Surrogate thrombopoietin

To evaluate the practical value of natural beta-carotene (NbetaC) and to elucidate the apparent discrepancy between epidemiological observations and intervention trials on the role of beta-carotene (betaC) in tumor prevention, the genotoxicity and the antigenotoxicity of NbetaC and synthetic betaC crystal (SbetaCC) stereoisomers were studied comparatively using chromosome aberration analysis and the micronucleus test in human lymphocytes in vitro. NbetaC was extracted from the halotolerant algae Dunaliella salina. The NbetaC crystal (NbetaCC) preparation is about 70% all-trans (TbetaC) and 8% 9-cis (CbetaC). The NbetaC oil (NbetaCO) preparation is about 40% all-trans and 38% 9-cis. SbetaCC is more than 97% all-trans, and the 9-cis can not be detected. The mixture of betaC (betaCM) preparation is 74% SbetaCC and 26% NbetaC. Our results show no genotoxicity of 1-30 microg/ml NbetaCC, but this concentration of NbetaCC inhibited significantly gamma-ray-induced micronucleus formation in human lymphocytes in vitro. One to thirty microg/ml NbetaCO was most effective against both gamma-ray-induced and spontaneous micronucleus formation. However, no influence of NbetaCO on spontaneous chromosome aberrations in human lymphocytes in vitro was observed. NbetaCO suppressed significantly mitomycin C (MMC)-induced chromosome aberrations. One to thirty microg/ml SbetaCC induced a dose-dependent increase in micronucleus frequency, and also inhibited gamma-ray-induced micronucleus formation. No effect of betaCM on spontaneous chromosome aberrations was found. One to thirty microg/ml betaCM is more effective against MMC-induced chromosome aberrations than NbetaCO. These results suggest that CbetaC might play a critical role in the genotoxicity and antigenotoxicity of SbetaCC and NbetaC. The genotoxic activity of SbetaCC might be involved in carcinogenesis. NbetaC or betaCM could be of practical value in tumor prevention and supplementary treatment.     

Evaluation of 9-cis retinoic acid for a new remedy of human retinoblastoma

We investigated the effect of two isomers of retinoic acid (RA), all-trans RA and 9-cis RA, on the proliferation of Y79 human retinoblastoma cells. The two isomers inhibited the cell proliferation in a concentration-dependent manner. The IC50 for this inhibition by all-trans RA and 9-cis RA was 1.50 and 0.15 microM, respectively. The inhibitory effect of 9-cis RA on Y79 cell growth was observed within 24 hr, thereafter the cell number was gradually decreased. In contrast, no inhibition by all-trans RA of Y79 cell growth was observed within 24 hr, thereafter the cell number was slightly increased. In these cases, the cell viability at 4 days after the addition of 9-cis RA and all-trans RA was more than 90% and 95%, respectively. These results indicate that the two RA inhibit the proliferation of Y79 human retinoblastoma cells without inducing the cell death and that the effect of 9-cis RA on the inhibition of Y79 cell growth is much greater than that of all-trans RA.     

Gene expression and neuroblastoma cell differentiation in response to retinoic acid: differential effects of 9-cis and all-trans retinoic acid

Retinoic acid has considerable potential for the chemoprevention and chemotherapy of cancer. Neuroblastoma cells differentiate in response to retinoic acid in vitro, an observation that has led to clinical trials using either the 13-cis or all-trans isomers of retinoic acid. We review the effects of retinoic acid on neuroblastoma, and the potential involvement of nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). 9-cis retinoic acid is a ligand for RXRs, and we review recent data on the differential effects of 9-cis and all-trans retinoic acid on neuroblastoma differentiation and proliferation in vitro, and possible mechanisms of action via hetero- and homodimers of RARs and RXRs. Although there is uncertainty whether or not 9-cis retinoic acid produces its biological effects primarily via RXR homodimers, in vitro data suggest that this isomer of retinoic acid or stable analogues may have considerable potential for the treatment of resistant, disseminated neuroblastoma.     

A critical role for transforming growth factor-beta in donor transfusion-induced allograft tolerance

To evaluate the relationship between carotenoid concentrations in serum and breast tissue, we measured serum carotenoid concentrations and endogenous carotenoid levels in breast adipose tissue of women with benign breast tumor (n = 46) or breast cancer (n = 44). Before extraction, serum was digested with lipase and cholesterol esterase, and breast adipose tissue was saponified. Serum and tissue carotenoids were extracted with ether/hexane and measured by using HPLC with a C30 column. Serum retinoic acid was extracted with chloroform/methanol and measured using HPLC with a C18 column. There were no significant differences in serum carotenoids [lutein, zeaxanthin, cryptoxanthin (both alpha- and beta-), alpha-carotene, all-trans beta-carotene, 13-cis beta-carotene and lycopene], retinoids (retinol, all-trans and 13-cis retinoic acids), and alpha- and gamma- tocopherol concentrations between benign breast tumor patients and breast cancer patients. A substantial amount of 9-cis beta-carotene was present in adipose tissue and was the only carotenoid that had a significantly lower level in benign breast tumor patients than in breast cancer patients. Correlations between carotenoid concentrations in serum and in breast adipose tissue were determined by combining the data of the two groups. Concentrations of the major serum carotenoids except cryptoxanthin showed significant correlations with breast adipose tissue carotenoid levels. When the concentrations of serum carotenoids were adjusted for serum triglycerides or LDL, correlations between serum carotenoid concentrations and breast adipose tissue carotenoid levels markedly increased, including that of cryptoxanthin (P <0. 001). The strong correlation between serum carotenoid concentrations and endogenous breast adipose tissue carotenoid levels indicate that dietary intake influences adipose tissue carotenoid levels as well as serum concentrations, and that adipose tissue is a dynamic reservoir of fat-soluble nutrients.     

Growth of single HL60 cells in liquid culture: analysis of the influences of differentiative agents

Treatment of serum-free grown HL60 cells with certain combined amounts of retinoic acid (9-cis or all-trans RA) and 1 alpha 25 dihydroxyvitamin D3 (D3) results in differentiation of 71-77% of cells towards either neutrophils or monocytes. Studies of the differentiation of HL60 cells in flask cultures does not reveal: (i) the extent to which selective growth of cells might have occurred; and (ii) the overall level of cell survival. This information can be obtained by monitoring the effects of differentiative agents on individual cells. Serum-free grown HL60 cells were cultured as single cells in microtitre wells in conditioned medium obtained from exponentially growing and serum-free cultures of HL60. This resulted in a cloning efficiency of 85% and HL60 cells doubled every 24 h. During a period of exponential growth < 0.5 to 2% of the cells generated died. Single HL60 cells were treated with 9-cis and all-trans RA (5 x 10(-7) M) together with a small amount of D3 (3.9 x 10(-14) M) to promote neutrophil differentiation. D3 alone (10(-7) M) and D3 (5 x 10(-9) M) in combination with 9-cis RA (10(-8) M) were used to promote monocyte differentiation. The growth kinetics of HL60 cell cultures that were differentiating to neutrophils and to monocytes were similar. Single-cell experiments have revealed that: (i) differentiating HL60 cells undergo a variable number of divisions (two to five) prior to arresting their growth; and (ii) up to 33% of the cells that are generated (by day 5) die. Seventy to eighty per cent of the cells in each of the wells had matured. These findings have important implications in regard to whether retinoids and D3 provide signals that determine the choice of maturation pathway or that merely facilitate selective survival and/or expansion of cells that have independently determined their differentiation fates.     

Protection against UVA damage and effects on neutrophil-derived reactive oxygen species by beta-carotene

OBJECTIVE: Phagocyte-derived reactive oxygen species (ROS) are involved in microbicidal activities as well as in tissue damage at sites of inflammation. Carotenoids play an important function in protecting cells from oxidant damage. We investigated the in vitro and in vivo effect of 13-cis and 9-cis-beta-carotene on human neutrophils. METHODS: Neutrophils from healthy donors in the presence of 0.25 mumol/L-1 mumol/l beta-carotene and from subjects under beta-carotene supplementation and UVA or UVA/B exposure were stimulated by opsonized zymosan and the generation of ROS was measured by electron spin resonance spectroscopy. RESULTS: Our in vitro results show different effects of the two isomers on stimulated neutrophils. 9-cis-beta-carotene did not produce any change, whereas 13-cis-beta-carotene significantly and concentration-dependent inhibited the ROS generation by stimulated neutrophils. Further, in a controlled study, we were able to demonstrate an in vivo protective effect of beta-carotene on neutrophils against UVA damage by beta-carotene supplemented subjects.     

Training in pregnant women: effects on fetal development and birth

Vitamin A (VA) deficiency is the leading cause of blindness in children in developing countries. Dietary intervention with foods rich in provitamin A carotenoids, such as beta-carotene (betaC), has been suggested as one solution to this problem. The objective of the two studies described in this paper was to examine the utilization of betaC as a source of VA at different stages of VA depletion using the Mongolian gerbil as a model. Male 4- to 5-wk-old Mongolian gerbils were fed powdered betaC-free semipurified diets either with or without VA for 26 d (Study 1), or without VA for 8-10 wk (Study 2). Gerbils were then fed diets with or without VA (20.9 nmol/g diet) and/or betaC [(67.0 micromol/g diet (Study 1) and 145.9 micromol/g diet (Study 2)] for variable periods. Two (Study 1) or three (Study 2) days before termination of the study, 3-4 gerbils per group were dosed orally with 14C-betaC. Tissues were evaluated for VA and betaC content by HPLC. Liver was extracted with and without saponification to evaluate 14C-betaC and 14C-VA content. The results demonstrate the following: 1) the gerbil is an appropriate animal model to study betaC utilization; 2) 20.9 nmol VA/g diet is more than sufficient for this species; 3) the daily VA utilization rate for this species is calculated to be 3.1 microg/100 g body weight; 4) a highly bioavailable source of betaC at a 6:1 weight ratio of betaC:VA is sufficient to reverse marginal VA status in this model; and 5) a highly bioavailable source of betaC fed between a 6:1 and 13:1 weight ratio to VA provides equivalent VA status as preformed VA in Mongolian gerbils.     

Divergent pathways in photobleaching of 7,9-dicis-rhodopsin and 9,11-dicis-12-fluororhodopsin: one-photon-two-bond and one-photon-one-bond isomerization

Through low-temperature photochemistry, UV/vis spectroscopy, and chromophore extraction experiments, we have established that 7,9-dicis-rhodopsin undergoes one-photon-two-bond photoisomerization to a batho intermediate (its absorption maximum is slightly blue shifted from that of bathorhodopsin) containing the all-trans geometry, while 9,11-dicis-12-fluororhodopsin undergoes one-photon-one-bond isomerization to the corresponding 9-cis isomer and then the all-trans batho intermediate. The difference in the photochemical properties of the two dicis pigment analogs was rationalized by possible local protein perturbation, lability of the 11-cis geometry, and photochemical properties of the chromophores.     

Specific-heat critical behavior of the structural random-field system Dy(AsxV1-x)O4

Vitamin D3 derivative 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) exerts various biological effects in cells that possess vitamin D3 receptor (VDR), including enhancement of cell differentiation and inhibition of cell proliferation. These activities of 1,25(OH)2D3 might be responsible for its anti-neoplastic effects, as shown in various experimental systems. The aim of this study was to compare the anti-angiogenic activity of 1,25(OH)2D3, retinoids, and interleukin-12 (IL-12) in an experimental tumor cell-induced angiogenesis assay in mice. Tumor cell-induced angiogenesis assay was performed in x-ray immunosuppressed BALB/c mice by intradermal injections of human tumor cell lines of different origin. The injections resulted within 3 d in a local formation of new blood vessels, and the intensity of angiogenesis correlated with the number of injected cells. Systemic treatment of the mouse recipients with 1,25(OH)2D3 significantly decreased angiogenesis, comparable to the effect of retinoids (all-trans retinoic acid [RA], 9-cis RA and 13-cis RA) and of IL-12. In vitro preincubation of the cells with all compounds (except IL-12) led to the inhibition of their angiogenic capability in vivo. Moreover, combination of 1,25(OH)2D3 and retinoids resulted in a synergistic inhibition of angiogenesis. The results strongly suggest that anti-angiogenic compounds with relatively low toxicity (e.g., 1,25(OH)2D3, retinoids, and IL-12) and their combinations could be beneficial in the treatment of some angiogenesis-associated malignancies.     

Effects of light adaptation on the purple membrane structure of Halobacterium halobium

Absorption, circular dichroism and optical rotatory dispersion of the bacteriorhodopsin containing purple membrane form Halobacterium halobium were studied in regard to the structural stability of this membrane during the photoisomerization of the retinal of the bacteriorhodopsin from the 13-cis to the all-trans configuration. The following conclusions were reached: (a) the macromolecular structure (protein-protein interaction which may result in the possible exciton interaction of the retinal pi-pi* (NV1) transition moments and protein-lipid interaction) are not significantly altered, (b) possibilities of delocalized conformation changes of the apoprotein involving secondary and/or tertiary structure can be ruled out, (c) localized secondary structure conformation changes of the apoprotein must be limited to the involvement of no more than one or two amino acid residues and localized tertiary structure conformation changes of the apoprotein must be limited to a very short segment of the protein chain containing only a few aromatic amino acid residues, and (d) the interaction between the apoprotein and retinal seems to be relatively more pronounced when the retinal is in the all-trans form than the 13-cis from and also the apoprotein seems to impose a more pronounced dissymmetric constraint on the retinal in the all-trans form than in the 13-cis form.     

TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor

We investigated the potential for 9-cis-retinoic acid in the differentiation therapy of neuroblastoma using an N-type neuroblastoma cell line, SH SY 5Y, as an experimental model. In these cells, 9-cis-retinoic acid is more effective than other isomers at inducing the expression of RAR-beta. An RAR-alpha-specific antagonist inhibited the induction of RAR-beta in response to all-trans-but not to 9-cis-retinoic acid. This indicates that the mechanism of gene induction by 9-cis-retinoic acid differs markedly from all-trans-retinoic acid. 9-cis-retinoic acid is also better than all-trans at producing sustained morphological differentiation and inhibition of proliferation of SH SY 5Y cells. Although N-type neuroblastoma cells are not thought to undergo apoptosis in response to all-trans-retinoic acid, we observed a significant degree of apoptosis in SH SY 5Y cells treated with 9-cis-retinoic acid for 5 days and then cultured in the absence of retinoid, an effect not observed in cells treated with the all-trans isomer. These results suggest that 9-cis- and all-trans-retinoic acid have distinct biological properties and that 9-cis retinoic acid may be clinically effective in neuroblastoma by inducing both differentiation and apoptosis under an appropriate treatment regimen.     

The role of vitamin D derivatives and retinoids in the differentiation of human leukaemia cells

The capabilities of 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3), and two novel vitamin D analogues, EB1089 and KH1060, to induce the differentiation of two established leukaemia cell lines, U937 and HL-60, were assessed alone or in combination with the retinoid compounds, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (ATRA). The vitamin D derivatives acted to increase the differentiation of U937 and HL-60 cell cultures in a dose-dependent manner, as determined by nitroblue tetrazolium (NBT) reduction, with EB1089 and KH1060 being more effective than the native hormone. As an additional index of leukaemic cell differentiation, induction of expression of the phenotypic cell surface antigen, CD14, and the beta2-integrins, CD11b and CD18 by the vitamin D and retinoid compounds were monitored using fluorescence activated cell sorting (FACS) analyses. Following 96-hr treatment of U937 and HL-60 cells with 5 x 10(-10) M of the vitamin D derivatives, a striking increase in CD14 antigen expression was apparent, indicating the promotion by these compounds of a monocyte/macrophage lineage of cells. CD11b and CD18 antigen expression were also raised above control levels. In contrast, both retinoid compounds used at the higher concentration of 1 x 10(-8) M were not effective inducers of CD14 antigen expression. However, CD11b and CD18 were both readily increased in U937 and HL-60 cell cultures. Treatment of U937 cell cultures with the vitamin D compounds and the retinoids resulted in cooperative effects on induction of differentiation, with correlation by both NBT reduction and FACS analyses of CD14 antigen expression. The presence of 9-cis RA or ATRA appeared to contribute to the further increase of CD14 in these cells. HL-60 cell cotreatment with these compounds also displayed enhanced cooperative effects in phagocytic function by NBT reduction. However, analysis of CD14 revealed a dramatic diminution in HL-60 cells treated with the combinations of the vitamin D derivatives and the retinoids. Assessment of HL-60 cell morphology treated with these combinations demonstrated the presence of a mixed population of monocytes and granulocytes. CD11b and CD18 antigen expression was also enhanced in both cell lines with cotreatment. The ability of EB1089 and KH1060 to induce leukaemic cell differentiation may provide an additional option for therapeutic use alone or together with other differentiation agents such as 9-cis RA or ATRA.     

An analysis of the effects of retinoic acid and other retinoids on the development of adrenergic cells from the avian neural crest

In the present work, we have investigated the role of all-trans-retinoic acid (all-trans RA), and several other natural and synthetic retinoids, in the development of adrenergic cells in quail neural crest cultures. Dose response studies using all-trans RA and 13-cis RA revealed a dose-dependent increase in the number of adrenergic cells in neural crest cultures. Similar dose response studies using RA isomers and other natural retinoids did not result in the same increases. In order to determine the receptor mediating the effects of all-trans RA in the neural crest, we tested several synthetic analogs which specifically bind to a particular RA receptor (RAR) subtype. We found that the compound AM 580, which activates the RAR-alpha, produced an increase in adrenergic cells similar to that seen with all-trans RA. The compound TTNPB, which activates all RAR subtypes, also resulted in an increase in adrenergic cells. We conclude that the increase in adrenergic cells seen with all-trans RA is mediated by RAR-alpha and possibly RAR-beta. To further define the actions of all-trans RA on the neural crest we incubated cultures with 5-bromo-2'-deoxyuridine (BrdU) to determine whether all-trans RA could affect the rate of proliferation. The results show that while all-trans RA did not increase the fraction of cells incorporating BrdU into their nuclei at early time points (24 h), it did increase BrdU incorporation by tyrosine hydroxylase (TH) positive cells at 5 days in culture. These findings demonstrate that the increase in adrenergic cells seen with all-trans RA in neural crest cultures is likely due to an increase in the proliferation of cells already expressing TH.     

Enhancing communication with the Passy-Muir valve

Effective copper and cadmium concentrations which limited the growth of two chlorophytes by 50%, EC(50)s, after 96 h of static exposure were determined. EC(50)s were 5.94 microM copper and 4.55 microM cadmium for Dunaliella salina, and 0.78 microM copper and 0.025 microM cadmium for Chlamydomonas bullosa. The relationship of the two cations was synergistic towards the growth of both species. Chronic exposure to 4.5 x 10(-6) microM cadmium or 4.9 x 10(-4) microM copper increased the sensitivity of C. bullosa by 26% and 29% towards cadmium and copper, respectively. Changes in co-tolerance were not observed. Cd-treated D. salina was 50% more tolerant towards this cation, whereas Cu-treated cultures showed extreme sensitivity towards copper and "co-sensitivity" towards cadmium. Furthermore, the phylogenetic hypothesis, predictive of toxic response, failed to hold at the familial level.     

A case cohort study of suicide in relation to exposure to electric and magnetic fields among electrical utility workers

BACKGROUND: All-trans retinoic acid is currently used in clinical trials in combination with tamoxifen to treat breast cancer, and 13-cis retinoic acid is used with a-interferon to treat metastatic endometrial cancer. We examined the effects of all-trans retinoic acid and 13-cis RA alone on endometrial adenocarcinoma (RL95-2) cells to investigate the cell biological mechanisms by which retinoic acid may reduce the metastatic phenotype and induce differentiation. METHODS: RL95-2 cells were seeded onto 4-chamber plastic slides and treated with 13-cis retinoic acid or all-trans retinoic at 0.5 microM, 1 microM and 5 microM doses for 90 minutes at 37 degrees C and stained for F-actin. RESULTS: Untreated RL95-2 cells exhibited staining of disrupted aggregates of F-actin only near the cell periphery. Cells treated with the three doses of 13-cis retinoic acid exhibited a dramatic reorganization of F-actin throughout the cells. When cells were treated with 0.5 microM all-trans retinoic acid, actin filaments reorganized. Cells treated with 1 microM all-trans retinoic acid and 5 microM all-trans retinoic acid displayed increased organization of F-actin and cell size increased. The percentage of S-phase cells increased at the high doses of retinoic acid treatment. This effect was apparently transient, since retinoic acid did not significantly affect cell growth. CONCLUSION: An organized cytoskeleton and an increase in cell size are associated with differentiation. We suggest that retinoic acid exerts its effects on these transformed cells by reorganizing actin filaments, and inducing differentiation, thus inducing a more stationary phenotype.     

Apoptosis induction of POS canine osteosarcoma cells by vitamin D and retinoids

Vitamin D3: 1-alpha, 25(OH)2D3 (calcitriol), 22-oxa-1,25(OH)2D3 (OCT), cholecalciferol (vitamin D3), and retinoids: all-trans retinoic acid (ATRA) and 9-cis retinoic acid, induced morphological changes in POS canine osteosarcoma cells into elongated, spindle or fibroblast like-shaped cells, and apoptotic like cell death characterized by cell shrinkage, condensation and margination of the nucleus for all drugs at 10(-6)M-10(-9)M after 72 to 120 hr culture. Apoptosis as shown by DNA laddering was induced at 48 hr by all drugs at 10(-6)M, 10(-7)M at 96 hr, 10(-8)M and 10(-9)M at 120 hr respectively. These vitamins are suggested to adjunct antineoplastic agents in canine osteosarcoma therapy by induction of apoptosis.