Biomedical subjects

T T Wang

Publications and source records attributed to T T Wang.

At least 19 recordsLinked to original sources

PubMed2000

Low-dose genistein induces cyclin-dependent kinase inhibitors and G(1) cell-cycle arrest in human prostate cancer cells.

Genistein, a naturally occurring isoflavone found chiefly in soy products, reportedly has antiprostate cancer effects, but the mechanisms underlying these effects are unknown. We studied the antiproliferative and apoptosis-inducing effects of genistein in the androgen-sensitive human prostate cancer cell line LNCaP. Viable cell number was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay; cell-cycle progression and apoptosis were evaluated by flow cytometry; apoptosis was also assessed by a histone enzyme-linked immunosorbent assay; and the expression of several cell-cycle- and apoptosis-related genes and their gene products was determined by northern blot analysis, western blot analysis, and/or assays based on polymerase chain reaction. Physiologic concentrations of genistein (< or = 20 microM) decreased LNCaP viable cell number in a dose-dependent manner, induced a G(1) cell-cycle block, decreased prostate-specific antigen mRNA expression, and increased p27(KIP1) and p21(WAF1) (mRNA and protein) but had no effect on apoptosis or the mRNA expression of the apoptosis- and cell-cycle-related markers bcl-2, bax, Rb, and proliferating cell nuclear antigen. Higher concentrations of genistein (> 20 microM) did induce apoptosis. We conclude that genistein (at physiologic concentrations) exerts potent antiproliferative effects on LNCaP cells by inducing a G(1) cell-cycle block. The antiproliferative effects of genistein may be mediated by increased levels of p27(KIP1) and p21(WAF1), which are negative cell-cycle regulators that act as cyclin-dependent kinase inhibitors and that have been recently linked with prostate carcinogenesis. These findings may provide insights into the mechanisms underlying the apparent antiprostate cancer effects of soy consumption observed in epidemiologic studies.

Antineoplastic Agents↗

PubMed2000

Coordinated regulation of two TRAIL-R2/KILLER/DR5 mRNA isoforms by DNA damaging agents, serum and 17beta-estradiol in human breast cancer cells.

A search of the Genebank database revealed that there are two distinct gene sequences with the common name of TRAIL-R2/Killer/DR5. Using reverse transcription-polymerase chain reaction (RT-PCR), we confirmed the existence of two isoforms of TRAIL-R2/Killer/DR5 mRNA, which we have designated the long and short isoforms based on their electrophoretic mobility. We found that both the long and short mRNA isoforms are ubiquitously expressed in human tissues and cell lines. The long form generally predominates, but the proportion of the two isoforms varies depending on the tissue type. Treatment of MCF-7 human breast cancer cells with the DNA damaging drugs adriamycin, campthothecin, or etoposide causes a coordinated up-regulation of both isoforms. Treatment of the p53-mutant T-47D breast cancer cell line with adriamycin also results in up-regulation of both isoforms, suggesting that adriamycin up-regulates TRAIL-R2/Killer/DR5 expression independent of functional p53. The expression of both mRNA isoforms are increased in MCF-7 cells cultured in charcoal-stripped fetal bovine serum compared to normal serum, suggesting that sex steroid hormones may play a role in the negative regulation of their expression. This was confirmed in MCF-7 cells cultured in stripped serum supplemented with 17beta-estradiol, which also resulted in a decrease in the mRNA expression of both isoforms. These results demonstrate that the TRAIL-R2/Killer/DR5 gene gives rise to two distinct forms of mRNA, and that these two forms are coordinately regulated by DNA damage and 17beta-estradiol in human breast cancer cells. The functional significance of the two isoforms remains to be determined.

Antineoplastic Agents↗

PubMed2000

Inhibition of aromatase activity and expression in MCF-7 cells by the chemopreventive retinoid N-(4-hydroxy-phenyl)-retinamide.

The effect of the chemopreventive synthetic retinoid N-(4-hydroxyphenyl)-retinamide (4-HPR) on aromatase activity and expression was examined. 4-HPR caused a dose-dependent inhibition of aromatase activity in microsomes isolated from JEG-3 human placental carcinoma cells. The kinetics of inhibition were analysed by double-reciprocal plot. The Km of the substrate increased and the Vmax of the reaction decreased in the presence of 4-HPR, indicating that enzyme inhibition involved both competition for the substrate-binding site and non-competitive mechanisms. To determine whether 4-HPR would also inhibit aromatase activity in intact cells, MCF-7 human breast cancer cells were incubated with or without cAMP in the presence of 4-HPR. 4-HPR inhibited both basal and cAMP-induced aromatase activity in intact MCF-7 cells. The induction of aromatase mRNA expression in MCF-7 cells by cAMP was inhibited in cells treated with 4-HPR. These results indicate that 4-HPR inhibits both the enzymatic activity and expression of aromatase. These activities may play an important role in the known chemopreventive effect of 4-HPR towards breast cancer.

Anticarcinogenic Agents↗

PubMed2000

Effects of calorie restriction on thymocyte growth, death and maturation.

We previously reported that calorie restriction (CR) significantly delays the spontaneous development of thymic lymphomas and other neoplasms in p53-deficient mice and their wild-type littermates. The purpose of the present study was to further characterize the anti-lymphoma effects of CR by assessing thymocyte growth, death and maturation in response to acute (6 day) and chronic (28 day) CR regimens. Male C57BL/6J mice fed a CR diet (restricted to 60% of control ad libitum intake) for 6 days displayed a severe reduction in thymic size and cellularity, as well as a decrease in splenic size and cellularity; these declines were sustained through 28 days of CR. Mice maintained on a CR diet for 28 days also displayed a significant depletion in the cell numbers of all four major thymocyte subsets defined by CD4 and CD8 expression. Analysis within the immature CD4(-)8(-) thymocyte subset further revealed an alteration in normal CD44 and CD25 subset distribution. In particular, CR for 28 days resulted in a significant decrease in the percentage of the proliferative CD44(-)25(-) subset. In addition, a significant increase in the percentage of the early, pro-T cell CD44(+)25(-) population was detected, indicative of a CR-induced delay in thymocyte maturation. Taken together, these findings suggest that CR suppresses (through several putative mechanisms) lymphomagenesis by reducing the pool of immature thymocytes that constitute the lymphoma-susceptible subpopulation.

Animals↗

PubMed2000

Bcl-2 is not reduced in the death of MCF-7 cells at low genistein concentration.

Soy consumption has been associated with a lower incidence of breast cancer in Southeast Asia. Among the phytochemicals in soy, genistein has been suggested to be chemopreventive. Because genistein is an estrogen-receptor (ER) agonist, the chemopreventive mechanism has been attributed to its ability to compete with estrogen for receptor binding. In this study, we used an ER-positive cell line to investigate the effects of different genistein concentrations on the apoptotic response. The threshold concentration at which a significant number of cells underwent apoptosis was titrated to be 25 micromol/L. At or above this concentration, c-jun N-terminus kinase was activated and Bax and Bcl-2 expression were both elevated. The elevated Bcl-2 protein might neutralize the proapoptotic effect of Bax. Therefore, the mechanism of genistein-induced apoptosis at this concentration might rely largely on the stress pathway rather than the pathway mediated by the Bcl-2 family of proteins.

Antineoplastic Agents↗

PubMed2000

Catecholamines and angiotensinogen gene expression in kidney proximal tubular cells.

To investigate the molecular mechanism(s) of action of catecholamines on the expression of the angiotensinogen (ANG) gene in kidney proximal tubular cells, we used opossum kidney (OK) cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (rANG N-1498/+18), permanently integrated into their genomes. The level of expression of the ANG-GH fusion gene was quantified by the amount of immunoreactive-hGH (IR-hGH) secreted into the medium. The addition of norepinephrine (NE), isoproterenol (a beta1/beta2-adrenergic receptor (AR) agonist) and iodoclonidine (an alpha2-AR agonist) stimulated the expression of the ANG-GH fusion gene in a dose-dependent manner, whereas the addition of epinephrine and phenylephrine (alpha1-AR agonist) had no effect. The stimulatory effect of NE was blocked by the presence of propranolol (beta-AR blocker), atenolol (beta1-AR blocker), yohimbine (alpha2-AR blocker), Rp-cAMP (an inhibitor of cAMP-dependent protein kinase AI & AII) and staurosporine (an inhibitor of protein kinase C), but was not blocked by ICI 118, 551 (beta2-AR blocker) and prazosin (alpha1-AR blocker). The addition of a combination of isoproterenol and iodoclonidine or a combination of 8-Bromo-cAMP (8-Br-cAMP) and phorbol 12-myristate (PMA) synergistically stimulated the expression of the ANG-GH fusion gene as compared to the addition of isoproterenol, iodoclonidine, 8-Br-cAMP or PMA alone. Furthermore, the addition of NE, 8-Br-cAMP or PMA stimulated the expression of pOGH (rANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+18) in OK cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. Gel mobility shift assays revealed that the ANG-CRE binds with the DNA-binding domain (bZIP254-327) of the cAMP-responsive binding protein (CREB). The binding of the labeled ANG-CRE to CREB (bZIP254-327) was displaced by unlabeled ANG-CRE and the CRE of the somatostatin gene but not by the mutants of the ANG-CRE. Finally, NE stimulated the phosphorylation of CREB in OK cells. These studies demonstrate that the molecular mechanism(s) of NE action on the expression of the ANG gene in OK cells may be mediated via both the PKA and PKC signalling pathways and via the phosphorylation of CREB. The phosphorylated CREB then interacts with the CRE in the 5'-flanking region of the ANG gene and subsequently stimulates the gene expression.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

Mechanistic studies of the effects of the retinoid N-(4-hydroxyphenyl)retinamide on prostate cancer cell growth and apoptosis.

To explore the mechanisms underlying the chemopreventive effects of the synthetic retinoid N-(4-hydroxyphenyl)retinamide (4-HPR) in prostate cancer, we evaluated the anti-proliferative and apoptosis-inducing effects of 4-HPR in the androgen-sensitive human prostate cancer cell line LNCaP. 4-HPR decreased the number of viable LNCaP cells (as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay) in a dose-dependent manner. Although 4-HPR exerted a modest G1 cell-cycle block (as determined by flow cytometry), its effect on reduced cell number appeared to result primarily from induction of apoptosis (as measured by an enzyme-linked immunosorbent assay and flow-cytometric assays). The mitogenic effects of R1881, a non-metabolizable androgen that potently induces LNCaP cell proliferation, was completely blocked by greater than 0.5 microM 4-HPR. Furthermore, increasing the R1881 concentration in the presence of 2.0 microM 4-HPR increased apoptotic cell death. 4-HPR decreased prostate-specific antigen (PSA) protein levels in conditioned medium and decreased PSA mRNA expression. 4-HPR also decreased the ratio of bcl-2 to bax mRNA expression in LNCaP cells by approximately 45%, indicating that the apoptotic effects of 4-HPR may be mediated, at least in part, by alterations in the bcl-2/bax-regulated apoptotic pathway. N-acetylcysteine (4 mM) completely blocked the anti-proliferative and apoptotic-inducing effects of 4-HPR, suggesting that an oxidative mechanism may be involved. We concluded that (i) 4-HPR exerts growth-suppressive and apoptotic effects on LNCaP cells, (ii) 4-HPR can interact with androgen to suppress proliferation and induce apoptosis, (iii) the apoptotic effects of 4-HPR may be mediated in part by the bcl-2/bax pathway, and (iv) a pro-oxidant mechanism may contribute to the anti-proliferative and apoptotic-inducing effects of 4-HPR.

Androgens↗

PubMed1999

Differential effects of chemotherapeutic agents on the Bcl-2/Bax apoptosis pathway in human breast cancer cell line MCF-7.

The present study explored the effects of three commonly used chemotherapeutic agents on the Bcl-2/Bax apoptosis pathway and the interaction of these chemotherapeutic drugs with the estradiol-mediated regulation of this pathway. Our results showed that: (1) Treatment of MCF-7 cells with Adriamycin resulted in time- and concentration-dependent decreases in Bcl-2 and increases in Bax mRNA and protein levels. (2) Camptothecin elicited similar trends on Bcl-2 and Bax as Adriamycin, while etoposide, at 50-100 fold (1-5 microM) the effective concentration of Adriamycin and camptothecin, only resulted in an increase in Bax mRNA levels. (3) Adriamycin and camptothecin, but not etoposide, were effective in suppressing estradiol-stimulated increases in Bcl-2 mRNA levels. Our study provides evidence that the Bcl-2/Bax apoptosis pathway may be differentially regulated by chemotherapeutic agents. In addition, interaction between these agents and estradiol on the Bcl-2/Bax apoptosis pathway may also exist.

Antineoplastic Agents↗

PubMed1999

Paradoxical regulation of Bcl-2 family proteins by 17beta-oestradiol in human breast cancer cells MCF-7.

Tumorigenesis is related to the dysregulation of cell growth or cell death pathways. Hence, elucidation of the mechanisms involved in the modulation of pro- or anti-apoptotic proteins is important in furthering understanding of breast cancer aetiology and may aid in designing prevention and treatment strategies. In the present study, we examined the role of 17beta-oestradiol on the regulation of apoptosis in the breast cancer cell line MCF-7. Using multi-probe RNAase protection assays, we found changes in the mRNA levels of several Bcl-2 family proteins upon treatment of MCF-7 cells with 17beta-oestradiol. Unexpectedly, we found a paradoxical effects of 17beta-oestradiol on two anti-apoptotic proteins Bcl-2 and Bcl-x. Treatment with 17beta-oestradiol resulted in up-regulation of Bcl-2 mRNA and protein, but down-regulated Bcl-x(L) mRNA and protein. The effect of 17beta-oestradiol on Bcl-x(L) occurred at concentration-dependent fashion. The effect was specific to 17beta-oestradiol since other steroid hormones exert no effect on Bcl-x(L). Tamoxifen, an anti-oestrogen, blocked the down-regulation of Bcl-x(L) by 17beta-oestradiol demonstrating this effect is oestrogen receptor-dependent. We speculate that different members of the Bcl-2 family proteins may be regulated through different pathway and these pathways may be modulated by 17beta-oestradiol.

Adenocarcinoma↗

PubMed1999

Molecular mechanism(s) of action of isoproterenol on the expression of the angiotensinogen gene in opossum kidney proximal tubular cells.

BACKGROUND: beta-adrenoceptors are present in the renal proximal tubules. We have previously reported that isoproterenol stimulates the accumulation of intracellular cAMP and the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta 1-adrenoceptor. We hypothesized that the molecular mechanism(s) of action of isoproterenol on the expression of the ANG gene is mediated via the interaction of the phosphorylated cAMP-responsive element binding protein (CREB) and the cAMP-responsive element (CRE; that is, ANG N-806/-779) in the 5'-flanking region of the rat ANG gene. METHODS: The fusion genes containing the putative ANG-CRE of the rat ANG gene inserted upstream of the rat ANG basal promoter (ANG N-53/+18) fused to a human growth hormone (hGH) gene as reporter were stably cotransfected, with or without the plasmid containing the cDNA for 43 kDa CREB, into the OK cells. The effect of various agonists and antagonists of adrenoceptors on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH secreted into the culture medium. The interactions of OK cellular nuclear protein(s) with the ANG N-806/779 were determined by gel mobility shift assays and by Southwestern and Western blot analysis. RESULTS: The addition of isoproterenol, forskolin, or 8-Bromo-cAMP (8-Br-cAMP) stimulated the expression of pOGH (ANG N-806/-779/-53/+18) by 135, 150, and 160%, respectively, but not mutants of the ANG N-806/-779. The stimulatory effect of isoproterenol was blocked in the presence of propranolol, Rp-cAMP, and atenolol, but not by the presence of stauro-sporine, U73122, and ICI 118,551. Transient transfection of the plasmid containing the cDNA for the catalytic subunit of protein kinase A further enhanced the stimulatory effect of 43 kDa CREB on the expression of the fusion gene. The gel mobility shift assays revealed the the nuclear protein(s) of OK cells binds to the radioactive-labeled ANG N-806/-779. The binding of the labeled ANG N-806/-779 to the OK cell nuclear protein(s) was displaced by unlabeled ANG N-806/-779, but not by the CRE of the somatostatin gene, the CRE of the tyrosine amino-transferase gene, or the mutants of the ANG N-806/-779. Southwestern blot analysis revealed that the labeled ANG N-806/-779 binds to two nuclear species of 43 and 35 kDa proteins. Western blot analysis, however, revealed that rabbit polyclonal antibodies against the 43 kDa CREB interacted with only the 43 kDa molecular species but not with the 35 kDa species. CONCLUSION: These studies demonstrate that the stimulatory effect of isoproterenol on the expression of the ANG gene may be mediated, at least in part, via the interaction of the phosphorylated CREB and the CRE in the 5'-flanking region of the rat ANG gene. The novel 35 kDa nuclear protein that is immunologically different from the 43 kDa CREB may also play a role in the expression of the ANG gene.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1999

The molecular biology of Schwanniomyces occidentalis klocker.

This review describes the molecular studies of Schwanniomyces occidentalis (Debaryomyces occidentalis) concerning transformation, genome, gene cloning, gene structure, gene expression and its characteristics to application. Schw. occidentalis appears to have at least five or seven chromosomes and no native plasmid from the yeast has been reported. Four transformation systems based on complement of Schw. occidentalis auxotrophic mutants were established. Vectors with the replicon of 2-micron plasmid and autonomous replication sequences (ARS) of Saccharomyces cerevisiae and Schw. occidentalis ARS replicated extrachromosomally in Schw. occidentalis transformants, without modification of the transformed vector DNA. So far, at least 21 Schw. occidentalis genes encoding 14 different proteins have been cloned. Most of the Schw. occidentalis genes have shown homologies (45 to 91%) with the corresponding genes of other organisms, especially of S. cerevisiae. However, some Schw. occidentalis genes possess other unique structures for their operators, promoters, transcription initiation sites, and terminators. Some foreign genes were expressed in Schw. occidentalis, while Schw. occidentalis genes functioned in other yeasts and bacteria, Escherichia coli, and Streptomyces lividans. Due to a strong ability of secretion and low level of glycosylation, Schw. occidentalis might be a promising host to produce heterologous proteins.

Amino Acid Sequence↗

PubMed1998

Effect of curcumin on the aryl hydrocarbon receptor and cytochrome P450 1A1 in MCF-7 human breast carcinoma cells.

We examined the interaction of curcumin, a dietary constituent and chemopreventive compound, with the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 mammary epithelial carcinoma cells. Curcumin caused a rapid accumulation of cytochrome P450 1A1 (CYP1A1) mRNA in a time- and concentration-dependent manner, and CYP1A1 monooxygenase activity increased as measured by ethoxyresorufin-O-deethylation. Curcumin activated the DNA-binding capacity of the AhR for the xenobiotic responsive element of CYP1A1 as measured by the electrophoretic-mobility shift assay (EMSA). Curcumin was able to compete with the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin for binding to the AhR in isolated MCF-7 cytosol, indicating that it interacts directly with the receptor. Although curcumin could activate the AhR on its own, it partially inhibited the activation of AhR, as measured by EMSA, and partially decreased the accumulation of CYP1A1 mRNA caused by the mammary carcinogen dimethylbenzanthracene (DMBA). Curcumin competitively inhibited CYP1A1 activity in DMBA-treated cells and in microsomes isolated from DMBA-treated cells. Curcumin also inhibited the metabolic activation of DMBA, as measured by the formation of DMBA-DNA adducts, and decreased DMBA-induced cytotoxicity. These results suggest that the chemopreventive effect of curcumin may be due, in part, to its ability to compete with aryl hydrocarbons for both the AhR and CYP1A1. Curcumin may thus be a natural ligand and substrate of the AhR pathway.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Diosmin and diosmetin are agonists of the aryl hydrocarbon receptor that differentially affect cytochrome P450 1A1 activity.

We investigated the effect of the chemopreventive compound diosmin and its aglycone form, diosmetin, on the carcinogen activation pathway mediated by the aryl hydrocarbon receptor (AhR) in MCF-7 human breast epithelial cancer cells. Treatment of the cells with diosmin caused a dose-dependent increase in the metabolism of the mammary carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), as assessed by increased formation of DMBA-DNA adducts and by DMBA-induced cytotoxicity. In contrast, treatment of the cells with diosmetin decreased both parameters. Diosmetin, but not diosmin, directly inhibited cytochrome P450 1A1 (CYP1A1) activity in a noncompetitive manner in microsomes isolated from DMBA-treated cells, as assayed by ethyoxyresorufin-O-deethylase activity. Treatment of the cells with diosmin or diosmetin, on the other hand, caused a dose- and time-dependent increase in CYP1A1 activity in intact cells that was comparable to that induced by DMBA or by the aryl hydrocarbon benzo(a)pyrene. Both diosmin and diosmetin caused an increase in the transcription of the CYP1A1 gene, as measured by increased levels of CYP1A1 mRNA. Both compounds caused the activation of the DNA-binding capacity of the AhR for the xenobiotic-responsive element of CYP1A1. These results indicate that diosmin and diosmetin are natural dietary agonists of the AhR, causing a potent increase in CYP1A1 transcription and CYP1A1 activity; however, only diosmetin is capable of inhibiting CYP1A1 enzyme activity, thus inhibiting carcinogen activation.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Increased UDP-glucuronosyltransferase activity and decreased prostate specific antigen production by biochanin A in prostate cancer cells.

Our laboratory has characterized androgen metabolism in an androgen-responsive prostate cancer cell line (LNCaP) and showed that these cells accumulated intracellular testosterone primarily as glucuronidated metabolites. Using a cell-free assay with testosterone as substrate, we showed that LNCaP had UDP-glucuronosyltransferase (UDPGT) activity. Because dietary factors, such as flavonoids in soy products, may reduce the risk for hormone-dependent cancers, we studied the effects of flavonoids on testosterone-UDPGT activity. LNCaP cells were exposed to selected flavonoids for up to 6 days. The increase in UDPGT-specific activity was linear over this period. Of the compounds tested, biochanin A was the most potent, with increased activity at concentration range 0.5-50 microM. Activities were linear for time and protein and were unaffected by flavonoids added directly to the assay. Kinetics studies showed no change in Km for testosterone in the face of these large increases in specific activity. Cellular metabolism of testosterone reflected the increase in enzyme activity. Intact cells treated with biochanin A produced testosterone-glucuronide from testosterone at twice the rate of controls. The steroid form of the UDPGT transcript was expressed in LNCaP cells and was enhanced in biochanin A-treated LNCaP cells. Additionally, biochanin A markedly decreased prostate specific antigen (PSA) level against the effect of testosterone on PSA production. Biochanin A significantly decreased the testosterone-stimulated release of PSA, presumably because biochanin A increased UDPGT and increased the intracellular glucuronidation of testosterone. These studies suggest that the modulation of hormone metabolism by dietary factors may be important in the prevention and treatment of prostate cancer.

Anticarcinogenic Agents↗

PubMed1998

Regulation of death promoter Bak expression by cell density and 17 beta-estradiol in MCF-7 cells.

In the current study we examined the regulation of Bak, a death promoter of an apoptotic pathway, in the human breast cancer cell line MCF-7. We observed a time-dependent increase in both Bak mRNA and protein levels which appeared to correlate well with the increase in cell density. We also found that treatment of cells with 17beta-estradiol resulted in inhibition of the time-dependent increases in Bak mRNA and protein. The effects of estradiol appeared to be via estrogen receptor as treatment of cells with progesterone did not effect Bak expression. Our study provides additional molecular evidence for (1) a link between apoptosis pathways and cell-cell and/or cell-cell matrix interactions and (2) a role for estradiol in the modulation of signals between apoptosis pathways and cell-cell and/or cell-cell matrix interactions.

Apoptosis↗

PubMed1998

A simple program to calculate codon bias index.

A computer program (PCBI) was developed to quickly calculate codon bias index (CBI). PCBI can analyze a gene containing introns. The 22 preferred codons defined from Saccharomyces cerevisiae were used in PCBI as the standard to measure the CBI values. However, users can modify the preferred codons to suit each organism. The data PCBI provides include DNA sequence of open reading frame without introns, amino acid sequence of gene product, a table of amino acid composition, a table of codon usage and (G + C) content, parameters for calculating CBI, and the value of CBI. PCBI runs on a DOS or Windows environment, but results can be saved in ASCII text format.

Amino Acids↗

PubMed1998

Concomitant alterations in distribution of 70 kDa heat shock proteins, cytoskeleton and organelles in heat shocked 9L cells.

Maintenance of cell architecture and positioning of organelles are major functions of the cytoskeleton. On the other hand, induction of heat shock proteins (HSPs) and reorganization of the cytoskeleton are the most significant changes in heat-shocked mammalian cells. We examine the alterations in HSP70 and its constitutively expressed cognate, HSC70, as well as the cytoskeleton and organelles in 9L rat brain tumor cells upon heat shock. We employed fluorescence microscopy and scanning electron microscopy to follow these changes. Levels of HSP70s were quantified by Western blotting. Accumulation of HSC70 was more transient and the protein translocated to and subsequently exited from the nucleus more rapidly than HSP70. Changes in actin microfilaments include the nuclear localization of actin fraction and disappearance of cytoplasmic microfilament bundles, while the cortical actin microfilaments were almost unaffected. Furthermore, microtubules retracted slightly from the cell periphery but remained largely unchanged. In contrast, the intermediate filaments collapsed into the perinuclear region. The mitochondria converted from filamentous into granular forms and clustered in a region overlapping with the collapsed intermediate filaments. All of the above alterations are reversible and largely reverted after 8 h of recovery. The effect on Golgi organization was very transient and the apparatus assumed a normal appearance within 4 h after the heat treatment. The ER, on the other hand, was totally unaffected by the heat treatment. These observations help correlate the sequential events following a stress like heat shock and suggest possible physiological functions of these essential constituents of a cell under stress.

Animals↗

PubMed1998

Molecular mechanism(s) of action of norepinephrine on the expression of the angiotensinogen gene in opossum kidney cells.

BACKGROUND: Norepinephrine (NE) is the major endogenous neurotransmitter of the renal sympathetic nerves interacting with both the alpha- and beta-adrenoceptors in the renal proximal tubules. We have previously reported that isoproterenol and iodoclonidine stimulate the expression of the angiotensinogen (ANG) gene in opossum kidney (OK) proximal tubular cells via the beta1-adrenoceptor and alpha2-adrenoceptor, respectively. We hypothesized that NE may interact with the beta- and/or alpha2-adrenoceptors to stimulate the expression of the ANG gene in OK cells. METHODS: The fusion genes containing the various lengths of the 5'-flanking regulatory sequence of the rat ANG gene fused with a human growth hormone (hGH) gene as a reporter were stably transfected into the OK cells. The stimulatory effect of NE on the expression of the fusion genes was evaluated by the amount of immunoreactive hGH (IR-hGH) secreted into the culture medium. RESULTS: The addition of NE stimulated the expression of the fusion gene, pOGH (ANG N-1498/+18) in a dose-dependent manner. The stimulatory effect of NE was inhibited in the presence of propranolol, atenolol, Rp-cAMP, yohimbine, staurosporine, H-7 and U73122 but not in the presence of ICI 118,551 and prazosin. The addition of a combination of isoproterenol and iodoclonidine synergistically stimulated the expression of pOGH (ANG N-1498/+18) as compared to the addition of isoproterenol and iodoclonidine alone. Furthermore, the addition of NE, forskolin, 8-Br-cAMP or phorbol 12-myristate (PMA) stimulated the expression of pOGH (ANG N-806/-779/-53/+18), a fusion gene containing the putative cAMP responsive element (CRE, ANG N-806/-779) upstream of the ANG promoter (ANG N-53/+ 18) in OK 95 cells, but had no effect on the expression of fusion genes containing the mutant of the CRE. CONCLUSION: These studies demonstrate that the stimulatory effect of NE on the expression of the ANG gene in OK cells may be mediated via both the beta1- and alpha2-adrenoceptors and via the CRE (ANG N-806/-779) in the 5'flanking region of rat ANG gene.

8-Bromo Cyclic Adenosine Monophosphate↗