p53 protein subcellular localization and apoptosis in rodent corneal epithelium cell culture following ultraviolet irradiation.

The tumor-suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. p53 expression and function have been documented in malignancy, apoptosis and the aging processes. Recently, p53 has been mapped and characterized in the normal cornea across different species. In the present study, high levels of cytoplasmic p53 protein were noted in normal primary corneal epithelium cultures by immunohistochemistry and western blot analysis. Following ultraviolet (UV) irradiation, the level of cytoplasmic p53 protein expression was increased beginning from 30 min and lasting until 6 h post-irradiation and then returned close to control levels by 24 h. Cytoplasmic p53 phosphorylation was detected from 30 min following UV treatment until 6 h post-irradiation. p53 protein became apparent in the nucleus in a fraction of these cultured cells beginning 30 min following UV irradiation and was still present 24 h later. We also found that p53 colocalized with mitochondria 2 h following UV irradiation in some of the cells and remained there up to 24 h. As the expression levels of p53 transcription following UV irradiation were not significantly altered, the increase in cytoplasmic p53 protein expression may be conditional only upon post-translational stabilization. We also observed that the apoptotic index increased following UV irradiation in the same time frame as the p53 nuclear transfer and was partially suppressed by pifithrin-α, which is a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription. The present study offers new evidence suggesting that cytoplasmic p53 in rodent corneal epithelium is functionally active.

Growth inhibition of human thyroid carcinoma and goiter cells in vitro by the isoflavone derivative 7-(O)-carboxymethyl daidzein conjugated to N-t-boc-hexylenediamine.

BACKGROUND: Estrogens may enhance thyroid cancer cell growth. We have recently reported that a novel isoflavone-derived anti-estrogenic compound developed in our laboratory, the N-t-boc-hexylenediamine derivative of 7-(O)-carboxymethyl daidzein (cD-tboc), can induce apoptosis and retard growth in human thyroid carcinoma cell lines through inhibitory interaction on estrogen receptor ß. Here we tested the hypothesis that cD-tboc can likewise retard cell growth in cultured human thyroid papillary carcinoma cells, normal thyroid cells, and goiter cells removed during thyroidectomy. METHODS: In vitro experiments in cultured human thyroid normal, goiter, and papillary thyroid carcinoma (PTC) cells were performed. Estrogen receptors α and ß (ERα and ERß), DNA synthesis and creatine kinase (a marker of estrogenic genomic response), and the effects of cD-tboc on DNA synthesis in cultured human PTC cells were assessed. RESULTS: First, all cell types thus harvested and grown in culture expressed both ERα and ERß, with a variably higher abundance of ERß over ERα seen in the goiter and PTC cells, but not in the normal thyroid cells. Second, DNA synthesis and creatine kinase were increased in response to estradiol-17ß (E2), the ERα agonist propyl-pyrazole-trisphenol as well as the ERß agonist diarylpropionitrile. Third, cD-tboc dose-dependently inhibited DNA synthesis in cultured human PTC cells (-65%) and to a lesser extent in goiter cells (∼-30%). CONCLUSION: This study provides the first evidence that cD-tboc can act to inhibit growth in primary cultures of human PTC cells and goiter cells removed during thyroidectomy. Whether this can be utilized for the treatment of human thyroid cancer and/or goiter remains to be explored.

In vitro mitochondrial effects of PK 11195, a synthetic translocator protein 18 kDa (TSPO) ligand, in human osteoblast-like cells.

The role of the TSPO in metabolism of human osteoblasts is unknown. We hypothesized that human osteoblast metabolism may be modulated by the TSPO. Therefore we evaluated the presence of TSPO in human osteoblast-like cells and the effect of its synthetic ligand PK 11195 on these cells. The presence of TSPO was determined by [(3)H]PK 11195 binding using Scatchard analysis: Bmax 7682 fmol/mg, Kd 9.24 nM. PK 11195 did not affect significantly cell proliferation, cell death, cellular viability, maturation, [(18)F]-FDG incorporation and hexokinase 2 gene expression or protein levels. PK 11195 exerted a suppressive effect on VDAC1 and caused an increase in TSPO gene expression or protein levels. In parallel there was an increase in mitochondrial mass, mitochondrial ATP content and a reduction in ΔΨm collapse. Thus, it appears that PK11195 (10(-5) M) stimulates mitochondrial activity in human osteoblast-like cells without affecting glycolytic activity and cell death.

Aldosterone up-regulates 12- and 15-lipoxygenase expression and LDL oxidation in human vascular smooth muscle cells.

Several lines of evidence suggest that aldosterone excess may have detrimental effects in the cardiovascular system, independent of its interaction with the renal epithelial cells. Here we examined the possibility that aldosterone modulates 12- and/or 15-lipoxygenase (LO) expression/activity in human vascular smooth muscle cells (VSMC), in vitro, thereby potentially contributing to both vascular reactivity and atherogenesis. Following 24 h treatment of VSMC with aldosterone (1 nmol/L), there was a approximately 2-fold increase in the generation rate of 12 hydroxyeicosatetraenoic acid (12-HETE), 70% increase in platelet type 12-LO mRNA expression (P < 0.001) along with a approximately 3-fold increase in 12-LO protein expression, which were blocked by the mineralocorticoid receptor (MR) antagonists spironolactone (100 nmol/L) and eplerelone (100 nmol/ml). Additionally, aldosterone (1 nmol/L; 24 h) increased the production of 15-HETE (50%; P < 0.001) and the expression of 15-LO type 2 mRNA (50%; P < 0.05) (in VSMC). Aldosterone also increased the 12- and 15-LO type 2 mRNA expression in a line of human aortic smooth muscle cells (T/G HA-VSMC) (60% and 50%, respectively). Aldosterone-induced 12- and 15-LO type 2 mRNA expressions were blocked by the EGF-receptor antagonist AG 1478 and by the MAPK-kinase inhibitor UO126. Aldosterone-treated VSMC also showed increased LDL oxidation, (approximately 2-fold; P < 0.001), which was blocked by spironolactone. In conclusion, aldosterone increased 12- and 15-LO expression in human VSMC, in association with increased 12- and 15-HETE generation and enhanced LDL oxidation and may directly augment VSMC contractility, hypertrophy, and migration through 12-HETE and promote LDL oxidation via the pro-oxidative properties of these enzymes.

Estradiol modulates uterine 18 kDa translocator protein gene expression in uterus and kidney of rats.

We examined the effect of ovariectomy, with and without estradiol treatment, on 18 kDa translocator protein (TSPO) gene expression and its binding density in the uterus and kidney of rats. Ovariectomy causes a significant decrease in uterine, but not renal TSPO binding density, while estradiol treatment of ovariectomized rats restored TSPO binding density in the uterus. These TSPO density levels did not correlate with steady state or new RNA transcription. Our in vivo study suggests that estradiol is responsible for the maintenance of uterine TSPO density via transcriptional mechanisms. Our in vivo study also suggests that in the kidney estradiol appears to operate via post-transcriptional mechanisms to maintain TSPO density.

The 18-kDa translocator protein, formerly known as the peripheral-type benzodiazepine receptor, confers proapoptotic and antineoplastic effects in a human colorectal cancer cell line.

OBJECTIVE: The involvement of the 18-kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, in apoptosis regulation of HT29 colorectal cancer cells was studied in-vitro. In-vivo TSPO involvement in tumor growth of HT29 cells xenografted into SCID mice was studied. METHODS: Knockdown of TSPO expression in the human HT29 cell line was established by stable transfection with vectors containing the TSPO gene in the antisense direction. Successful TSPO knockdown was characterized by reduction of 20% in TSPO RNA levels, 50% in protein expression of the TSPO, and 50% in binding with the TSPO ligand, [3H]PK 11195. Subsequently, in-vitro cell viability and proliferation assays were applied. In addition, transient transfecton with short interfering RNA (siRNA) directed against human TSPO was studied in this way. Furthermore, we also grafted HT29 cells subcutaneously into the right thighs of SCID mice to examine the effects of the putative TSPO agonist, FGIN-1-27, on tumor growth in-vivo. RESULTS: In-vitro TSPO knockdown established by stable transfection of TSPO antisense gene resulted in HT29 clones displaying significantly lower levels of cell death as determined with trypan blue (50% less), lower apoptotic rates (28% less), and higher proliferation rates (48% more one week after seeding and 27% more two weeks after seeding). Transient transfection with anti-human TSPO siRNA resulted in similar viability and antiapoptotic effects. In-vivo, the proapoptotic TSPO ligand, FGIN-1-27 significantly reduced the growth rate of grafted tumors (40% less), in comparison with vehicle-treated mice. CONCLUSION: TSPO knockdown by genetic manipulation transforms the human HT29 cancer line to a more malignant type in-vitro. In-vivo pharmacological treatment with the putative TSPO agonist FGIN-1-27 reduces tumor growth of the HT29 cell line. These data suggest that TSPO involvement in apoptosis provides a target for anticancer treatment.

Lipoxygenase-derived metabolites are regulators of peroxisome proliferator-activated receptor gamma-2 expression in human vascular smooth muscle cells.

BACKGROUND: Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor family that has been implicated in cell differentiation and proliferation, glucose metabolism, macrophage development, and inflammatory responses. PPAR-gamma can be activated by a range of synthetic substances and also by products of lipid oxidation such as oxidized low-density lipoprotein, 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Since 12- and 15-lipoxygenase (12- and 15-LO) are expressed in human vascular smooth muscle cells (VSMCs), we set out to investigate the possible relation between PPAR-gamma and LO system in these cells. METHODS: In vitro experiments in human VSMC using standard methods. RESULTS: The LO products, 12-HETE (10(-7) mol/l), 15-HETE (10(-7) mol/l) and 13-HODE (10(-7) mol//l) increased the expression of PPAR-gamma-2 messenger RNA (mRNA) in VSMC (by 100, 50, and 100%, respectively. Rosiglitazone (1-10 micromol/l) was found to upregulate both the mRNA expression of two LO enzymes, platelet-type 12-lipoxygenase (12-LO; +70%) and 15-lipoxygenase type 2 (15-LO; +60%), and the secretion of their eicosanoid products 12- and 15-HETE. In addition, rosiglitazone-induced a threefold increase in PPAR-gamma-2 mRNA expressions and modest 50% rise in PPAR-gamma-1 mRNA expression. The effect of rosiglitazone on PPAR-gamma-2 could be entirely blocked by the LO inhibitor baicalein and restored by the addition of exogenous 12-HETE. CONCLUSIONS: These results suggest a novel amplification cycle in which PPAR-gamma activation induces production of 12- and 15-LO-derived metabolites which in turn feed back to upregulate PPAR-gamma-2's own expression. The implications of this link in VSMC pathophysiology remain to be elucidated.

12S-lipoxygenase protein associates with alpha-actin fibers in human umbilical artery vascular smooth muscle cells.

The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to alpha-actin, a component of the cytoplasmic myofilaments. 12-LO/alpha-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to alpha-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein alpha-actin.

The 5 lipoxygenase system in the vasculature: emerging role in health and disease.

Activation of the 5 lipoygenase (5LO) system within the vascular bed requires the presence of several cell types with distinct transcellular cross-talk mechanisms, resulting in the generation of 5LO produced metabolites and increased expression of receptors for these metabolites in vascular cells. The key products in this system, the leukotriens LTB4, LTC4 and LTD4, are potent mediators of vascular inflammation initiated by white blood cells and sustained or propagated thereafter through amplified metabolite generation and direct effects in endothelial and vascular smooth muscle cells. Leukotrienes act to enhance cell permeability and increase oxidative stress, vascular smooth muscle cell migration and arterial tone. 5LO activation is highly regulated, and is apparently both model/species-specific and region-specific. 5LO activation is also linked to plaque progression, plaque stability, activation of matrix metalloproteinases, propensity to coronary and cerebrovascular events and the evolution of aortic aneurysms. Genetic variants in the 5LO activating protein are strongly linked to increased cardiovascular risk and may serve as useful markers for future therapy targeting down regulation of 5LO expression and activity as a means to combat cardiovascular disease.

Identification of cytoplasmic p53 protein in corneal epithelium of vertebrates.

The tumour suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. It's expression and function have been documented in malignancy, apoptosis and the aging processes. Recently, p53 expression has been demonstrated in normal murine tissues, including whole eye. Currently, we intend to map and to characterize p53 expression in the normal cornea across different species. To do this, eyes of animals were enucleated after sacrifice by CO(2) narcosis and then p53 expression in whole eyes (cornea) was mapped by indirect immunohistochemical staining techniques using the anti-p53 monoclonal antibodies PAb 248, PAb 421 and PAb 240 (alternatively called mAb 248, mAb 421 and mAb 240, respectively). Additionally, eyes were freshly dissected to separate the corneas, for quantitating p53 expression, using Western blot analysis. We found strong cytoplasmic p53 expression in the corneal epithelium of various vertebrate species by immunohistochemistry and by Western analysis. High levels of cytoplasmic p53 protein were normally found in normal corneal epithelium of various vertebrate species. Hence, these data may indicate that p53 may have a new evolutionary significant function in the eye.

The peripheral-type benzodiazepine receptor and tumorigenicity: isoquinoline binding protein (IBP) antisense knockdown in the C6 glioma cell line.

Peripheral-type benzodiazepine receptors (PBR) are constituted by three protein components, the isoquinoline binding protein (IBP), the voltage-dependent anion channel (VDAC), and the adenine nucleotide transporter (ANT). Recently, we found that high levels of PBR ligand binding in glioma cell lines correlate with in vitro tumorigenicity. To study whether enhanced PBR expression is causative or in response to cancer, we genetically modified C6 glioma cells. Antisense knockdown of the IBP resulted in more than 50% reductions in PBR ligand binding both in the mitochondrial and whole cell fractions, accompanied by similar reductions in IBP levels in these respective fractions. The IBP knockdown was accompanied by a 25% increase in cell number in confluent cultures. This correlated with an 8-fold increase in in vitro tumorigenicity, as assessed by anchorage independent growth. Cell cycle analysis indicated that knockdown of the IBP resulted in a 60% reduction in the number of cells in the pre-G1 apoptosis phase. This paralleled the reduction seen in apoptosis and cell death shown by DNA fragmentation and Trypan blue assays, respectively. Furthermore, knockdown of the IBP appeared to prevent induction of apoptosis by the antineoplastic agent, erucylphosphocholine. In addition, IBP knockdown prevented processing of the caspase 3 component of the apoptosis cascade by the erucylphosphocholine congener, erucylphospho-N,N,N-trimethylammonium. In conclusion, our results suggest that enhanced IBP expression, including enhanced PBR ligand binding, such as occurring in untreated C6 glioma cells, may provide a mechanism to increase apoptotic rates of cancer cells.

Angiotensin II increases the expression of lectin-like oxidized low-density lipoprotein receptor-1 in human vascular smooth muscle cells via a lipoxygenase-dependent pathway.

BACKGROUND: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a membrane protein that can act as a surface endocytosis receptor for oxidized LDL (ox-LDL). As increased cellular uptake of ox-LDL by macrophages and activated smooth muscle cells may transform these cells into foam cells, potential interactions among LDL oxidation, ox-LDL uptake, and regulators of vascular smooth muscle cell function are of obvious interest. The objective of this study was to examine the effect of angiotensin II (AII) on the expression of LOX-1 and ox-LDL degradation in human vascular smooth muscle cells (VSMC) METHODS: We performed in vitro experiments in a human VSMC line (T/G HA-VSMC) derived from normal aortic VSMC, using standards methods. RESULTS: We found that AII (10(-7) mol/L) increased the expression of LOX-1 (approximately 2.5-fold, P < .0001) in association with higher degradation of ox-LDL by HA-SMC (from 4019 +/- 529 ng/mg cell protein to 6207 +/- 287 ng/mg cell protein; P = .0033). AII also increased the expression of 12-lipoxygenase (12-LO) and 15-lipoxygenase (15-LO) by approximately 2.2-fold (P = .03) and approximately 3-fold (P = .006), respectively. In addition, AII (10(-7) mol/L) increased the release of 12- and 15-hydroxyeicosatetraenoic acid from VSMC within 10 min approximately 3-fold (P = .03) and 50% (P < .05), respectively. CONCLUSIONS: Our study findings provide evidence that angiotensin II upregulates LOX-1 and 12-LO and 15-LO expression in human VSMC, thereby potentially providing mechanisms for both accelerated LDL oxidation within the cell and the internalization of exogenous ox-LDL, two processes that could increase the susceptibility of human VSMC to further transformation into foam cells.

Peripheral benzodiazepine receptor antisense knockout increases tumorigenicity of MA-10 Leydig cells in vivo and in vitro.

Peripheral benzodiazepine receptors (PBR), first described more than 20 years ago, have been attributed with many putative functions including ones in cellular proliferation and cellular respiration. Hence, it is quite conceivable that deregulation of this receptor could lead to pathology. We and others have reported the existence of PBR overexpression in different human and nonhuman malignancies, but it has never been made clear whether this aberrant malignant PBR expression is a cause or consequence of the cancer. In the current study we induced PBR underexpression by downregulating one critical subunit of the PBR complex, the isoquinoline-binding protein (IBP), using the stable antisense knockout approach, in the MA-10 Leydig cell line. Resultant clones, showing PBR deregulation, also demonstrated increased tumorigenicity, using both in vitro (loss of contact inhibition and growth in soft agar) and in vivo (increased mortality on grafting back into isogenic mice) assays. We suggest that this type of deregulation could be a later event in natural tumor progression. Consequently, PBR deregulation should be more closely studied in human malignancy.

Peripheral-type benzodiazepine receptor density and in vitro tumorigenicity of glioma cell lines.

The peripheral-type benzodiazepine receptor is found primarily on the outer mitochondrial membrane and consists of three subunits: the 18kDa isoquinoline binding protein, the 32kDa voltage-dependent anion channel, and the 30kDa adenine nucleotide transporter. The current study evaluates the potential importance of peripheral-type benzodiazepine receptor expression in glioma cell tumorigenicity. While previous studies have suggested that peripheral-type benzodiazepine receptor-binding may be relatively increased in tumor tissue and cells, so far, little is known about the relationships between peripheral-type benzodiazepine receptor density and factors underlying tumorigenicity. In the present study, we found in glioma cell lines (C6, U87MG, and T98G), that peripheral-type benzodiazepine receptor ligand-binding density is relatively high for C6 and low for T98G, while U87MG displays intermediate levels. Cell growth of these cell lines in soft agar indicated that high levels of peripheral-type benzodiazepine receptor-binding were associated with increased colony size, indicative of their ability to establish anchorage independent cell proliferation. Potential causes for differences in tumorigenicity between these cell lines were suggested by various cell death and proliferation assays. Cell death, including apoptosis, appeared to be low in C6, and high in T98G, while U87MG displayed intermediate levels in this respect. Cell proliferation appeared to be high in C6, low in T98G, and intermediate in U87MG. In conclusion, our study suggests that relatively high peripheral-type benzodiazepine receptor-binding density is associated with enhanced tumorigenicity and cell proliferation rate. In particular, apoptosis appears to be an important tumorigenic determinant in these glioma cell lines. Moreover, application of PBR-specific ligands indicated that PBR indeed are functionally involved in apoptosis in glioma cells.

p53 expression in the normal murine eye.

PURPOSE: The tumor-suppressor gene p53 encodes a phosphoprotein involved in the control of cell growth. Its expression and function have been documented in malignancy, apoptosis, and other abnormal cell proliferation processes. Recently, expression of p53 has been demonstrated in certain normal tissues, including whole eye. The purpose of the study was to map and to characterize expression of p53 in the normal murine eye. METHODS: Eyes of adult C57BL/6 mice were enucleated after death by CO2 narcosis. Expression of p53 in frozen sections of whole cryoprotected eyes was mapped by indirect immunofluorescence microscopy using the anti-p53 monoclonal antibodies 248 and 421 and the polyclonal antibody FL-393. Additionally, eyes were freshly dissected to separate the various ocular tissues. In these ocular tissues, expression of p53 was quantitated with ELISA and Western blot analyses. RESULTS: Strong expression of p53 was observed in various normal ocular tissues. The corneal and conjunctival epithelium exhibited very high cytoplasmic p53 protein levels. High nuclear p53 protein staining was seen in the lens epithelial cells of the central and pre-equatorial zones and in the lens fiber nuclear bow, situated posterior to the epithelial germinative zone. Cells of the actual lens germinative zone did not stain for p53 protein. Low levels of p53 protein were expressed in retinal tissue. CONCLUSIONS: High levels of p53 protein are found in various normal murine ocular tissues, especially the corneal and conjunctival structures and the lens epithelium. Each of these tissues demonstrate unique patterns of staining.

GENETICALLY MODIFIED PRIMARY ASTROCYTES AS CELLULAR VEHICLES FOR GENE THERAPY IN THE BRAIN.

Combining genetic engineering and cell transplantation has been proposed as one way to overcome the limited availability of donor tissue that may restrict the application of graft therapy to neurological diseases. Important issues in this approach concern the choice of a suitable cellular vehicle, and the method of gene insertion. In this regard, we have investigated the use of brain-derived primary astrocytes as cellular vehicles for gene therapy, because they can be transfected, divide in culture, are brain-region specific, possess a secretory mechanism, and may migrate several mm from the transplant site. To address the issue of gene insertion, we have generated stably transfected primary rat astrocytes using the nonviral calcium phosphate method to co-transfect a reporter construct (RSV-chloramphenicol acetyltransferase (CAT), or human enkephalin promoter CAT, plus a neomycin resistance plasmid (pRSVNeo). Modified astrocytes were then propagated by transfer to selective media containing G418 (300 µg/mL) for 3 wk. The presence of the reporter gene product (CAT) was demonstrated by immunocytochemistry, and by biochemical assay of CAT enzyme catalytic activity. These genetically modified astrocytes were followed for up to 3 wk after transplantation into the rat striatum. Criteria used to distinguish transplanted astrocytes included histological evidence of abundant nuclei interrupting the normal cytoarchitecture of the striatum, astrocyte morphology, and the presence of CAT enzyme activity. Our data indicates that genetically modified astrocytes are an important candidate vehicle for use in transplantation therapy in neurological diseases. We suggest that genetically modified astrocytes can also be used for studying the human enkephalin promoter, other promoters, and expressed proteins using this paradigm.