THE DIFFERENTIAL EFFECT OF ACTINOMYCIN D IN NORMAL AND VIRUS-TRANSFORMED CELLS

Actinomycin D (AMD) at concentrations up to 0,25 µg/ml shows a differential effect on cell RNA synthesis and on the replication of an influenza virus in normal and virally transformed cells, both functions being more resistant to AMD in the transformed cell. A possible explanation for these differences in AMD sensitivity is provided by the observation that isotopically labeled AMD is maintained at a lower concentration in transformed BHK 21/13 (BHK) cells. There is evidence that the decreased sensitivity of the transformed cells to AMD is a result of maintenance of a lower internal concentration of the drug, since a correlation exists for a number of polyoma virus-transformed clones between sensitivity to and uptake of AMD.     

Mechanism of Actinomycin Resistance in SV40-Transformed Hamster Cells

Mechanisms of actinomycin D (AMD) resistance were studied in a hamster-derived SV₄₀-transformed cell line which is able to grow continuously in presence of 2 μg/ml of AMD. Resistant cells acquire differentiated phenotypic properties induced by continuous growth in presence of AMD. These properties include impermeability to AMD, cross-resistance with puromycin and slower growth rate. The uptake of ³H-AMD is 100 times greater in sensitive cells than in resistant cells. There is no difference between resistant and sensitive cells in RNA polymerase sensitivity to AMD, nor in the capacity of chromatin to bind AMD. These and other results suggest that modifications of the permeability at the plasma membrane or cell surface level comprise the major factor for AMD resistance.     

Contribution of organic anion transporting polypeptide OATP2B1 to amiodarone accumulation in lung epithelial cells

The accumulation mechanisms of amiodarone (AMD) involving transporters in lung alveolar epithelial type II cells were studied. The uptake of AMD was examined using human alveolar epithelial-derived cell line A549 as a model. AMD was transported by the carrier-mediated system, and the apparent K_m and V_max values were 66.8 ± 30.3 μM and 49.7 ± 9.7 nmol/mg protein/5 min, respectively. The uptake of AMD by A549 cells was Na⁺-independent and was inhibited by substrates of human organic anion transporting polypeptide (OATP). The inhibition profiles were similar to the inhibitory effects of several compounds on OATP2B1-mediated E-3-S transport, and RT-PCR analysis showed mRNA expression of OATP2B1 and 1B3 in A549 cells. SiRNAs targeted to the OATP2B1 gene decreased the OATP2B1 mRNA expression level in A549 cells up to about 50% and reduced the uptake of AMD up to about 40%. These results indicate that AMD uptake mediated by carriers, including OATP2B1, might lead to accumulation of AMD in the lung and AMD-induced pulmonary toxicity (AIPT).     

Changes in the uptake of 2-deoxy-D-glucose in BALB-3T3 cells chemically transformed in culture

Balb/3T3 cells transformed in culture by chemical carcinogens were shown to multiply in a medium supplemented with 2% calf serum or with 10% agamma new-born calf serum. The cell lines that multiply well in medium supplemented with 10% agamma serum produced a higher incidence of tumors in X-irradiated weanling mice than the lines that multiply poorly. The difference in 2-deoxy-D-glucose uptake into exponentially growing transformed and un-transformed cells was 50–100%. In crowded cultures untransformed Balb/3T3 cells ceased taking up the sugar, while chemically transformed cells continued at the same rate even at high cell densities; thus, the difference became greater in crowded cultures. When the serum concentration in the media was reduced from 10% to 2%, untransformed Balb/3T3 cells took up the sugar at a reduced rate, while chemically transformed cells were only slightly affected; agamma new born calf serum supplemented medium had no effect on sugar uptake in any of the cells. When the serum concentration was changed from 2% to 10%, untransformed cells increased sugar uptake followed by cell division. The immediacy (within 15 min) of the response in the sugar uptake to 10% serum concentration suggested that the increased uptake rate and the consequent higher concentration of the sugar (D-glucose in normal situation) within Balb/3T3 cells triggered the cell cycle. Chemical carcinogens appear to alter permanently the uptake mechanism for a key nutrient.     

Transformed BHK cells exhibiting normal or subnormal sugar uptake

The uptake of deoxyglucose was compared in BHK cells and in DMN4B cells, a conditionally transformed line of BHK cells which exhibits transformed behavior at 38.5° but not at 32°. At 32°, DMN4B cells took up deoxyglucose more slowly than BHK cells, reflecting a higher Km for uptake of this sugar. When both cell lines were grown at 38.5°, the Km for DMN4B cells was reduced to a level only slightly greater than for BHK cells, and deoxyglucose uptake became similar in the two cell lines. Growth in glucose-free medium for 22 hours stimulated deoxyglucose uptake in both BHK and DMN4B cells; under these conditions, uptake was equal in the two cells lines, both at 32° and 38.5°. Glycolysis, as measured by lactic acid production, was slower in DMN4B than BHK cells, but in contrast to deoxyglucose uptake, this difference was observed at 38.5° rather than 32°. The observation that the subnormal deoxyglucose uptake of DMN4B cells in the untransformed state (32°) can be normalized by growth at 38.5°, a temperature permissive for transformation, suggests that membrane changes facilitating sugar uptake, which have been found in other transformed cells, are associated with transformation in DMN4B cells as well. However, the failure of uptake to exceed normal in these cells indicates that their transformed behavior is not attributable to excessive sugar uptake per se.     

Influence of retinoic acid on protein synthesis and transport of l-methionine in cultured L cells

The effects of retinoic acid (RA) on cell proliferation, activity of acid phosphatase, protein synthesis and methionine uptake were studied in transformed murine LPA cells. Early inhibition of protein synthesis was demonstrated under experimental conditions in which the rate of cell proliferation was diminished and non-specific effects of vitamin action could be excluded. Measurements of l-methionine uptake revealed a decrease to approximately one-half of that in control cultures after treatment with RA at the concentrations of 5 × 10^?5 M and 10^?5 M.     

Differences in transferrin receptor function between normal developing and transformed myogenic cells as revealed by differential effects of phorbol ester on receptor distribution and rates of iron uptake

The effects of the tumor promotor, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), on the intra- and extracellular distribution of transferrin receptors and rates of iron uptake were studied in normal developing myogenic cells and myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus. In normal developing cells PMA was found to increase the rate of iron uptake by 15-30%. There was, however, no effect on transferrin receptor distribution, suggesting that the increase in iron uptake was due to stimulation of the rate of receptor cycling. In contrast, in transformed myogenic cells, PMA had no effect even at concentrations 10 times those effective in normal myogenic cells. The specificity of PMA was demonstrated by comparison with 4 alpha-phorbol which had no effect compared with the control cells which were incubated with dimethyl sulfoxide, the solvent used to dissolve the phorbols. These results indicate a functional difference in the transferrin receptor between normal and transformed myogenic cells. The data for normal myogenic cells are similar to those previously reported for normal erythroid cells, but differ from those for some transformed cell lines in which phorbol esters were shown to cause internalization of transferrin receptors.     

Pleiotropic drug resistance and gene amplification in a SEWA mouse tumor cell line : Complex relations revealed by drug uptake data, and lipid and protein analysis

SEWA mouse lines resistant to actinomycin D (AMD) or vincristine (VCR) exhibit the pleiotropic drug resistance (PDR) phenotype, and express a low-MW protein (p21) and numerous double minutes (DM). In drug uptake studies these lines were compared with the non-resistant parental line and with a methotrexate (MTX)-resistant line, not exhibiting PDR. On treatment with labelled AMD or VCR the two PDR lines displayed a highly reduced intracellular content of drug, whereas uptake of MTX was unchanged. Uptake of AMD was shown to be temperature-dependent. The MTX-resistant line did not exhibit any significant change in AMD or VCR uptake. Other workers have emphasized the role of a high-MW glycoprotein in the development of PDR. A search for a similar glycoprotein in our cells was unsuccessful. Since all indications point to membrane factors being important in the development of PDR, the lines were also subjected to lipid analysis. Compared with control cells distinct differences were detected in the lipid composition of all resistant lines (including the MTX-resistant line). In the course of our experiments, the DM in our most AMD-resistant line were replaced by two homogeneously staining regions (HSR). Simultaneously, the overproduction of p21 ceased, but the PDR phenotype persisted. This event tends to implicate a minor role for the p21 protein in PDR, but similar transitions from DM to HSR in other AMD-resistant SEWA lines were not accompanied by a decrease in p21 over-production. Our data point to a complex genetic control of multi-drug resistance.     

Transformation by the oncogene v-fms: The effects of castanospermine on transformation-related parameters

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.     

Smoking,Antioxidant Supplementation and Dietary Intakes among Older Adults with Age-Related Macular Degeneration over 10 Years

We aimed to compare the micronutrient usage and other lifestyle behaviors over 10 years among those with and without age-related macular degeneration (AMD). 1612 participants aged 49+ years at baseline were re-examined over 10 years, west of Sydney, Australia. AMD was assessed from retinal photographs. Dietary data were collected using a semi-quantitative food frequency questionnaire. Smoking status was self-reported. 56 participants had any AMD at baseline, of these 25% quit smoking at 5 years and were still not smoking at 10-year follow-up. Among participants who had below the recommended intake of vitamins A, C or E supplements at baseline, those who did compared to those who did not develop late AMD over 10 years were more likely to report vitamins A (total), C or E supplement intake above the recommended intake at 10-year follow-up: multivariable-adjusted OR 4.21 (95% CI 1.65-10.73); OR 6.52 (95% CI 2.76-15.41); and OR 5.71 (95% CI 2.42-13.51), respectively. Participants with compared to without AMD did not appreciably increase fish, fruit and vegetable consumption and overall diet quality. Adherence to smoking and dietary recommendations was poor among older adults with AMD. However, uptake of antioxidant supplements increased significantly among those with late AMD.     

Effects of Tyrosine Kinase Inhibitors and CXCR4 Antagonist on Tumor Growth and Angiogenesis in Rat Glioma Model: MRI and Protein Analysis Study

The aim of the study was to determine the antiangiogenic efficacy of vatalanib, sunitinib, and AMD3100 in an animal model of human glioblastoma (GBM) by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumor protein expression analysis. Orthotopic GBM-bearing animals were randomly assigned either to control group or vatalanib, sunitinib, and AMD3100 treatment groups. Following 2 weeks of drug treatment, tumor growth and vascular parameters were measured using DCE-MRI. Expression of different angiogenic factors in tumor extracts was measured using a membrane-based human antibody array kit. Tumor angiogenesis and invasion were determined by immunohistochemistry. DCE-MRI showed a significant increase in tumor size after vatalanib treatment. AMD3100-treated group showed a significant decrease in a number of vascular parameters determined by DCE-MRI. AMD3100 significantly decreased the expression of different angiogenic factors compared to sunitinib or vatalanib; however, there were no significant changes in vascular density among the groups. Sunitinib-treated animals showed significantly higher migration of the invasive cells, whereas in both vatalanib- and AMD3100-treated animals the invasive cell migration distance was significantly lower compared to that of control. Vatalanib and sunitinib resulted in suboptimal therapeutic effect, but AMD3100 treatment resulted in a significant reduction in tumor growth, permeability, interstitial space volume, and invasion of tumor cells in an animal model of GBM.     

Effect of recombinant tumor necrosis factor on HL-60 cells: cell-cycle specificity and synergism with actinomycin D

The tumor necrosis factor (TNF) exhibits a multitude of activities depending on the type of target cells. We characterized the cytostatic and cytotoxic effects of recombinant TNF, alone and in combination with actinomycin D (AMD), on the human leukemic cell line HL-60. Because HL-60 cells, when triggered to monocytic differentiation by phorbol esters, are known to produce and secrete TNF, their sensitivity to the factor could indicate an autocrine function of TNF in this cell system. Indeed, HL-60 cells were affected by TNF; their doubling time was increased by about 50% and progression through the cell cycle was perturbed. Initially, (up to 8 h) TNF induced a temporary arrest in G2 while later (24-48 h) it delayed progression through the G1 phase. Also, a transient increase in RNA content peaking at 6-8 h was apparent. The cytotoxicity of TNF alone was low. Thus, TNF may be involved in the regulation of the cell cycle of HL-60 cells during early stages of their differentiation. The cytotoxicity of TNF was markedly potentiated in the presence of AMD; the effect was AMD but not TNF concentration-dependent. Whereas at 20 and 50 ng/ml of AMD alone nonviable cells did not exceed 20% during the first 24 h of treatment, their proportion increased to 80 and 90%, respectively, in the presence of TNF. The most sensitive were cells in the S phase of the cell cycle. The observed synergistic effect of TNF and AMD does not appear to be caused by the action of TNF increasing the permeability of the cell membrane to AMD. The results indicate that HL-60 cells, ordinarily resistant to the cytotoxic action of TNF, can be rendered sensitive by treatment with AMD. This implies that a combination of TNF and AMD may be considered in oncology for treatment of tumors otherwise nonresponding to TNF alone.     

Control of the uptake of amino acids by serum in chick embryo cells,untransformed or transformed with rous sarcoma virus

Summary Forty to fifty minutes after removal of serum, the net total uptake of amino acids in growing secondary cultures of normal or virus-transformed chick embryo cells, stopped or proceeded only at a highly reduced rate. In both normal and transformed cells, theinitial (0–40 min) rate of the above uptake was the same in the absence of serum as in its presence. The initial rate of the total uptake of amino acids in growing transformed cells was about the same as in growing normal cells. Neither in the normal nor in the transformed cells was the rate of the total uptake of amino acids reduced by cell confluence alone. In highly dense, hyperconfluent cultures of normal cells in which cell growth was arrested, the rate of uptake in the absence or in the presence of serum was four- to fivefold lower than the rate obtained in growing normal cells under similar conditions; in the absence of serum, the net uptake stopped after 40 min in the hyperconfluent cultures as well. It appears that cells growing in tissue culture require a serum factor for maintenance of the required high rates of uptake of amino acids and that the inhibition of growth at high cell densities is a result of depletion of this factor from serum, or the inability of the cells in a dense culture to respond to the factor. A serum factor is apparently also required for maintenance of the reduced rates of uptake of amino acids observed in hyperconfluent cultures.     

The pigmentation of human iris influences the uptake and storing of zinc

Age-related macular degeneration (AMD) is more prevalent among the elderly Caucasians than in Africans. A significant association between light iris colour, fundus pigmentation and incidence of AMD is reported, suggesting a possible correlation with melanin pigment. Zinc is known to bind to melanin in pigmented tissues and to enhance antioxidant capacity by function as a cofactor or gene expression factor of antioxidant enzymes in the eye. In this in vitro study, we investigated the uptake and storage of zinc in human irides. Irides of blue and brown human eyes were used. The number of melanocytes was measured. Tissues without any treatment served as controls. The irides were incubated with 100 microM zinc chloride in culture medium for 24 h. Specimens of the tissues were stored for the uptake examination. The remained pieces were further incubated for 3 and 7 d to investigate the storage of zinc. The concentration of zinc was measured by inductively coupled plasma mass spectrometry (ICP-MS). Melanocytes count was significantly higher in the brown tissues (P < 0.0001). Zinc concentration of blue coloured irides after 24 h zinc treatment was close to the controls. We did not observe any significant storing. In contrast, the concentration of zinc in brown irides was significantly increased after 24 h (P < or = 0.01) and remained at a high level for 7 d. The uptake of zinc is likely dependent on the amount of pigmentation in human iris. Therefore, we assume that in patients suffering from AMD the degree of pigmentation of the irides and eventually fundi should be under consideration when the patients are treated with zinc supplementation.     

Mobilization of endogenous stem cell populations enhances fracture healing in a murine femoral fracture model

Background aimsDelivery of bone marrow–derived stem and progenitor cells to the site of injury is an effective strategy to enhance bone healing. An alternate approach is to mobilize endogenous, heterogeneous stem cells that will home to the site of injury. AMD3100 is an antagonist of the chemokine receptor 4 (CXCR4) that rapidly mobilizes stem cell populations into peripheral blood. Our hypothesis was that increasing circulating numbers of stem and progenitor cells using AMD3100 will improve bone fracture healing.MethodsA transverse femoral fracture was induced in C57BL/6 mice, after which they were subcutaneously injected for 3 d with AMD3100 or saline control. Mesenchymal stromal cells, hematopoietic stem and progenitor cells and endothelial progenitor cells in the peripheral blood and bone marrow were evaluated by means of flow cytometry, automated hematology analysis and cell culture 24 h after injection and/or fracture. Healing was assessed up to 84 d after fracture by histomorphometry and micro–computed tomography.ResultsAMD3100 injection resulted in higher numbers of circulating mesenchymal stromal cells, hematopoietic stem cells and endothelial progenitor cells. Micro-computed tomography data demonstrated that the fracture callus was significantly larger compared with the saline controls at day 21 and significantly smaller (remodeled) at day 84. AMD3100-treated mice have a significantly higher bone mineral density than do saline-treated counterparts at day 84.ConclusionsOur data demonstrate that early cell mobilization had significant positive effects on healing throughout the regenerative process. Rapid mobilization of endogenous stem cells could provide an effective alternative strategy to cell transplantation for enhancing tissue regeneration.     

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance in Escherichia coli

Properties of putrescine uptake by PotFGHI and PuuP and their physiological significance were studied using a polyamine biosynthesis and uptake deficient Escherichia coli KK3131 transformed with pACYC184 containing potFGHI or puuP. Putrescine uptake activity of E. coli KK3131 transformed with pACYC184-PotFGHI was higher than that of E. coli 3131 transformed with pACYC-PuuP when cells were cultured in the absence of putrescine. Putrescine uptake by PotFGHI was both ATP and membrane potential dependent, while that by PuuP was membrane potential dependent. Feedback inhibition by polyamines occurred at the PotFGHI uptake system but not at the PuuP uptake system. Expression of PuuP was reduced in the presence of PuuR, a negative regulator for PuuP, and expression of PuuR was positively regulated by glucose, which reduces the level of cAMP. The complex of cAMP and CRP (cAMP receptor protein) inhibited the expression of PuuR in the absence of glucose. Thus, the growth rate of E. coli KK3131 in the presence of both 0.4 % (22.2 mM) glucose and 10 mM putrescine was in the order of cells transformed with pACYC-PotFGHI > pACYC-PuuP > pACYC-PuuP + PuuR, which was parallel with the polyamine content in cells. The results indicate that PotFGHI is necessary for rapid cell growth in the presence of glucose as an energy source. When glucose in medium was depleted, however, PuuP was absolutely necessary for cell growth in the presence of putrescine, because accumulation of putrescine to a high level by PuuP was necessary for utilization of putrescine as an energy source.     

Vegetation and Soil Development in Compost‐Amended Iron Oxide Precipitates at a 50‐Year‐Old Acid Mine Drainage Barrens

Acid mine drainage (AMD) barrens result from destruction of vegetation within AMD flow paths. When exposed to air, soluble iron in AMD undergoes oxidation and hydrolysis to form ferric iron (oxyhydr)oxides which accumulate on soil surfaces. A restoration experiment was conducted at a 50‐year‐old AMD barrens created by discharge from an abandoned underground coal mine. The objective was to determine whether vegetation could be established by altering rather than removing surface layers of acidic precipitates at a site representative of other mining‐degraded areas. Three zones in the barrens were identified based on moisture content, pH (2.7–3.3), and thickness of precipitates (0–35 cm). Our hypothesis was that application of the same reclamation method to all zones would fail to sustain >70% vegetative cover in each zone after four growing seasons. The method consisted of applying 11 t/ha lime and 27 or 54 t/ha compost before rototilling (top 15 cm) and mulching with oat straw containing viable seeds for a nurse crop. Lime‐only plots were included for comparison, and all amended plots were sown with a mine reclamation seed mix. Oats, sown species, and indigenous species dominated cover in the first, second, and fourth growing seasons, respectively. In the fourth year following reclamation, compost‐amended plots had >70% cover and improved soil properties in all three zones, providing evidence to reject our hypothesis. Vegetative restoration of AMD barrens did not require removal of highly acidic precipitates, since they could be transformed at low‐cost into a medium that supports indigenous plants.     

Selective Enhancement of Donor Hematopoietic Cell Engraftment by the CXCR4 Antagonist AMD3100 in a Mouse Transplantation Model

The interaction between stromal cell-derived factor-1 (SDF-1) with CXCR4 chemokine receptors plays an important role in hematopoiesis following hematopoietic stem cell transplantation. We examined the efficacy of post transplant administration of a specific CXCR4 antagonist (AMD3100) in improving animal survival and in enhancing donor hematopoietic cell engraftment using a congeneic mouse transplantation model. AMD3100 was administered subcutaneously at 5 mg/kg body weight 3 times a week beginning at day +2 post-transplant. Post-transplant administration of AMD3100 significantly improves animal survival. AMD3100 reduces pro-inflammatory cytokine/chemokine production. Furthermore, post transplant administration of AMD3100 selectively enhances donor cell engraftment and promotes recovery of all donor cell lineages (myeloid cells, T and B lymphocytes, erythrocytes and platelets). This enhancement results from a combined effect of increased marrow niche availability and greater cell division induced by AMD3100. Our studies shed new lights into the biological roles of SDF-1/CXCR4 interaction in hematopoietic stem cell engraftment following transplantation and in transplant-related mortality. Our results indicate that AMD3100 provides a novel approach for enhancing hematological recovery following transplantation, and will likely benefit patients undergoing transplantation.