Thermodynamic stability of wild-type and mutant p53 core domain

Some 50% of human cancers are associated with mutations in the core domain of the tumor suppressor p53. Many mutations are thought just to destabilize the protein. To assess this and the possibility of rescue, we have set up a system to analyze the stability of the core domain and its mutants. The use of differential scanning calorimetry or spectroscopy to measure its melting temperature leads to irreversible denaturation and aggregation and so is useful as only a qualitative guide to stability. There are excellent two-state denaturation curves on the addition of urea that may be analyzed quantitatively. One Zn²⁺ ion remains tightly bound in the holo-form of p53 throughout the denaturation curve. The stability of wild type is 6.0 kcal (1 kcal = 4.18 kJ)/mol at 25°C and 9.8 kcal/mol at 10°C. The oncogenic mutants R175H, C242S, R248Q, R249S, and R273H are destabilized by 3.0, 2.9, 1.9, 1.9, and 0.4 kcal/mol, respectively. Under certain denaturing conditions, the wild-type domain forms an aggregate that is relatively highly fluorescent at 340 nm on excitation at 280 nm. The destabilized mutants give this fluorescence under milder denaturation conditions.     

Deregulation of cell proliferation by polycyclic aromatic hydrocarbons in human breast carcinoma MCF-7 cells reflects both genotoxic and nongenotoxic events.

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are carcinogens suggested to be involved in development of human cancer. Several recent studies have reported that PAHs can activate estrogen receptors (ER), either directly or indirectly by producing estrogenic metabolites. We hypothesized that the activation of ER by PAHs or their metabolites could induce cell proliferation in estrogen-sensitive cells. In the present study, we found that two PAHs, benz[a]anthracene (BaA) and BaP, can stimulate proliferation of human breast carcinoma MCF-7 cells at concentrations 100 nM and higher. This effect was ER-dependent, because it was blocked by the pure antiestrogen ICI 182,780. Although both PAHs partially inhibited S-phase entry and DNA synthesis induced by 17beta-estradiol, they stimulated S-phase entry when applied to MCF-7 cells synchronized by serum deprivation. This was in contrast with model antiestrogenic aryl hydrocarbon receptor ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which fully suppressed S-phase entry. BaP, which is a strong mutagen, was found to induce p53 tumor suppressor expression, a partial S-phase arrest and at higher concentrations also cell death. Pifithrin-alpha, a synthetic inhibitor of p53 activity, abolished both S-phase arrest and apoptosis induced by genotoxic PAHs, and it potentiated the proliferative effect of BaP. Thus, both genotoxic and nongenotoxic events seem to interact in the effects of BaP on cell proliferation. Taken together, our data indicate that both BaA and BaP can stimulate cell proliferation through activation of ER. The proliferative effects of these carcinogenic compounds might contribute to tumor promotion in estrogen-sensitive tissues.     

The effect of single and repeated UVB radiation on rabbit cornea

Background Cumulative effect of ultraviolet radiation (UVR) is an important aspect of UV corneal damage. The purpose of this study was to apply high resolution magic angle spinning proton nuclear magnetic resonance (HR-MAS ¹H NMR) spectroscopy to evaluate the effect of single and repeated UV radiation exposure of the same overall dose on the rabbit cornea.Methods Corneal surfaces of 24 normal rabbit eyes were examined for the effects of UVB exposure (312 nm). In the first group (UVB1), animals were irradiated with a single dose (3.12 J/cm²; 21 min) of UVB radiation. The animals in the second group (UVB2) were irradiated three times for 7 min every other day (dose of 1.04 J/cm²; days 1, 3, 5) to give the same overall dose (3.12 J/cm²). The third group served as an untreated control group. One day after the last irradiation, the animals were sacrificed, and the corneas were removed and frozen. HR-MAS ¹H NMR spectra from intact corneas were obtained. Special grouping patterns among the tissue samples and the relative percentage changes in particular metabolite concentrations were evaluated using modern statistical methods (multivariate analysis, one-way ANOVA).Results The metabolic profile of both groups of UVB-irradiated samples was significantly different from the control corneas. Substantial decreases in taurine, hypo-taurine and choline-derivatives concentrations and substantial elevation in glucose and betaine levels were observed following the UVR exposure. There was no significant difference between the effect of a single and repeated UVB irradiation of the same overall dose.Conclusions For the first time, the effects of single and repeated UVR doses on the metabolic profile of the rabbit cornea were analysed and compared. The combination of HR-MAS ¹H NMR spectroscopy and modern statistical methods (multivariate analysis, one-way ANOVA) proved suitable to assess the overall view of the metabolic alterations in the rabbit corneal tissue following UVB radiation exposure.     

The cellular uptake of sensitizers bound to cyclodextrin carriers

Photodynamic therapy of cancer uses the interaction of sensitizers and light to destroy cancer cells. In this study we tested the cellular uptake of meso-tetrakis(4-sulfonatophenyl)porphine (TPPS4) and its complex PdTPPS4 in the presence or absence of 2-hydroxypropyl-cyclodextrins (hpCDs) on G361 human melanoma cells. Self-fluorescence in G361 cells were measured by Perkin-Elmer LS50B luminometer equipped with well plate reader accessory. Morphological changes in cells have been evaluated using inversion fluorescent microscope Olympus IX 70 and image analysis. The uptake of the sensitizer PdTPPS4 at the given time interval from 1 to 48 hours is markedly higher than the uptake of TPPS4. The highest uptake was found for sensitizer PdTPPS4 in combination with hpbetaCD. TPPS4 and PdTPPS4 especially in the supramolecular complex with nontoxic cyclodextrin carriers represent efficient sensitizers for photodynamic therapy in vitro on G361 cells.     

Malignant fibrous histiocytoma arising in a recurrent chordoma

Summary We present the case of a sacrococcygeal chordoma which recurred 15 years after the radical removal as a soft tissue tumor in the gluteal musculature. This tumor consisted of two parts: a chordoma without symptoms of aggressive cellular proliferation and a malignant fibrous histiocytoma. During the following 4 years several local recurrences of the malignant fibrous histiocytoma occurred in the gluteal musculature. The patient finally died of lung metastases. No chordoma tumor tissue was found in the lungs, in the gluteal musculature or in the sacrococcygeal bone area. Histology including electron microscopy revealed no proof of a transition of chordoma into malignant fibrous histiocytoma. It must be assumed that the secondary soft tissue tumor originated from residual chordoma cells which were implanted during the operation of the primary tumor. It remains unclear whether the malignant fibrous histiocytoma arose from mesenchymal stromal cells within the chordoma or directly from primitive neuroectodermal chorda cells which possess the ability to differentiate into a variety of cell types including mesenchymal cells.     

Über die Wechselwirkung des Katalysators und Cokatalysators mit dem Monomeren und dem Polymeren bei der kationischen Polymerisation des Isobutylens. II. Modellstudium des Polymerisations-Depolymerisations-Gleichgewichts im System Triisobutylen/AlBr3/DBr/Heptan

In the reaction of triisobutylene with AlBr₃/DBr system, backbone isomerization and formation of higher oligomers takes place, in addition to deuteration. Both reactions are explained by the cleavage of the backbone bonds of the intermediate carbonium ions. The fragments formed can react with other components of the reaction system. The possible role of these reactions in the mechanism of termination and chain transfer in the cationic polymerization of isobutylene is discussed briefly.