Oxygen- and temperature-dependent cytotoxic and radiosensitizing effects of cis-dichlorodiammineplatinum(II) on human NHIK 3025 cells in vitro

The radiosensitizing effect of the chemotherapeutic drug cis-dichlorodiammineplatinum(II) (cis-DDP) was tested on human NHIK 3025 cells cultivated in vitro. cis-DDP was found to exert a radiomodifying effect under hypoxic but not under aerobic conditions. These results confirm that cis-DDP may act as a radiosensitizer of hypoxic cells; however, the radiosensitizing effect was seen only at concentrations of cis-DDP having a considerable cytotoxic activity, and for practical reasons concerning survival level the highest drug concentration that was investigated was 15 microM at 37 degrees C. The radiosensitizing effect was of a dose-modifying type and with a dose-modifying factor (DMF) of 1.2 at 15 microM in hypoxic cells. The radiosensitizing as well as the cytotoxic effect of cis-DDP was found to be strongly temperature dependent. Isoeffect doses of cis-DDP was reduced with a factor of 3 at 22 as compared to 37 degrees C. We also found that hypoxic cells were less sensitive to cis-DDP than cells treated in the presence of oxygen. To test the correlation between cytotoxicity and radiosensitization on the one hand and cellular uptake of cis-DDP on the other, cell-associated Pt was measured by atomic absorption spectroscopy. From these studies the cytotoxicity of cis-DDP at 22 and 37 degrees C under aerobic conditions was found to be the same as long as the amount of cell-associated Pt (i.e., the cellular uptake) was the same. However, whether the cells were treated under hypoxic or aerobic conditions, the cellular uptake of Pt was the same. While the radiosensitizing effect was present at 37 and at 40 degrees C, no such effect could be found at 22 degrees C. Since the cytotoxicity of cis-DDP as well as the drug uptake was reduced about three times at 22 as compared to 37 degrees C, we increased the concentration threefold, to 50 microM at 22 degrees C. Still no radiosensitizing effect was found at this temperature.     

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.     

In search of a correlation between the electron affinity and the radiosensitizing activity of some glyoxylic compounds.

The electron affinity of some glyoxylic compounds with radiosensitizing properties was evaluated using different approaches. The half-wave reduction potentials were measured by polarography, and the molecular structures in solution were determined by NMR spectroscopy. The electron affinity of the unhydrated and hydrated molecules was theoretically evaluated by means of a semi-empirical quantum mechanical procedure, the MNDO. The possible correlations between these values and the radiosensitizing efficiency of these glyoxylic compounds is discussed.     

An experimental study of the amides of aromatic carboxylic acids as radiosensitizing agents]

In experiments with mice a study was made of the radiosensitizing efficacy of 10 aromatic carbonic acid amides, benzene, naphthalene, pyridine and quinoline derivatives. It has been found, for this group of substances, that there is a direct correlation between the ability of the substance to reduce the splenic endocolonies production and the inhibitory activity with regard to poly(ADP-ribose) polymerase of thymocyte nuclei. Within the group of substances under study, new agents are found and described and new relationships are revealed between their chemical structure and biological activity.     

Potent radiosensitizing agents: 5-methylselenyl- and 5-phenylselenyl-methyl-2'-deoxyuridine

This Letter describes the novel radiosensitizing agents based on nucleoside base modification. In addition to the known 5-phenylselenide derivative, 5-methylselenide modified thymidine, which has a van der Waals radius smaller than the phenyl group, was newly synthesized. The similar monomer activity of 5-methylselenide derivative under oxidation condition was confirmed by NMR experiments. The cytotoxicity tests and radiosensitizing experiments of both compounds were carried out using the H460 lung cancer cell line. Both the 5-phenylselenide and the 5-methylselenide derivatives showed a relatively low toxicity to the cells. However, in combination with γ-radiolysis, both exerted good radiosensitizing effects to the lung cancer cell lines in vitro. This result confirms that 5-methylselenide modified thymidine could be a useful candidate as a potential radiosensitizing agent in vivo.     

Some complexes of cobalt(III) and iron(III) are radiosensitizers of hypoxic EMT6 cells

The radiosensitizing potential in hypoxic EMT6 cells of several complexes of Co(III) and Fe(III) has been examined. The cytotoxicity of each of the agents toward oxygenated and hypoxic EMT6 cells was tested over the concentration range of 1 to 500 micron for 1-h drug exposure. There was no statistically significant difference between the cytotoxicity of these complexes toward oxygenated and hypoxic cells. Based on these findings, 100 micron was selected as the drug concentration for the initial assessment of radiosensitizing potential. The radiation survival of EMT6 cells in the presence of 100 microM drug for a series of Co(III) complexes in which the number of nitro ligands was varied showed that the hexanitro and the triamine-trinitro complexes are very effective radiosensitizers. The trans-tetrammine dinitro complex was a more effective radiosensitizer than the corresponding cis-dinitro complex. The diethylenetriamine and 1,10-phenanthroline complexes were very effective radiosensitizers, producing dose-modifying factors of 2.4. The trans-tetrammine dichloro complex was moderately effective, giving a dose-modifying factor of 1.9. On the other hand, the hexammine and triammine tricyano complexes and the trans-dinitro complex with negatively charged acetylacetonate ligands were ineffective as radiosensitizers in this system. Finally, three complexes with cyclopentadienyl ligands were examined. The ferricenium salt itself was a moderately effective radiosensitizer, giving a dose-modifying factor of 2.0. However, both the dimethylferricenium salt and the analogous cobalt complex were ineffective. The FSaIIC fibrosarcoma was used to study radiosensitizing potential in vivo. The trans-tetramminedinitro complex was administered at doses of 100, 200, or 300 mg/kg as a single ip injection 1 h prior to irradiation or as three daily ip injections. There was increasing dose modification with increasing drug dosage. With a fractionated radiation protocol in which five daily fractions of 2, 3, or 4 Gy were administered to the tumor-bearing limb with ip drug injections of 100 or 200 mg/kg given 1 h prior to irradiation, a dose-modifying effect of 1.6 was observed with 5 X 200 mg/kg of the drug.     

Analysis of matched mRNA measurements from two different microarray technologies

MOTVIATION: The existence of several technologies for measuring gene expression makes the question of cross-technology agreement of measurements an important issue. Cross-platform utilization of data from different technologies has the potential to reduce the need to duplicate experiments but requires corresponding measurements to be comparable. METHODS: A comparison of mRNA measurements of 2895 sequence-matched genes in 56 cell lines from the standard panel of 60 cancer cell lines from the National Cancer Institute (NCI 60) was carried out by calculating correlation between matched measurements and calculating concordance between cluster from two high-throughput DNA microarray technologies, Stanford type cDNA microarrays and Affymetrix oligonucleotide microarrays. RESULTS: In general, corresponding measurements from the two platforms showed poor correlation. Clusters of genes and cell lines were discordant between the two technologies, suggesting that relative intra-technology relationships were not preserved. GC-content, sequence length, average signal intensity, and an estimator of cross-hybridization were found to be associated with the degree of correlation. This suggests gene-specific, or more correctly probe-specific, factors influencing measurements differently in the two platforms, implying a poor prognosis for a broad utilization of gene expression measurements across platforms.     

Effect of radiosensitizing agents on electron transport systems

Experiments have been carried out to study the interaxtion between chemical radiosensitizing agents and model electron transport systems. Using an NAD(P)H:O2 oxidoreductase enzyme as such a model, it was demonstrated that radiosensitizers can act as intermediates in the transfer of electrons from NADH to O2, even in the presence of classical inhibitors of electron transport, with anefficiency related to both their redox potentials and their radiosensitizing abilities. This work which was further confirmed in mammalian mitochondria and microsomes as well as in a cultured cell system indicated that these sensitizers can accept electrons from a variety of organelle systems. This action was shown to be related to the concentration of reduced pyridine nucleotides present both in vivo and in vitro. Of the electron-affinic agents tested, those whose redox potential was more negative than -0.39 V may possibly serve as better radiotherqpeutic mediators.     

Normal Values for Segmental Bioimpedance Spectroscopy in Pediatric Patients

Introduction

Localized limb edema is a clinically relevant sign in diseases such as post-thrombotic syndrome and lymphedema. Quantitative evaluation of localized edema in children is mainly done by measuring the absolute difference in limb circumference, which includes fat and fat-free mass. Bioimpedance spectroscopy (BIS) provides information on the fluid volume of a body segment. Our objective was to determine normal ranges for segmental (arm and leg) BIS measurements in healthy children. Additionally, we determined the normal ranges for the difference in arm and ankle circumference and explored the influence of handedness and the correlation between techniques.

Methods

Healthy children aged 1-18 years were recruited. The ratio of extracellular fluid content between contralateral limbs (estimated as the inter-arm and inter-leg extracellular impedance ratio), and the ratio of extracellular to intracellular fluid content for each limb (estimated as the intracellular to extracellular impedance ratio) were determined with a bioimpedance spectrometer. Arm and ankle circumference was determined with a Gulick II tape.

Results

We recruited 223 healthy children (48 infants, 54 preschoolers, 66 school-aged children, and 55 teenagers). Normal values for arm and leg BIS measurements, and for the difference in arm and ankle circumference were estimated for each age category. No influence of handedness was found. We found a statistically significant correlation between extracellular impedance ratio and circumference difference for arms among teenagers.

Conclusion

We determined normal BIS ranges for arms and legs and for the difference in circumference between arms and between ankles in children. There was no statistically significant correlation between extracellular impedance ratio and difference in circumference, except in the case of arms in adolescents. This may indicate that limb circumference measures quantities other than fluid, challenging the adequacy of this technique to determine the presence of localized edema in most age groups.     

Physicochemical properties of Shiga toxigenic Escherichia coli

AIMS: To investigate the physicochemical surface properties, such as cellular surface charge, hydrophobicity and electron donor/acceptor potential of a selection of Shiga toxigenic Escherichia coli (STEC) isolates grown in broth and agar culture. METHODS AND RESULTS: Cellular surface charge was determined using zeta potential measurements. Hydrophobicity of the isolates was determined using bacterial adhesion to hydrocarbons assay, hydrophobic interaction chromatography and contact angle measurements. Microbial adhesion to solvents was used to determine the electron donor/acceptor characteristics. No differences of surface charge measurements were found between broth and agar grown cultures. Isolates belonging to serogroup O157 and serotypes O26:H11 and O111:H- were significantly (P < 0.05) less negatively charged than other STEC serotypes tested. All strains were hydrophilic with most methods and demonstrated a lower hydrophobicity in agar culture compared with broth culture. All strains demonstrated a strong microbial adhesion to chloroform indicating that STEC possess an electron donor and basic character. A relationship between serogroup O157 and other STEC serotypes was apparent using principal-component analysis (PCA). CONCLUSIONS: Combining the results for physicochemical properties using PCA differentiated between strains belonging to the O157 serogroup and other STEC/non-STEC strains. PCA found similar results for broth and agar grown cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Particular serotypes of STEC possess similar physicochemical properties which may play a role in their pathogenicity or potential attachment to various surfaces.     

Comparison between orientational and conformational orders in fluid lipid bilayers.

The orientational order as determined by 2H NMR and the infrared frequencies of the C--H stretching modes of the methylene groups have been measured for several systems (POPC, POPC/cholesterol and POPE), all in the fluid phase, and then were compared; this work reveals an unexpected linear correlation between them. This experimental result shows that both measurements are essentially sensitive to a common motion, most likely trans/gauche isomerisation. This new correlation with those already found in the literature suggest that several measurements related to the hydrophobic core of the fluid bilayer describe different aspects of a universal behavior. The correlation presented here does not extend to the lipid in gel phase where slower motions affect the NMR lineshape.     

Radioprotection of bovine erythrocytes to haemolysis

The effect of several free-radical scavengers on the radiation-induced haemolysis of bovine erythrocytes (50 krad, 60Co, cell concentration: 0.05 v/v) was studied. A two-phase effect of the additives was found with a maximum of the protective action in the millimolar concentration range. A correlation was established between the degree of protection at a fixed concentration (5mM) and the rate constants of reactions of respective compounds with OH. A radiosensitizing effect of diethyldithiocarbamate, an inhibitor of superoxide dismutase (SOD) was not confirmed and no specific protective effect of SOD was seen.     

Serum and hair selenium levels in hereditary nonpolyposis colorectal cancer

Since low selenium (Se) levels have been identified in some individuals with colon cancer, we evaluated Se levels as a potential marker for this malignancy in a kindred subject to hereditary nonpolyposis colorectal cancer, an autosomal dominant disease. Unaffected family members and spouses were selected randomly for testing. Serum Se levels were performed on dialyzed sera using the neutron activation technique. Hair Se assays were determined by a spectrofluorometric method. Family members were classified as having low, intermediate, or high risk for colon cancer based on family history. There was no correlation between serum and hair Se measurements. There was also no significant difference in hair or serum Se levels between any of the groups, suggesting that serum Se levels do not correlate with hereditary risk for colon cancer. Prospective studies are in progress to evaluate tissue Se levels and serial Se measurements in high risk patients to establish whether Se levels change with the development of colon cancer.     

Noninvasive fluorescent imaging reliably estimates biomass in vivo

Whole-body optical imaging of small animals has emerged as a powerful, user friendly, and high-throughput tool for assaying molecular and cellular processes as they occur in vivo. As with any imaging method, the utility of such technology relies on its ability to provide quantitative, biologically meaningful information about the physiologic or pathologic process of interest. Here we used an animal tumor model to evaluate the extent of correlation between noninvasively measured fluorescence and more traditional measurements of biomass (tumor volume and tumor weight). C57/BL6 mice were injected subcutaneously with murine colon adenocarcinoma cells that were engineered to express GFP. Serial measurements of fluorescence intensities were performed with a macroscopic in vivo fluorescence system. The progressive increases in intensity correlated strongly with growth in tumor volume, as determined by caliper measurements (R2 = 0.99). A more stringent correlation was found between fluorescence intensity and tumor weight (R2 = 0.97) than between volume and weight (R2 = 0.89). In a treatment experiment using tumor necrosis factor-alpha, fluorescence intensity (but not tumor volume) was able to differentiate between treated and control groups on day 1 post-treatment. These results validate the ability of noninvasive fluorescent imaging to quantify the number of viable, fluorescent cells in vivo.     

cIAP1/2 are involved in the radiosensitizing effect of birinapant on NSCLC cell line in vitro

Tumour radioresistance is a major problem for cancer radiation therapy. To identify the underlying mechanisms of this resistance, we used human non-small cell lung cancer (NSCLC) cell lines and focused on the Inhibitor of Apoptosis Protein (IAP) family, which contributes to tumourigenesis and chemoresistance. We investigated the possible correlation between radioresistance in six NSCLC cell lines and IAP protein levels and tested the radiosensitizing effect of birinapant in vitro, a molecule that mimics the second mitochondria-derived activator of caspase. We found that birinapant-induced apoptosis and inhibited the proliferation of NSCLC cells after exposure to radiation. These effects were induced by birinapant downregulation of cIAP protein levels and changes of cIAP gene expression. Overall, birinapant can inhibit tumour growth of NSCLC cell lines to ironizing radiation and act as a promising strategy to overcome radioresistance in NSCLC.     

Flow cytometry of uveal melanomas

Tumor cell kinetic parameters were determined for 36 uveal melanomas retained in fixed tissue sections using flow cytometric techniques and computerized morphometry. By flow cytometry the majority of cells comprising the 36 tumors were in the G0/G1 phase (55.7%). The DNA index was 1.40 +/- 0.57 units. Using Spearman's rank order correlation test, the correlation between DNA index and the inverse standard deviation of nucleolar area was found to be statistically significant. The potential usefulness of flow cytometry in predicting tumors of high malignant potential is discussed.     

Monitoring ovarian response of Booroola Assaf ewe lambs to PMSG, using ultrasonography and serum estradiol

Ovarian size was determined for 30-day-old ewe lambs treated with pregnant mare serum gonadotropin (PMSG; 1000 IU) using ultrasonography and was compared with direct measurement at laparotomy. Ultrasonography was found to be an accurate and reliable method for estimating ovarian size, and a significantly high correlation was found between the methods (R=0.96, P<0.01). Forty-eight hours following PMSG treatment, serum estradiol levels increased from below detectable levels to a value of 72.8 +/- 27.4 pg/ml (mean +/- SEM). A high correlation was found also between estradiol levels and ovarian size measured at laparotomy and by ultrasound, with a correlation coefficient of 0.94 and 0.80, respectively. It is suggested that a differential increase in ovarian size and estradiol secretion in immature ewe lambs treated with PMSG may reflect the different stages of ovarian development in individual animals. Noninvasive methods such as ultrasonography and measurements of serum estradiol may be utilized in the estimation of ovarian response.     

Spermagglutinins in serum and seminal fluid of bulls and their relationship to fertility classification

Twenty-five satisfactory and 25 unsatisfactory potential breeder bulls were examined with a modified Shulman spermagglutination test for the presence of spermagglutinating antibodies in their serum and seminal fluid. This was done to determine whether antibodies to spermatozoa were a contributing factor to classification of the bull as an unsatisfactory potential breeder. No correlation was found between classification as an unsatisfactory potential breeder and the presence of spermagglutinating antibodies. In addition, indirect fluorescent antibody tests were performed to detect antisperm antibodies in the serum and seminal fluid of the bulls. Again, no correlation was found between antisperm antibodies and breeding soundness classification. Finally, there was no correlation between the age of the bulls and occurrences of spermagglutination antibodies in serum or seminal fluid.     

Glyoxylic compounds as radiosensitizers of hypoxic cells

The radiosensitizing effect of five glyoxal derivatives on the survival of TC-SV40 cells has been measured, under aerobic and hypoxic conditions. A toxicity study was previously performed in order to use nontoxic concentrations. The OER for the TC-SV40 cells was 2.74. None of the glyoxylic compounds showed radiosensitizing activity under aerobic conditions while in hypoxia their radiosensitizing factors decreased in the order phenylglyoxylic acid (1.68 at 8 x 10(-3) mole dm-3) greater than phenylglyoxal (1.55 at 5 x 10(-6) mole dm-3) greater than 2-2' furil (1.48 at 5 x 10(-5) mole dm-3) greater than glyoxylic acid (1.39 at 1 x 10(-3) mole dm-3) greater than glyoxal (1.30 at 5 x 10(-5) mole dm-3). The dose-modifying factors were also determined at two equimolar concentrations 5 x 10(-5) and 5 x 10(-6) mole dm-3. A concentration effect was noticed for all the compounds although their relative radiosensitizing activity kept, independently of the concentration, the same order noted above. Glyoxals with aromatic or heterocyclic rings exert a greater radiosensitization than the others. The acidic compounds have less radiosensitizing activity than their aldehydic counterparts. Interaction of these glyoxals with NPSH cellular groups was tested and the low degree of inhibition shows that this mechanism would contribute very little, if any, to the radiosensitization effect.     

Negative correlation between protozoal and bacterial levels in rumen samples and its relation to the determination of dietary effects on the rumen microbial population.

The bacterial protein content and protozoal protein content of unfractionated samples from the liquid-small particle phase of the rumen were determined on the basis of direct microscopic measurement of bacteria numbers and protozoa numbers and cell volumes. Standard values of 8.7 X 10(-11) mg of protein per bacterial cell and 5.9 X 10(-11) mg/micron 3 of protozoa cell volume, obtained from analysis of isolated cells, were used to convert the microscopic measurements to an estimate of the protein content of the rumen sample. When the correlation between bacterial and protozoal protein levels was examined within groups of animals, a highly significant negative correlation between these two parameters was found (P less than 0.001). The variation among animals for total (bacterial plus protozoal) microbial protein was smaller than the variation among animals for bacterial or protozoal protein alone. There was also a highly significant positive correlation (P less than 0.001) between protozoal protein level and total microbial protein level. The variation found among animals in total microbial protein level could be reduced by using a regression equation determined for bacterial versus protozoal protein to correct for the different population dynamics of the two groups.