Internalization and processing of the EGF receptor in the induction of DNA synthesis in cultured fibroblasts: the endocytic activation hypothesis.

The addition of EGF to cultured murine 3T3 cells produces a decrease in EGF binding activity with concomitant internalization and degradation of the initially bound EGF. When the EGF receptor on cultured 3T3 cells is affinity labeled with high specific activity 125I-EGF, and the fate of the affinity labeled EGF-receptor complex determined, the loss in binding activity was accounted for by receptor internalization and subsequent proteolytic processing of the EGF receptor molecules in the lysosomes. Studies of the effects of EGF concentration on EGF binding by cells, EGF-induced receptor internalization and EGF-induced stimulation of 3H-thymidine uptake into cellular DNA show that there is a direct correlation between EGF-induced receptor internalization and EGF-induced stimulation of DNA synthesis, but not between EGF binding and EGF-induced stimulation of DNA synthesis. This correlation is lost at high EGF concentrations, where stimulation of DNA synthesis is suboptimal. Optimal stimulation of DNA synthesis requires a minimum of 6 h of incubation of EGF with cells, and the suboptimal stimulation of DNA synthesis at high EGF concentration is intensified when the period of incubation of EGF with cells is less than 6 h. These data are consistent with a model of hormone signal transmission by Endocytic Activation, wherein the activation of EGF-induced processes requires constant EGF-induced internalization of receptor for a requisite 6-8 h period as an obligatory step in production of "second messenger" in the action of this hormone.     

Effects of glucocorticoids on deoxyribonucleic acid (DNA) synthesis stimulated by growth factors in cultured rat skin fibroblasts

The effects of glucocorticoids on deoxyribonucleic acid (DNA) synthesis were studied by using confluent cultured rat skin fibroblasts prepared by enzymatic dispersion and expanded up to passage 3. Dexamethasone caused the inhibition of the DNA synthesis stimulated by 10% fetal calf serum (FCS) in a dose dependent manner. Maximum inhibition (90%-100%) was achieved by the concentration of 10(-7)M. A similar dose dependent inhibition was also obtained in the experiment using epidermal growth factor (EGF) (1 ng/ml) as a stimulant. Dexamethasone (10(-7)M) also inhibited the DNA synthesis stimulated by somatomedin C (100 ng/ml) or platelet derived growth factor (1 half-maximum unit/ml) almost to control levels. Binding studies with 125I-labeled EGF suggested that dexamethasone caused this inhibitory action without modulation of cell surface receptors for EGF. Furthermore, the effects of a variety of glucocorticoids on the DNA synthesis were studied to clarify the structural requirement of glucocorticoids for the inhibition of the DNA synthesis. The results showed that 11 beta-hydroxyl and 21-hydroxyl groups on the steroid nucleus were necessary for the inhibition of the growth factor-stimulated DNA synthesis. Meanwhile, the inhibitory action on the DNA synthesis was markedly diminished by the replacement of a 16 alpha-methyl group by a 16 beta-methyl group in the presence of a bulky group at C-17 (e.g. 17 alpha-valerate). For further elucidation of mechanisms of action of glucocorticoids on the inhibition of the growth factor-stimulated DNA synthesis, the relationships between the structural features of glucocorticoids and their binding ability to the glucocorticoid receptor ([3H]-dexamethasone-binding receptor) were studied.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival, excision repair and inhibition of DNA synthesis in normal human skin fibroblasts exposed to simulated sunlight.

The responses of normal human skin fibroblasts exposed to simulated sunlight produced by a solar simulator were examined. The parameters investigated were cellular survival, excision repair and the inhibition and recovery of DNA synthesis. The latter two effects were examined using the bromodeoxyuridine photolysis assay and the alkaline step elution assay respectively. The results of these experiments are consistent with the conclusion that the lesions induced by simulated sunlight represent a mixture of damage which elicits cellular responses and repair mechanisms similar to those manifested by cells irradiated with UVC and UVA radiation.     

Effects of diazepam administration on melatonin synthesis in the rat pineal gland in vivo.

The effect of diazepam (DZP) on melatonin synthesis in rat pineal gland was investigated in vivo. Subcutaneous injection of DZP (3 mg/kg) 1 h before the start of darkness significantly suppressed nocturnal elevations of pineal N-acetylserotonin (NAS) and melatonin contents in rats, and caused a 2-h delay in reaching the maximum melatonin level in the dark phase. DZP treatment also markedly suppressed the dark-induced increase of pineal N-acetyltransferase activity, which catalyzes the rate-limiting step in melatonin synthesis, but had no effect on hydroxyindole-O-methyltransferase activity, which catalyzes the final step of melatonin formation. Pineal norepinephrine and dopamine contents, in contrast, were not altered by DZP injection. The distribution rate of DZP to the brain reached the highest level 30 min after a single injection, while that to the pineal gland was observed 5 h later (i.e., 4 h after the start of darkness). It is clear that the inhibitory effect of DZP on melatonin synthesis in rat pineal gland appears concomitantly with the increase in the distribution volume of DZP into this gland. These results suggest that the inhibitory effect of DZP on melatonin synthesis results from the drug's direct action on the rat pineal gland.     

助剂Cu、K对F-T合成铁基催化剂作用的表征研究

采用连续共沉淀和喷雾干燥技术相结合的方法制备了一组Cu、K助剂单独或同时加入的微球状Fischer-Tropsch（F-T）合成铁基催化剂,借助低温N2吸附、MES、XRD、H2-TPR、CO-TPR研究了Cu和K助剂对催化剂织构、还原性能以及还原和炭化过程中的物相变化的影响。结果表明,K助剂的加入能明显提高催化剂的比表面积和铁物相在催化剂中的分散程度,增加了Fe2O3与SiO2间的相互作用;当催化剂在H2和合成气中还原时,Cu助剂的加入有利于催化剂的还原和Fe3O4的生成,在CO中还原时,Cu助剂的加入则有利于α-Fe的生成和稳定化。在H2和合成气中,单独K助剂的加入会抑制催化剂的还原或炭化,而Cu和K助剂的同时加入在H2、CO和合成气下均可使催化剂的还原或炭化能力明显提高,表明Cu和K助剂间存在一定的协同作用。     

DNA synthesis in cultured human fibroblasts: regulation by 3':5'-cyclic AMP.

The addition of serum to density-inhibited human fibroblast cultures induced a wave of DNA synthesis, measured as [3H] thymidine incorporation into acid-precipitable material, beginning after 8-12 hr and reaching maximum levels of 16-24 hr. Addition of dibutyryl-3':5'-cyclic AMP (DBcAMP) together with serum inhibited [3H] thymidine incorporation by 75-95%. When DBcAMP was added for the first 4 hr of serum stimulation and then removed, the wave of DNA synthesis was not delayed. This suggested that serum could induce DNA synthesis even though cyclic AMP concentrations were maintained at high levels by DBcAMP during this initial period. These results are inconsistent with the hypothesis that it is the immediate transient reduction in 3':5'-cyclic AMP concentration following the addition of serum that triggers DNA synthesis. By contrast, DBcAMP added 8 hr after serum inhibited [3H] thymidine incorporation to the same extent as DBcAMP added at the same time as serum. This indicated that a step essential for DNA synthesis and occurring late in G1 was inhibited by high concentrations of 3':5'-cyclic AMP.     

Effects of process parameters on ultrafine SiC synthesis using induction plasmas

A study is reported of the formation of ultrafine SiC powder through the reaction of elemental silicon and CH₄ in an induction plasma. The reaction route used involved in the first place the vaporization of a fine elemental silicon powder axially injected into the center of the discharge followed by the carburization reaction through the coinjection of CH₄. The powder obtained was composed of a mixture of α- and β-SiC with varying amounts of free carbon and free silicon. The particle size distribution was typically in the range of 40–60 nm with a corresponding specific surface area of 30–50 m²/g. A parametric study showed that the quality of the powder obtained varied with the plasma plate power and the position of the injection probe. The plasma gas composition employed was found to influence the proportions of α- and β-SiC in the synthesized SiC powder. With an Ar/N₂ mixture as the plasma gas, the ratio of the α to β phases was less than 1.0, whereas the ratio was greater than 1.5 when using a mixture of Ar/H₂ as plasma gas. The Si powder feed rate and the input C/Si molar ratio in the injected reactants significantly affected both the formation of the SiC and the free Si and free C content in the synthesized powder. Lining the cylindrical reactor wall with graphite resulted in improved conversion of Si to SiC. The weight fraction of the powder collected at different sections of the reactor system varied with the reactor operating conditions. The experimental results support the view that the formation mechanism for ultrafine SiC is dominated by the reaction of Si vapor with the thermal decomposition products of CH₄.     

Exacerbating effect of vitamin E supplementation on DNA damage induced in cultured human normal fibroblasts by UVA radiation

The effects of vitamin E supplementation were evaluated in cultured human normal fibroblasts exposed to ultraviolet A radiation (320-380 nm) (UVA). Cells were incubated in medium containing alpha-tocopherol, alpha-tocopherol acetate or the synthetic analog Trolox for 24 h prior to UVA exposure. DNA damage in the form of frank breaks and alkali-labile sites, collectively termed single-strand breaks (SSB), was assayed by the technique of single cell gel electrophoresis (comet assay), immediately following irradiation or after different repair periods. The generation of hydrogen peroxide (H2O2) and superoxide ion (O2.-) was measured by flow cytometry through the oxidation of indicators into fluorescent dyes. It was observed that pretreatment of cells with any form of vitamin E resulted in an increased susceptibility to the photoinduction of DNA SSB and in a longer persistence of damage, whereas no significant change was observed in the production of H2O2 and O2.- reactive oxygen species, compared to untreated controls. These findings indicate that in human normal fibroblasts, exogenously added vitamin E exerts a promoting activity on DNA damage upon UVA irradiation and might lead to increased cytotoxic and mutagenic risks.     

Extreme dark cytotoxicity of Nile Blue A in normal human fibroblasts.

Early reports using mouse models indicated that Nile Blue A (NBA) is taken up more efficiently by tumor cells than normal tissue and retards tumor growth. NBA also shows both dark toxicity and phototoxicity of human tumor cells in vitro. However, studies on the dark toxicity of NBA and the effects of NBA-mediated photodynamic treatment in normal human cells are lacking. In the current study we have examined the cytotoxicity of NBA in normal human fibroblasts, spontaneously immortalized Li-Fraumeni Syndrome (LFS) cells and three different human tumor cell lines. The normal human fibroblasts showed extreme sensitivity to NBA compared with LFS cells and the human tumor cell lines. Treatment with 0.1 microgram/mL of NBA for 1 h reduced the colony formation of normal human fibroblasts by greater than 95%, but had no significant effect on the colony formation of LFS cells. No significant numbers of apoptotic cells were detected in either normal human fibroblasts or LFS cells following this drug concentration. Thus, unlike photodynamic therapy with some other photosensitizers, the dark toxicity of NBA was not caused by apoptosis. Although the drug uptake was higher in normal human fibroblasts compared with LFS cells, the difference in sensitivity between normal human fibroblasts and LFS cells could not be accounted for by the difference in drug uptake alone. In addition, we could not detect any significant photocytotoxic effect of NBA in either normal human fibroblasts or LFS cells for a drug concentration of 0.05 microgram/mL at light exposures of up to 6.7 J/cm2. These data indicate an extreme sensitivity of normal human fibroblasts to NBA and an inability to produce a significant photocytotoxic effect on human cells using NBA concentrations that have relatively low toxicity for normal human fibroblasts.     

Effects of adrenergic blockers or bicuculline on diazepam induced changes in rat pineal melatonin synthesis in vivo and in vitro.

The effect of propranolol (PPL), phenoxybenzamine (PBZ) or bicuculline (BCL) on the diazepam (DZP)-induced changes of pineal melatonin synthesis in male rats was examined in vivo and in vitro. Administration of PBZ did not affect the inhibitory action of DZP on pineal melatonin synthesis in vivo. A single injection of PPL inhibited the pineal melatonin synthesis similarly to the administration of DZP alone, but the two drugs together did not exhibit additive or synergistic effects on the melatonin synthesis. Significant decreases in the N-acetyltransferase (NAT) activity and the N-acetylserotonin (NAS) and melatonin contents were observed in the BCL-injected group, being greater than those in the DZP-treated group. Unexpectedly, however, the combination treatment of DZP and BCL causes an increase in the NAT activity and melatonin content compared with the BCL-alone group. Incubation with DZP at higher concentrations resulted in an increase of pineal NAT activity in vitro, but this increase was inhibited by preincubation with PPL, PBZ or BCL. DZP treatment thus appeared to have different effects on pineal NAT activity in vivo and in vitro. These results suggest that both a GABAergic mechanism and peripheral benzodiazepine (BZP) receptors in rat pineal gland may be involved in the modulation of melatonin synthesis by DZP.     

DNA damage and repair in Gorlin syndrome and normal fibroblasts after aminolevulinic acid photodynamic therapy: a comet assay study

Using normal, untransformed, human fibroblasts, the effectiveness of aminolevulinic (ALA)-mediated photodynamic therapy (PDT) was investigated in terms of both clonogenic survival and DNA damage. The response of normal fibroblasts was then compared with Gorlin syndrome-derived fibroblasts (basal cell nevus syndrome [BCNS]). In terms of clonogenic survival, no significant differences were observed between the two groups of cells. Using the alkaline comet assay, initial DNA damage after PDT was measured. Some DNA damage was detected at higher doses, but this was fully repaired within 24 h of treatment. The BCNS-derived cells showed levels of initial damage that did not differ significantly from normal lines.     

Induction of DNA strand breaks and DNA-protein cross-links in normal human skin fibroblasts following exposure to 254 nm UV radiation

Levels of DNA strand breaks and DNA-protein cross-links (DPCs) were measured using the alkaline elution assay in normal human skin fibroblasts irradiated with 0-200 J m-2 of 254 nm UV radiation and incubated for 0-24 h. On incubation, the yields of both single-strand breaks (SSBs) and DPCs increased with similar kinetics and remained elevated. In addition, when SSBs were measured under conditions in which DPCs were not eliminated by treatment with proteinase K, a measurable yield of SSBs could not be detected. Hence, the SSBs that form in the UV-irradiated cells following incubation appear to be associated with the DPCs.     

The influence of photodynamic therapy on the ultrastructure of the normal rat bladder.

Reduced bladder capacity is a major side effect for patients receiving photodynamic therapy (PDT) for bladder cancer. A rat bladder model has been developed to address both the vascular and tissue effects of the photodynamic treatment of the urinary bladder. Bladders were exteriorized and positioned in a plexiglass tissue bath. Effects on microvasculature were assessed during PDT of the bladder by recording luminal diameter changes in arterioles and venules. Animals receiving Photofrin II (10 mg kg-1) 30 min prior to PDT scored a statistically significant reduction in the diameter of the red blood cell column in the vessels, whereas administration of Photofrin II 48 h prior to PDT was ineffective. Morphological changes included significant endothelial and vascular myocyte damage in the 30 min PDT group alone. Among the other tissue components, the mucosal lining was minimally affected and the response of the muscularis was highly variable. Smooth muscle cell changes ranged from mild contraction to frank necrosis with many of the affected cells located near the altered vascular beds. These data suggest that the clinical symptoms of reduced bladder capacity can be accounted for by vascular damage and myocyte sensitivity. Further refinements in the Photofrin II and light doses used in therapy may reduce bladder complications and allow for better management of bladder cancer.     

Synergistic stimulation of early events and DNA synthesis by phorbol esters, polypeptide growth factors, and retinoids in cultured fibroblasts

12-O-Tetradecanoyl-phorbol-13-acetate (TPA), in the absence of serum, acts synergistically with a range of polypeptide growth factors to stimulate DNA synthesis in quiescent Swiss 3T3 cells. These growth factors include epidermal growth factor (EGF), insulin, and the peptide produced by BHK cells transformed by SV-40 virus (fibroblast-derived growth factor, FDGF). Retinoids also show mitogenic synergism with TPA or polypeptide growth factors. The spectrum of mitogenic synergisms displayed by TPA are similar to those of vasopressin, a pituitary peptide. However, TPA and vasopressin do not synergistically interact to stimulate DNA synthesis in quiescent 3T3 cells. This suggests that TPA and vasopressin act via an identical biochemical pathway. Several lines of evidence suggest rapid postreceptor convergence of the mitogenic mechanisms of action of the hormone and the tumor promotor. Thus, vasopressin and TPA both inhibit EGF binding to cellular receptors. Furthermore, TPA and vasopressin induce a similar array of early events in quiescent cells--most strikingly, identical stimulation of Rb+ influx. Stimulation of ion flux is suggested as the possible convergence point of the pathway by which TPA and vasopressin act as mitogens.     

Study on the structure-activity relationships of phoxalone and induction of apoptosis in H446 tumor cells

A novel macrolide with dioxa and double ring pentadecanone, named as phoxalone, isolated from the culture broth of a myxobacterium Sorangium cellulosum WXNXJ-C, was well investigated. The chemical structure of Phoxalone was elucidated by spectroscopic analysis including UV, IR, (1)H NMR, MALDI-TOF-MS, and LC/MS spectra. The results of cytotoxic bioactivities on human non-small lung cancer H446, in vitro, showed that not only did phoxalone express higher antitumor bioactivity than many antitumor drugs, but it also displayed less cytotoxicity to normal human liver L02 cell lines (IC(50), 286 microg mL(-1)). Further cytotoxic bioactivity study indicated that phoxalone could induce H446 cell line apoptosis in vitro. More investigation results of flow-cytometric analysis suggested that phoxalone arrested the mitosis of H446 cell line at G2/M phase. Hence, phoxalone and its derivatives or analogs would reveal huge research value and fascinating foreground.