or cell proliferative inducing activity. The following experiment was designed to compare the tumor promoting activity of various forms of saccharin and to evaluate the role in promotion of urinary sodium, calcium, and pH as well as other factors. Twenty groups of 40 male F344 rats, 5 weeks of age, were fed either FANFT or control diet during a 6-week initiation phase followed by feeding of a test compound for 72 weeks in the second phase. The chemicals were administered to the first 18 groups in Agway Prolab 3200 diet and the last 2 groups were fed NIH-07 diet. The treatments were as follows: (a) FANFT----5% sodium saccharin (NaS); (b) FANFT----3% NaS; (c) FANFT----5.2% calcium saccharin (CaS); (d) FANFT----3.12% CaS; (e) FANFT----4.21% acid saccharin (S); (f) FANFT----2.53% S; (g) FANFT----5% sodium ascorbate; (h) FANFT----4.44% ascorbic acid; (i) FANFT----5% NaS plus 1.15% CaCO3; (j) FANFT----5.2% CaS plus 1.34% NaCl; (k) FANFT----5% NaS plus 1.23% NH4Cl; (l) FANFT----1.15% CaCO3; (m) FANFT----1.34% NaCl; (n) FANFT----control; (o) control----5% NaS; (p) control----5.2% CaS; (q) control----4.21% S; (r) Control----control; (s) FANFT----5% NaS (NIH-07 diet); (t) FANFT----control (NIH-07 diet). NaS, CaS and S without prior FANFT administration were without tumorigenic activity. NaS was found to have tumor promoting activity, showing a positive response at the 5 and 3% dose levels, with significantly greater activity at the higher dose. CaS had slight tumor promoting activity but without a dose response, and S showed no tumor promoting activity. In addition, NaCl showed weak tumor promoting activity, but CaCO3 was without activity. NH4Cl completely inhibited the tumor promoting activity of NaS when concurrently administered with it. NaCl administered with CaS or CaCO3 administered with NaS showed activity similar to that of NaS. Sodium ascorbate was also shown to have tumor promoting activity, with slightly less activity than NaS. Ascorbic acid showed no tumor promoting activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Liver tumour promotion by chemicals: models and mechanisms.

Tumour promotion is defined as the process whereby a carcinogen initiated organ develops focal proliferations such as nodules, polyps or papillomas, one or more of which become precursors for subsequent steps in the carcinogenic process. The available models for a sequential analysis of carcinogenesis in liver have been examined within the framework of this operational definition with respect to the models themselves as well as the promoted hepatocytes. Using initiation-promotion protocols, several promoters which differ in the biological responses they elicit have been identified. The key issue in promotion pertains to the mechanisms involved in the focal proliferation of the initiated hepatocytes. The resistant hepatocyte model and perhaps the phenobarbital model suggest that focal proliferation of the initiated hepatocyte is induced by exerting a selective mitoinhibitory effect on the surrounding cells while permitting the initiated hepatocyte to respond to the proliferative stimulus, whether it is exogenous or endogenous. The promoter orotic acid, on the other hand, is a natural precursor of pyrimidine nucleotide biosynthesis and is neither an inducer nor an inhibitor of liver cell proliferation but it creates an imbalance in cellular nucleotide pools. Since nucleotides are intermediates in DNA synthesis as well as in the glycosylation of proteins and lipids including that of membranes, it has been postulated that the promotional effect of orotic acid is mediated through this imbalance affecting both DNA and membranes. There is some experimental evidence for this. The importance of this hypothesis is that it raises the possibility of achieving promotion in several organs by disturbing the normal nucleotide pool patterns. Indeed orotic acid has also been found to promote duodenal cancer. The finding that hepatic nodules, regardless of how they have been promoted, exhibit a common biochemical pattern with resistance to several agents has raised some important issues. For example, what is the basis for the resistant phenotype and how is it related to cancer development? Are all initiated hepatocytes identical? If not, do all promoters exert their effect on the same population of initiated hepatocytes? Is the heterogeneity of the initiated hepatocyte population generated by the carcinogen due to the induction of more than one critical lesion: a primary lesion responsible for initiation and secondary lesions in which different types of initiated cells are selected by different promoters?(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗
PubMed1984

Utilization of a Bacillus subtilis sigma 37 promoter by Escherichia coli RNA polymerase in vivo.

The promoter region of Bacillus subtilis subtilisin E was found to be composed of two overlapping promoters with their transcription starting sites separated from each other by 15 base pairs (Wong, S.-L., Price, C. W., Goldfarb, D. S., and Doi, R. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1184-1188). At least one of the promoters is transcribed by a minor form of B. subtilis RNA polymerase with a sigma factor of 37,000 daltons. In vitro transcription analyses and in vivo studies with promoter probe plasmids pKO-1 and pCED-6 demonstrated that Escherichia coli RNA polymerase was able to initiate transcription from the subtilisin promoter cluster. S1 nuclease-mapping studies with both in vivo and in vitro transcribed RNA from E. coli and B. subtilis illustrate that E. coli can initiate transcription from both promoters with the same transcription start points as B. subtilis. The promoter strength of this promoter cluster in E. coli, as expressed in terms of galactokinase units, was 64 units and represents weak promoter activity in the E. coli system. These data indicate that either the single E. coli RNA polymerase is able to recognize the minor sigma 37 promoter or E. coli contains a hitherto unrecognized minor RNA polymerase holoenzyme which is capable of recognizing a B. subtilis sigma 37 promoter. On the other hand the B. subtilis RNA polymerase holoenzymes have been quite promoter-specific in our experiments to date.

Bacillus subtilis↗
PubMed1984

Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters.

During infection of Escherichia coli by bacteriophage T7, E. coli RNA polymerase utilizes only three promoters (A1, A2, and A3). In vitro, the A promoters predominate at very low polymerase concentration, but at higher polymerase concentration the minor B, C, D, and E promoters are used with equal efficiency. The binding constant for the initial association of polymerase with promoters and the forward rate of isomerization to an "open" complex capable of initiation have been measured for the A1, A3, C, and D promoters using the abortive initiation reaction. At 80 mM KCl, 37 degrees C, both major and minor promoters isomerize rapidly (t1/2 = 10 to 30 s). In contrast, initial binding to the minor promoters (KI = 10(7) ) is at least 10-fold weaker than binding to major promoters KI greater than or equal to 10(8) ), suggesting promoter selectivity in the T7 system occurs at the point of initial binding. Association kinetics of the A1 and C promoters on intact T7 were the same as measured on restriction fragments of length greater than or equal to 500 base pairs. All open complexes dissociated with half-lives longer than 1 h. Overall equilibrium binding constants estimated from kinetic measurements ranged from 10(10) to greater than or equal to 10(11) M-1 for minor and major promoters, respectively. Data on heparin attack and abortive initiation turnover rates indicate open complex polymerase conformation may be different at the A1 and A3 promoters.

Animals↗
PubMed1996

[Promotive factors of chronic subdural hematoma in relation to age].

To reveal the pathogenesis of chronic subdural hematoma (CSDH), 256 patients with CSDH over 1 year of age were studied in relation to various traumatic or nontraumatic promotive factors of CSDH including arachnoid cyst, implantation of a cerebrospinal fluid shunt, secondary cerebral atrophy, coagulopathies and past history of acute subdural hematoma or effusion. All patients were grouped according to decade of age. The incidences of associated promotive factors were individually analyzed in each group. The incidences of associated promotive factors in the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th decades of life were 71%, 89%, 56%, 33%, 9%, 11%, 8%, 8% and 5%, respectively. Thus, they were divided into two groups; youth (1st to 4th decade: n = 34) and elder (5th to 9th decade: n = 222). The incidences of associated promotive factors were 62% and 8%, respectively (P < 0.001). Severity of head injury received was also evaluated in these two groups. In the youth group, 15 out of 34 patients (44.1%) had received severe head injuries with loss of consciousness, while in the elder group, only 22 out of 222 patients (9.9%) had received severe head injuries (P < 0.001). The analysis was repeated after each group was divided into two subgroups regarding the coexistence of promotive factors. Among the youth group, 13 patients had no promotive factors and 21 had one or more promotive factors. In those without any promotive factor, 9 out of 13 had received severe head injuries with loss of consciousness, while in those with promotive factors, 6 out of 21 had received severe head injuries. On the other hand, majority of the elder group had not received severe head injuries irrespective of whether patients had promotive factors or not. The present study revealed that majority of young CSDH patient had some promotive factors or severe head injuries with loss of consciousness, and that majority of patients aged over 40 years had no promotive factors and did not receive severe head injuries.

Adolescent↗
PubMed1996

Differential effects of phosphorylation of rat p53 on transactivation of promoters derived from different p53 responsive genes.

The tumor suppressor protein p53 is phosphorylated at multiple sites in the amino-terminal transactivation domain and at several sites in the carboxy-terminal region. Phosphorylation appears to modulate its DNA binding activity. Here we demonstrate that phosphorylation of p53 also modulates its transcriptional activity. Okadaic acid treatment of cells resulted in enhanced phosphorylation of p53 and concomitantly in enhanced transactivation of an mdm2 promoter-linked luciferase reporter gene. This effect was cell type specific, however, since transactivation was enhanced in rat and mouse fibroblasts but reduced in the human Saos-2 cell line. Moreover, the effect was dependent on the promoter. In rat cells transcription from the mdm2, waf1 (cip1) and bax gene promoters, and the artificial PG13 promoter was enhanced by okadaic acid treatment whereas that from the cyclin G promoter was reduced. When various phosphorylation site mutants of p53 were tested for transactivation of these promoters, they behaved differently. Amino-terminal mutants exhibited reduced transcriptional activities on mdm2, waf1 and cyclin G promoters but enhanced activities with bax and PG13 promoters. On the other hand, a mutant at the cdk phosphorylation site, A313, showed reduced activity with mdm2 and waf1 promoters but enhanced activity with the cyclin G promoter, and finally, mutant A390 exhibited enhanced activity on waf1 and bax promoters, but reduced activity on the cyclin G promoter. These results suggest that phosphorylation of p53 may have different effects on its transcriptional activity, depending on the cellular environment and the particular response element. Moreover, both, amino- and carboxy-terminal phosphorylation sites seem to be involved in modulating the DNA-binding and the transactivation activities.

Animals↗
PubMed1999

A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model.

In cancer chemoprevention studies, the identification of better antitumor-promoting agents is highly desired because they may have a wider applicability against the development of clinical cancers. Both epidemiological and animal studies have suggested that microchemicals present in the diet and several herbs and plants with diversified pharmacological properties are useful agents for the prevention of a wide variety of human cancers. Silymarin, a flavonoid isolated from milk thistle, is used clinically in Europe and Asia as an antihepatotoxic agent, largely due to its strong antioxidant activity. Because most antioxidants afford protection against tumor promotion, in this study, we assessed the protective effect of silymarin on tumor promotion in the SENCAR mouse skin tumorigenesis model. Application of silymarin prior to each 12-O-tetradecanoylphorbol 13-acetate (TPA) application resulted in a highly significant protection against tumor promotion in 7,12-dimethylbenz(a)anthracene-initiated mouse skin. The protective effect of silymarin was evident in terms of reduction in tumor incidence (25, 40, and 75% protection, P < 0.001, X2 test), tumor multiplicity (76, 84, and 97% protection, P < 0.001, Wilcoxon rank sum test), and tumor volume (76, 94, and 96% protection, P < 0.001, Student's t test) at the doses of 3, 6, and 12 mg per application, respectively. To dissect out the stage specificity of silymarin against tumor promotion, we next assessed its effect against both stage I and stage II of tumor promotion. Application of silymarin prior to that of TPA in stage I or mezerein in stage II tumor promotion in dimethylbenz(a)anthracene-initiated SENCAR mouse skin resulted in an exceptionally high protective effect during stage I tumor promotion, showing 74% protection against tumor incidence (P < 0.001, X2 test), 92% protection against tumor multiplicity (P < 0.001, Wilcoxon rank sum test), and 96% protection against tumor volume (P < 0.001, Student's t test). With regard to stage II tumor promotion, silymarin showed 26, 63, and 54% protection in tumor incidence, multiplicity, and volume, respectively. Similar effect of silymarin to that in anti-stage I studies, were also observed when applied during both stage I and stage II protocols. In other studies, silymarin significantly inhibited: (a) TPA-induced skin edema, epidermal hyperplasia, and proliferating cell nuclear antigen-positive cells; (b) DNA synthesis; and (c) epidermal lipid peroxidation, the early markers of TPA-caused changes that are associated with tumor promotion. Taken together, these results suggest that silymarin possesses exceptionally high protective effects against tumor promotion, primarily targeted against stage I tumors, and that the mechanism of such effects may involve inhibition of promoter-induced edema, hyperplasia, proliferation index, and oxidant state.

Animals↗
PubMed2025

Identification of a robust promoter in mouse and human hepatocytes by in vivo biopanning of a barcoded AAV library.

Recombinant adeno-associated viruses (AAVs) are leading vectors for in vivo human gene therapy. An integral vector element is promoters, which control transgene expression in either a ubiquitous or cell-type-selective manner. Identifying optimal capsid-promoter combinations is challenging, especially when considering on- versus off-target expression. Here, we report a pipeline for in vivo promoter biopanning in AAV building on our AAV capsid barcoding technology and illustrate its potential by screening 53 promoters in 16 murine tissues using an AAV9 vector. Surprisingly, the 2.2-kb human glial fibrillary acidic protein (GFAP) promoter was the top hit in the liver, where it outperformed robust benchmarks such as the human &#x3b1;-1-antitrypsin promoter or the clinically used liver-specific promoter 1 (LP1). Analysis of hepatic cell populations revealed preferred GFAP promoter activity in hepatocytes. Notably, the GFAP promoter also surpassed the LP1 and cytomegalovirus promoters in human hepatocytes engrafted in an immune-deficient mouse. These findings establish the GFAP promoter as an exciting alternative for research and clinical applications requiring efficient and specific transgene expression in hepatocytes. Our pipeline expands the arsenal of technologies for high-throughput in vivo screening of viral vector components and is compatible with capsid barcoding, facilitating the combinatorial interrogation of complex AAV libraries.

Dependovirus↗
PubMed2000

Constitutive expression of recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1 promoter.

The methylotrophic yeast H. polymorpha is a popular system for the expression of recombinant proteins using the strong and regulatable methanol oxidase (MOX) promoter. Here we show that the constitutive PMA1 promoter can programme the expression of two heterologous proteins, glucose oxidase and human serum albumin. A constitutive promoter provides a useful additional facility to the H. polymorpha expression system because it allows a simplified fermentation regime, avoids the use of methanol, which is both toxic and an explosive hazard, and allows more flexibility for ectopic gene expression during the course of academic studies. A fragment previously isolated in a promoter screen, using glucose oxidase (GOD) as a reporter gene, was shown to consist of the promoter region and the first 659 bp of the H. polymorpha PMA1 gene, encoding the plasma membrane H(+)-ATPase. When the PMA1 promoter was optimally aligned with the GOD coding region, it produced 185 mg/l glucose oxidase in high cell density fed batch fermentations, whereas in previous experiments using the MOX promoter, a yield of 500 mg/l was recovered. The PMA1 promoter was also used to express recombinant human serum albumin (rHA) in H. polymorpha. In high cell density fermentations the PMA1 promoter produced 460 mg/l rHA, whereas 280 mg/l rHA was obtained using the MOX promoter. Taken together, these experiments show that the HpPMA1 programmes the constitutive expression of recombinant proteins and provides a yield comparable to that from the MOX promoter.

Alcohol Oxidoreductases↗
PubMed1990

Kinetic studies of the modulation of ada promoter activity by upstream elements.

We have determined the kinetics of initiation of transcription of the wild-type ada promoter by abortive initiation assays. In the absence of activation, it is a weak promoter, with an association constant KB and an isomerization rate constant k2 comparable to those obtained under the same conditions for other positively regulated promoters (0.36 x 10(7) M-1 and 1.7 x 10(-2) s-1, respectively, at 37 degrees C and 50 mM NaCl, on a supercoiled template). As already observed for other promoters, these constants are modulated by varying the supercoiling of the plasmid. However, the strength of the promoter (given by the KB.k2 product) remains almost constant, because the maximum value of KB and k2 are obtained for different values of the superhelical density. The ada promoter has a stretch of seven adenosine residues (A7) in its upstream region. We have analysed the effect of this upstream sequence on the efficiency of initiation of the ada promoter by comparing the wild-type sequence with an up-mutant promoter characterized by the inversion of the central base pair in the sequence (A7) leading to the sequence (A3TA3). Although the mutation, which is located outside the promoter consensus regions, has no effect on the isomerization step, it affects the equilibrium constant KB that characterizes the association step. In the mutant promoters, the supercoiling of the plasmid modulates the isomerization and association constants in such a way that both KB and k2 are maximum for the same superhelical density (-0.05), leading to a 12-fold increase of the strength of the promoter, on a supercoiled template.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2001

Identification of signal transduction pathways and promoter sequences that mediate parathyroid hormone 1-38 inhibition of osteoprotegerin gene expression.

Osteoprotegerin (OPG), a secreted member of the tumor necrosis receptor superfamily, is a potent inhibitor of osteoclast formation and bone resorption. Parathyroid hormone (PTH), a potent inducer of osteoclast formation, suppresses OPG mRNA expression in vitro and in vivo. To determine the molecular basis of this inhibition, we analyzed the effects of PTH on the human OPG promoter (-5917 to +19) fused with beta-galactosidase reporter gene in stable and transient transfections into rat osteoblast-like UMR106 cells. The effect of PTH on OPG promoter expression was biphasic and concentration-dependent. PTH (1-100 nM) induced the transcriptional activity of the OPG promoter (1.7-fold) at 8 h followed by a gradual decrease with maximal inhibition (6.6-fold) at 24-48 h. To ascertain the signal transduction pathways mediating PTH (1-38) effects on OPG gene expression, we compared the effects of PTH with PTH analogs, parathyroid hormone-related protein 1-34 (PTHrP 1-34), forskolin, 3-isobutyl-1-methylxanthine (IBMX), dibutyryl cAMP, phorbol-12-myristate-13-acetate (PMA), thapsigargin and calcium ionophore A23187. PTH 1-31 and PTHrP 1-34, which stimulate the cAMP/PKA pathway, and other activators of cAMP/PKA, forskolin, IBMX, N(6), O(2')-dibityryl adenosine 3',5'-cyclic monophosphate (dibutyryl cAMP), all elicited a similar biphasic response on OPG promoter expression. PTH analogs PTH 3-34 and PTH 7-34, that do not stimulate cAMP production, had no effect on OPG expression. In contrast, phorbol-12-myristate-13-acetate (PMA), an activator of PKC, stimulated OPG promoter expression, while thapsigargin and calcium ionophore A23187, which increase intracellular Ca(2+), showed a dose-dependent inhibition of OPG promoter expression. To delineate the promoter sequences that mediate the inhibitory effects of PTH on OPG transcription, we analyzed systematic deletions of the OPG promoter for responsiveness in transient transfection assays. The major inhibitory effects of PTH were localized to 391 bp (-372 to +19) of the proximal promoter. Deletions of the promoter region led to a complete loss of responsiveness. Taken together, these results demonstrate that the inhibitory effects of PTH on OPG are mediated at the transcriptional level through cis elements in the proximal promoter. The similar biphasic response of OPG to PTH, PTH 1-31, PTHrP 1-34, forskolin, IBMX and dibutyryl cAMP suggests that PTH regulates OPG transcription via activation of the cAMP/PKA signal transduction pathway.

1-Methyl-3-isobutylxanthine↗
PubMed2003

Myocardial injection of CA promoter-based plasmid mediates efficient transgene expression in rat heart.

BACKGROUND: Although naked plasmid injection is the safest and most convenient method for gene delivery, a major limitation of this approach is currently poor transgene expression. The CA promoter (chicken beta-actin promoter with cytomegalovirus, CMV, enhancer) is one of the strongest transcriptional control modules found; however, it is uncertain whether a CA promoter-based vector is efficient enough for naked gene therapy in a cardiovascular context. METHODS: The beta-galactosidase (LacZ) expression provided by CA promoter plasmid (pCAZ2) injection into the skeletal muscle or the heart of Lewis rats was compared with CMV promoter plasmid or adenoviral vector (AxCAZ3). The effect of Simian virus 40 of the replication origin (SV40ori) deletion from pCAZ2 on transgene expression was also evaluated. RESULTS: pCAZ2 showed the highest LacZ expression in both skeletal muscle and heart in comparison with the CMV promoter-based vector 5 days after naked plasmid injection. LacZ expression in the heart obtained using 20 micro g of pCAZ2 was almost equivalent to that shown with AxCAZ3 at 6.0 x 10(9) optical particle units. The time course of transgene expression driven by CMV and CA promoters in the heart were similar, with the CA promoter providing significantly higher gene expression than the CMV promoter across all time points examined. SV40ori deletion from pCAZ2 did not affect transgene expression in either skeletal muscle or heart. CONCLUSIONS: Transgene expression mediated by naked CA promoter-based plasmid injection was shown to be quite efficient in the heart. We propose that the CA promoter vector is suitable for myocardial gene therapy.

Actins↗
PubMed2005

Improved neuronal transgene expression from an AAV-2 vector with a hybrid CMV enhancer/PDGF-beta promoter.

BACKGROUND: Adeno-associated virus type 2 (AAV-2) vectors are highly promising tools for gene therapy of neurological disorders. After accommodating a cellular promoter, AAV-2 vectors are able to drive sustained expression of transgene in the brain. This study aimed to develop AAV-2 vectors that also facilitate a high level of neuronal expression by enhancing the strength of a neuron-specific promoter, the human platelet-derived growth factor beta-chain (PDGF) promoter. METHODS AND RESULTS: A hybrid promoter approach was adopted to fuse the enhancer of human cytomegalovirus immediately early (CMV) promoter to the PDGF promoter. In cultured cortex neurons, AAV-2 vectors containing the hybrid promoter augmented transgene expression up to 20-fold over that mediated by titer-matched AAV-2 vectors with the PDGF promoter alone and 4-fold over the CMV enhancer/promoter. Injection of AAV-2 vectors with the hybrid promoter into the rat striatum resulted in neuron-specific transgene expression, the level of which was about 10-fold higher than those provided by the two control AAV-2 expression cassettes at 4 weeks post-injection and maintained for at least 12 weeks. Gene expression in the substantia nigra through possible retrograde transport of the AAV-2 vectors injected into the striatum was not obvious. After direct injection of AAV-2 vectors into the substantia nigra, transgene expression driven by the hybrid promoter was observed specifically in dopaminergic neurons and its level was about 3 and 17 times higher than that provided by the PDGF promoter alone and the CMV enhancer/promoter, respectively. CONCLUSIONS: Enhanced transgene capacity plus neuron-specificity of the AAV-2 vectors developed in this study might prove valuable for gene therapy of Parkinson's disease.

Animals↗
PubMed2006

Increased glia-specific transgene expression with glial fibrillary acidic protein promoters containing multiple enhancer elements.

The ability to direct transgene expression to astrocytes has become increasingly important as the roles for these cells continue to expand. Promoters consisting of the 5'-flanking region of the human or mouse glial fibrillary acidic protein (GFAP) gene have generally proved satisfactory. However, a more powerful promoter would be advantageous for several applications, such as expression of dominant negative RNAs or proteins, or for gene therapy. We investigated the possibility of increasing the transcriptional activity of the human GFAP promoter by inserting into it one or three additional copies of putative GFAP enhancer regions. The promoters enhanced with three additional copies gave 75-fold higher LacZ expression levels upon plasmid transfection into GFAP-expressing U251 cells than the parental gfa2 promoter. Surprisingly, in a transgenic mouse model, the enhanced promoters resulted in no or only very low expression of marker genes, probably caused by toxicity. When various cell lines were infected with replication-deficient adenoviral vectors, the enhanced promoters gave LacZ expression levels that were approximately 10-fold higher than those with the parental gfa2 promoter, while retaining specificity for GFAP-expressing cells. Injection of the adenoviral vectors carrying the enhanced promoters into nude mouse brain showed that LacZ expression was limited to GFAP-positive cells. We conclude that gfa2 enhanced promoters are useful for production of short-term, glia-specific, high expression levels of genes in an adenoviral context. Adenoviral vectors containing these enhanced promoters may be useful in glioma gene therapy.

Adenoviridae↗
PubMed2002

Distinct promoter hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric carcinomas.

Hypermethylation of CpG islands in gene promoters is associated with silencing of various tumour suppressor genes. Recent studies of colorectal and gastric carcinomas have defined a CpG island methylator phenotype (CIMP), which involves the targeting of multiple genes by promoter hypermethylation. In this study, methylation-specific polymerase chain reaction (PCR) was performed to study methylation of CpG islands in the promoters of the p16(INK4a), cadherin 1 (CDH1), and retinoic acid receptor-beta (RAR-beta) genes in 45 gastric carcinomas and to investigate whether CDH1 and RAR-beta promoter hypermethylation is associated with CIMP-positive gastric carcinoma. CpG island hypermethylation of the p16(INK4a), CDH1, and RAR-beta promoters was detected in 12 (27%), 26 (58%), and 24 (53%) of the 45 gastric carcinomas, respectively. Hypermethylation of the p16(INK4a) promoter was more common in intestinal type than in diffuse type gastric carcinomas (p = 0.0023; Fisher's exact test) and was inversely associated with p53 mutations (p = 0.0225; Fisher's exact test). However, CDH1 and RAR-beta promoter hypermethylation was observed more frequently in diffuse-scattered type gastric carcinoma than in other types (intestinal and diffuse-adherent types) (p = 0.0175 and p = 0.0335, respectively; Fisher's exact test) and was not associated with p53 mutation status. Moreover, hypermethylation of the CDH1 and RAR-beta promoters occurred concordantly (p < 0.0001; Fisher's exact test). These results suggest that at least two types of promoter methylation status are involved in the development of the intestinal (p16(INK4a) promoter hypermethylation) and diffuse-scattered types (CDH1 and RAR-beta promoter hypermethylation) of gastric carcinoma.

Antigens, CD↗