ascorbic-acid and Cell-Transformation--Viral

Ascorbic acid has been shown to kill various cancer cell lines at pharmacologic concentrations. We found that Epstein-Barr virus (EBV)-positive Burkitt lymphoma (BL) cells were more susceptible to ascorbic acid-induced cell killing than EBV-negative BL cells or EBV-transformed lymphoblastoid cells (LCLs). Ascorbic acid did not induce apoptosis in any of the tested cells but did induce the production of reactive oxygen species and cell death. Previously, we showed that bortezomib, a proteasome inhibitor, induces cell death in LCLs and EBV-positive BL cells. We found that ascorbic acid is strongly antagonistic for bortezomib-induced cell death in LCLs and EBV-positive BL cells. Finally, ascorbic acid did not prolong survival of severe combined immunodefiency mice inoculated with LCLs either intraperitoneally or subcutaneously. Thus, while ascorbic acid was highly effective at killing EBV-positive BL cells and LCLs in vitro, it antagonized cell killing by bortezomib and was ineffective in an animal model.

Topics: Animals; Antineoplastic Agents; Apoptosis; Ascorbic Acid; Boronic Acids; Bortezomib; Burkitt Lymphoma; Cell Line, Transformed; Cell Line, Tumor; Cell Survival; Cell Transformation, Viral; Female; Herpesvirus 4, Human; Humans; Mice; Pyrazines; Reactive Oxygen Species; Virus Replication

The HIF family of hypoxia-inducible transcription factors are key mediators of the physiologic response to hypoxia, whose dysregulation promotes tumorigenesis. One important HIF-1 effector is the REDD1 protein, which is induced by HIF-1 and which functions as an essential regulator of TOR complex 1 (TORC1) activity in Drosophila and mammalian cells. Here we demonstrate a negative feedback loop for regulation of HIF-1 by REDD1, which plays a key role in tumor suppression. Genetic loss of REDD1 dramatically increases HIF-1 levels and HIF-regulated target gene expression in vitro and confers tumorigenicity in vivo. Increased HIF-1 in REDD1(-/-) cells induces a shift to glycolytic metabolism and provides a growth advantage under hypoxic conditions, and HIF-1 knockdown abrogates this advantage and suppresses tumorigenesis. Surprisingly, however, HIF-1 up-regulation in REDD1(-/-) cells is largely independent of mTORC1 activity. Instead, loss of REDD1 induces HIF-1 stabilization and tumorigenesis through a reactive oxygen species (ROS) -dependent mechanism. REDD1(-/-) cells demonstrate a substantial elevation of mitochondrial ROS, and antioxidant treatment is sufficient to normalize HIF-1 levels and inhibit REDD1-dependent tumor formation. REDD1 likely functions as a direct regulator of mitochondrial metabolism, as endogenous REDD1 localizes to the mitochondria, and this localization is required for REDD1 to reduce ROS production. Finally, human primary breast cancers that have silenced REDD1 exhibit evidence of HIF activation. Together, these findings uncover a specific genetic mechanism for HIF induction through loss of REDD1. Furthermore, they define REDD1 as a key metabolic regulator that suppresses tumorigenesis through distinct effects on mTORC1 activity and mitochondrial function.

Topics: Animals; Antibiotics, Antineoplastic; Antioxidants; Ascorbic Acid; Blotting, Western; Cell Transformation, Viral; Cells, Cultured; Feedback, Physiological; Fibroblasts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Knockout; Mice, Nude; Neoplasms, Experimental; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Sirolimus; Transcription Factors; Tumor Cells, Cultured

In the present study, we established a new adult human trabecular osteoblastic (AHTO) cell line, immortalized by SV-40 Large T (LT) oncogene. From seven proliferative colonies identified, we selected clone 7 with high alkaline phosphatase (ALP) activity for further analysis. AHTO-7 cells were able to grow for at least 8 months and 25 passages, with a doubling time of about 22 hours. Immunocytochemistry staining and RT-PCR analysis indicated that the extended life-span of AHTO-7 cells results in genomic insertion of SV-40 LT oncogene. The cells responded to PTH and PGE2 in terms of cAMP accumulation. The time course study, in the presence of 10(-8) M vitamin D3 (vit D3) showed a marked increase (fourfold) in ALP activity with a peak at day 3. Furthermore, in the presence of ascorbic acid (50 microg/ml) and inorganic phosphate (3 mM), AHTO-7 cells produced abundant calcified extracellular matrix, as examined by the von Kossa staining after 2 weeks of culture. Molecular analysis of mRNAs for phenotypic osteoblast markers at day 15 showed the expression of ALP, osteocalcin (OC), and collagen type I (Col I) mRNAs constitutively. Col I expression was inhibited by vit D3 and dexamethasone treatment. In contrast, treatment with vit D3 induced a marked increase of ALP and OC transcripts. Therefore, the immortalized AHTO-7 cells express osteoblast markers that are induced by calciotropic hormones, and constitute a suitable model for identifying specific osteoblastic genes and their regulation during human osteoblast differentiation.

Topics: Aged; Alkaline Phosphatase; Antigens, Polyomavirus Transforming; Ascorbic Acid; Bone and Bones; Calcification, Physiologic; Cell Differentiation; Cell Division; Cell Transformation, Viral; Cholecalciferol; Collagen; Dexamethasone; DNA Primers; DNA, Viral; Extracellular Matrix; Gene Transfer Techniques; Humans; Male; Osteoblasts; Osteocalcin; Phenotype; Phosphates; Reverse Transcriptase Polymerase Chain Reaction; RNA; Simian virus 40

The present data confirmed previous studies with other cell types that ascorbic acid and dehydroascorbic acid are transported through different transporters into SV-40-transformed retinal pigment epithelial cells. These experiments were performed on cells grown on 96-well culture plates. Ascorbic acid was taken up into the cell by a high-affinity transporter with Km = 0.041 mmol/l and a low Vmax of 2.74 pmol/min/well. Dehydroascorbic acid was taken up by a low-affinity transporter with Km = 5.67 mmol/l; however, the Vmax was 325.5 pmol/min/well. The uptake of both ascorbic acid and dehydroascorbic acid was dependent on the sodium concentration. The uptake of ascorbic acid does not involve oxidation-reaction steps because the uptake of [14C]-ascorbate was unaffected by the presence of an excess amount of unlabelled dehydroascorbic acid.

Topics: Ascorbic Acid; Biological Transport, Active; Cell Transformation, Viral; Cells, Cultured; Chromatography, High Pressure Liquid; Dehydroascorbic Acid; Humans; Kinetics; Oxidation-Reduction; Pigment Epithelium of Eye; Simian virus 40; Sodium

We have previously reported the isolation of an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma and immortalized by the SV 40 oncogenes. In this report we describe the kinetics of osteogenic differentiation of aggregated C1 cells by following the matrix deposition and mineralization and the expression of alkaline phosphatase. We show that after addition of beta-glycerophosphate and ascorbic acid, more than 95% of C1 aggregates synthesize a bone matrix which is deposited as early as 2 days and increases progressively with time in culture. Matrix calcification is evidenced by von Kossa staining and tetracycline incorporation into the mineral whereas no calcification appears in control cultures. Calcium is detectable in mineralizing aggregates at 2 days and calcium content increases linearly with time in culture, being 125-fold higher in mineralizing nodules than in control aggregates at 30 days. Aggregated C1 cells are characterized by a high activity of the bone type isoenzyme of alkaline phosphatase, a marker of osteoblast phenotype. Upon addition of inducers, alkaline phosphatase activity decreases by five-fold after the onset of mineralization and remains stable thereafter. The down-regulation of alkaline phosphatase activity is confirmed at the cellular level by histochemical staining. The mRNA levels for alkaline phosphatase decline during osteogenesis, following a pattern similar to the decrease in protein activity. Analysis of DNA synthesis by (3H)-thymidine incorporation and quantification of labelled nuclei on autoradiographs shows that C1 cells proliferation is not down-regulated during the time course of differentiation and that proliferating C1 cells still express alkaline phosphatase activity during osteogenic differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaline Phosphatase; Animals; Ascorbic Acid; Biomarkers; Cell Differentiation; Cell Transformation, Viral; Clone Cells; DNA; Extracellular Matrix; Glycerophosphates; Isoenzymes; Kinetics; Mice; Minerals; Osteoblasts; Osteogenesis; RNA, Messenger; Simian virus 40; Teratoma; Tumor Cells, Cultured

We have studied the action of ascorbate (vitamin C) on human immunodeficiency virus type 1 (HIV-1), the etiological agent clinically associated with AIDS. We report the suppression of virus production and cell fusion in HIV-infected T-lymphocytic cell lines grown in the presence of nontoxic concentrations of ascorbate. In chronically infected cells expressing HIV at peak levels, ascorbate reduced the levels of extracellular reverse transcriptase (RT) activity (by greater than 99%) and of p24 antigen (by 90%) in the culture supernatant. Under similar conditions, no detectable inhibitory effects on cell viability, host metabolic activity, and protein synthesis were observed. In freshly infected CD4+ cells, ascorbate inhibited the formation of giant-cell syncytia (by approximately 93%). Exposure of cell-free virus to ascorbate at 37 degrees C for 1 day had no effect on its RT activity or syncytium-forming ability. Prolonged exposure of virus (37 degrees C for 4 days) in the presence of ascorbate (100-150 micrograms/ml) resulted in the drop by a factor of 3-14 in RT activity as compared to a reduction by a factor of 25-172 in extracellular RT released from chronically infected cells. These results indicate that ascorbate mediates an anti-HIV effect by diminishing viral protein production in infected cells and RT stability in extracellular virions.

Topics: Antiviral Agents; Ascorbic Acid; Cell Line; Cell Survival; Cell Transformation, Viral; HIV-1; Humans; Kinetics; Protein Biosynthesis; RNA-Directed DNA Polymerase; T-Lymphocytes; Virus Replication

Antioxidants were found to protect against the genotoxic effects of chemical and physical mutagenic and clastogenic agents. This study focused on the capacity of antioxidants to reduce an intrinsic and persistent chromosome instability. As a model system, strains of C127 cells, which were transformed by bovine papillomavirus (BPV) DNA and which carry BPV DNA varying from 20 to 160 copies, were used. Transformed cells of 10 different strains showed a persistently high incidence of mitotic irregularities detectable at anaphase and telophase (27.3-58.9%), an elevated frequency of cells with micronuclei (6.6-34.7%), and a broad spectrum of nuclear sizes, as measured by image analysis. A 3-day exposure to retinoic acid, retinol, beta-carotene, canthaxanthin, ascorbic acid and ellagic acid greatly reduced the degree of chromosome instability, whereas catechin, eugenol and pyrogallol showed a smaller inhibitory effect, and curcumin had no detectable effect on the frequency of mitotic irregularities. After withdrawal of retinoic acid treatment, the high levels of chromosome instability reappeared. The possibility that the protective effect of the retinoids and carotenoids examined in the model system points to their beneficial administration to human cells with an intrinsic or acquired chromosome instability is discussed.

Topics: Antioxidants; Ascorbic Acid; beta Carotene; Bovine papillomavirus 1; Canthaxanthin; Carotenoids; Catechin; Cell Line, Transformed; Cell Transformation, Viral; Chromosomes; Curcumin; DNA, Viral; Ellagic Acid; Eugenol; Micronucleus Tests; Mitosis; Papillomaviridae; Pyrogallol; Tretinoin; Vitamin A

Collagen synthesis is a complex orchestration of intracellular and extracellular events. In addition to synthesis of the polypeptide chains more than a dozen modifications of the molecule occur; most of these are enzymatic and specific for collagen. Regulational control of collagen synthesis promises to be equally complex. Examples are described to 4 specific regulatory influences. Ascorbic acid markedly stimulates collagen synthesis without affecting synthesis of other proteins. This effect appears to be unrelated to its cofactor roles for hydroxylation of lysine and proline. Glucocorticoids at microM concentration specifically inhibit collagen synthesis. Tissues treated with glucocorticoids have diminished levels of mRNA for collagen. During collagen synthesis the aminoterminal propeptide of procollagen is cleaved by a specific protease. This peptide appears to be a feedback inhibitor of collagen synthesis. This effect can be demonstrated in cells and in cell-free synthesizing systems. A membrane receptor system may permit the peptide to be recognized and subsequently act as a translational control mechanism. Viral transformation of fibroblasts results in selectively decreased synthesis of collagen. Levels of cytoplasmic and nuclear mRNA are likewise selectively diminished consistent with transcriptional control.

Topics: Animals; Ascorbic Acid; Cattle; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Collagen; Fibroblasts; Glucocorticoids; Humans; Peptide Fragments; Procollagen; Rats; Skin

Ascorbic acid, at nontoxic concentrations, causes a substantial reduction in the ability of avian tumor viruses to replicate in both primary avian tendon cells and chicken embryo fibroblasts. The virus-infected cultures appear to be less transformed in the presence of ascorbic acid by the criteria of morphology, reduced glucose uptake, and increased collagen synthesis. The vitamin does not act by altering the susceptibility of the cells to initial infection and transformation, but instead appears to interfere with the spread of infection through a reduction in virus replication and virus infectivity. The effect is reversible and requires the continuous presence of the vitamin in the culture medium.

Topics: Ascorbic Acid; Avian Sarcoma Viruses; Biological Transport; Cell Division; Cell Transformation, Viral; Cells, Cultured; Collagen; Glucose; Virus Replication

Topics: Ascorbic Acid; Cell Transformation, Viral; Metronidazole; Simian virus 40