Fractionated radiation exposure amplifies the radioresistant nature of prostate cancer cells.

The risk of recurrence following radiation therapy remains high for a significant number of prostate cancer patients. The development of in vitro isogenic models of radioresistance through exposure to fractionated radiation is an increasingly used approach to investigate the mechanisms of radioresistance in cancer cells and help guide improvements in radiotherapy standards. We treated 22Rv1 prostate cancer cells with fractionated 2 Gy radiation to a cumulative total dose of 60 Gy. This process selected for 22Rv1-cells with increased clonogenic survival following subsequent radiation exposure but increased sensitivity to Docetaxel. This RR-22Rv1 cell line was enriched in S-phase cells, less susceptible to DNA damage, radiation-induced apoptosis and acquired enhanced migration potential, when compared to wild type and aged matched control 22Rv1 cells. The selection of radioresistant cancer cells during fractionated radiation therapy may have implications in the development and administration of future targeted therapy in conjunction with radiation therapy.

Carbonic anhydrase IX expression and outcome after radiotherapy for muscle-invasive bladder cancer.

AIMS: Carbonic anhydrase IX (CA IX) expression has been described as an endogenous marker of hypoxia in solid neoplasms. Furthermore, CA IX expression has been associated with an aggressive phenotype and resistance to radiotherapy. We assessed the prognostic significance of CA IX expression in patients with muscle-invasive bladder cancer treated with radiotherapy. MATERIALS AND METHODS: A standard immunohistochemistry technique was used to show CA IX expression in 110 muscle-invasive bladder tumours treated with radiotherapy. Clinicopathological data were obtained from medical case notes. RESULTS: CA IX immunostaining was detected in 89 ( approximately 81%) patients. Staining was predominantly membranous, with areas of concurrent cytoplasmic and nuclear staining and was abundant in luminal and perinecrotic areas. No significant correlation was shown between the overall CA IX status and the initial response to radiotherapy, 5-year bladder cancer-specific survival or the time to local recurrence. CONCLUSIONS: The distribution of CA IX expression in paraffin-embedded tissue sections seen in this series is consistent with previous studies in bladder cancer, but does not provide significant prognostic information with respect to the response to radiotherapy at 3 months and disease-specific survival after radical radiotherapy.

Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity.

In the UK, the two main treatments of invasive bladder cancer are radiotherapy or cystectomy. However, approximately 50% of patients undergoing radiotherapy fail to respond. If tumour radiosensitivity could be predicted in advance, it may be possible to improve control rates significantly by selecting for radiotherapy those patients whose tumours are radiosensitive. Additionally, patients who would benefit from surgery would be identified earlier. The alkaline comet assay (ACA) is a sensitive method for the detection of DNA strand break damage in cells. In the present study, using six bladder cancer cell lines of differing radiosensitivities, cell survival was compared to the manifestation of radiogenic DNA damage as assessed by ACA. For all the cell lines, the extent of comet formation strongly correlates with cell killing (R2>0.96), with a greater response being noted in radiosensitive cells. In repair studies, measures of residual damage correlate with survival fraction at 2 Gy (R2>0.96), but for only five of the cell lines. Finally, cells from human bladder tumour biopsies reveal a wide range of predicted radiosensitivies as determined by ACA. Overall, these studies demonstrate ACA to be a good predictive measure of bladder cancer cell radiosensitivity at low dose, with potential clinical application.

Evaluation of phosphodiesterase I-based protocols for the detection of multiply damaged sites in DNA: the detection of abasic, oxidative and alkylative tandem damage in DNA oligonucleotides.

It has been proposed that DNA multiply damaged sites (MDS), where more than one moiety in a local region ( approximately 1 helical turn, 10 bp) of the DNA is damaged, are lesions of enhanced biological significance. However, other than indirect measures, there are few analytical techniques that allow direct detection of MDS in DNA. In the present study we demonstrate the potential of protocols incorporating an exonucleolytic snake venom phosphodiesterase (SVPD) digestion stage to permit the direct detection of certain tandem damage, in which two lesions are immediately adjacent to each other on the same DNA strand. A series of prepared oligonucleotides containing either single or pairs of tetrahydrofuran moieties (F), thymine glycol lesions (T(g)) or methylphosphotriester adducts (Me-PTE) were digested with SVPD and the digests examined by either (32)P-end-labelling or electrospray mass spectrometry. The unambiguous observation of SVPD-resistant 'trimer' species in the digests of oligonucleotides containing adjacent F, T(g) and Me-PTE demonstrates that the SVPD digestion strategy is capable of allowing direct detection of certain tandem damage. Furthermore, in studies to determine the specificity of SVPD in dealing with pairs of lesions on the same strand, it was found mandatory to have the two lesions immediately adjacent to each other in order to generate the trimer species; pairs of lesions separated by as few as one or two normal nucleotides behave principally as single lesions towards SVPD.

Differential effects of the poly (ADP-ribose) polymerase (PARP) inhibitor NU1025 on topoisomerase I and II inhibitor cytotoxicity in L1210 cells in vitro.

The potent novel poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025, enhances the cytotoxicity of DNA-methylating agents and ionizing radiation by inhibiting DNA repair. We report here an investigation of the role of PARP in the cellular responses to inhibitors of topoisomerase I and II using NU1025. The cytotoxicity of the topoisomerase I inhibitor, camptothecin, was increased 2.6-fold in L1210 cells by co-incubation with NU1025. Camptothecin-induced DNA strand breaks were also increased 2.5-fold by NU1025 and exposure to camptothecin-activated PARP. In contrast, NU1025 did not increase the DNA strand breakage or cytotoxicity caused by the topoisomerase II inhibitor etoposide. Exposure to etoposide did not activate PARP even at concentrations that caused significant levels of apoptosis. Taken together, these data suggest that potentiation of camptothecin cytotoxicity by NU1025 is a direct result of increased DNA strand breakage, and that activation of PARP by camptothecin-induced DNA damage contributes to its repair and consequently cell survival. However, in L1210 cells at least, it would appear that PARP is not involved in the cellular response to etoposide-mediated DNA damage. On the basis of these data, PARP inhibitors may be potentially useful in combination with topoisomerase I inhibitor anticancer chemotherapy.

Resistance-modifying agents. Part 7: 2,6-disubstituted-4,8-dibenzylaminopyrimido[5,4-d]pyrimidines that inhibit nucleoside transport in the presence of alpha1-acid glycoprotein (AGP).

The synthesis and biological evaluation of potent 4,8-dibenzylaminopyrimidopyrimidine nucleoside transport inhibitors, with reduced binding to alpha1-acid glycoprotein, is reported.

A statistical-based approach to assessing the fidelity of combinatorial libraries encoded with electrophoric molecular tags. Development and application of tag decode-assisted single bead LC/MS analysis.

A statistical sampling protocol is described to assess the fidelity of libraries encoded with molecular tags. The methodology, termed library QA, is based on the combined application of tag decode analysis and single bead LC/MS. The physical existence of library compounds eluted from beads is established by comparing the molecular weight predicted by tag decode with empirical measurement. The goal of sampling is to provide information on overall library fidelity and an indication of the performance of individual library synthons. The minimal sampling size n for library QA is l0 x the largest synthon set. Data are reported as proportion (p) +/- lower and upper boundary (lb-ub) computed at the 95% confidence level (alpha = 0.05). As a practical demonstration, library QA was performed on a 25,200-member library of statine amides (size = 40 x 63 x 10). Sampling was conducted three times at n approximately 630 beads per run for a total of 1902 beads. The overall proportions found for the three runs were consistent with one another: p = 84.4%, lb-ub = 81.5-87.2%; p = 83.1%, lb-ub = 80.2-85.95; and p = 84.5%, lb-ub = 81.8-87.3%, suggesting the true value of p is close to 84% compound confirmation. The performance pi of individual synthons was also computed. Corroboration of QA data with biological screening results obtained from assaying the library against cathepsin D and plasmepsin II is discussed.

Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced alpha1-acid glycoprotein binding.

Dipyridamole has been shown to enhance the in vitro activity of antimetabolite anticancer drugs through the inhibition of nucleoside transport. However, the clinical potential of dipyridamole has not been realized because of the avid binding of the drug to the plasma protein alpha1-acid glycoprotein (AGP). Dipyridamole analogues that retain potent nucleoside transport inhibitory activity in the presence of AGP are described and their ability to enhance the growth inhibitory and cytotoxic effects of thymidylate synthase (TS) inhibitors has been evaluated. Three dipyridamole analogues (NU3026, NU3059 and NU3060) were shown to enhance the growth inhibitory activity of the TS inhibitor CB3717 and block thymidine rescue in L1210 cells. The extent of potentiation at a fixed analogue concentration (10 microM) was related to the potency of inhibition of thymidine uptake. A further analogue, NU3076, was identified, which was more potent than dipyridamole with a Ki value for inhibition of thymidine uptake of 0.1 microM compared to 0.28 microM for dipyridamole. In marked contrast to dipyridamole, inhibition of thymidine uptake by NU3076 was not significantly affected by the presence of AGP (5 mg ml(-1)). NU3076 and dipyridamole produced equivalent potentiation of the cytotoxicity of the non-classical antifolate TS inhibitor, nolatrexed, in L1210 cells with both compounds significantly reducing the LC90, by > threefold in the absence of salvageable thymidine. Thymidine rescue of L1210 cells from nolatrexed cytotoxicity was partially blocked by both 1 microM NU3076 and 1 microM dipyridamole. NU3076 also caused a significant potentiation of FU cytotoxicity in L1210 cells. These studies demonstrate that nucleoside transport inhibition can be maintained in the absence of AGP binding with the dipyridamole pharmacophore and that such analogues can enhance the cytotoxicity of TS inhibitors.

Potentiation of anti-cancer agent cytotoxicity by the potent poly(ADP-ribose) polymerase inhibitors NU1025 and NU1064.

The ability of the potent poly(ADP-ribose) polymerase (PARP) inhibitor, NU1025 (8-hydroxy-2-methyl-quinazolin-4-[3H]one) to potentiate the cytotoxicity of a panel of mechanistically diverse anti-cancer agents was evaluated in L1210 cells. NU1025 enhanced the cytotoxicity of the DNA-methylating agent MTIC, gamma-irradiation and bleomycin 3.5-, 1.4- and 2-fold respectively. The cytotoxicities of the thymidylate synthase inhibitor, nolatrexed, and the cytotoxic nucleoside, gemcitabine, were not increased. Potentiation of MTIC cytotoxicity by a delayed exposure to NU1025 was equally effective as by a simultaneous exposure to NU1025, indicating that the effects of NU1025 were mediated by an inhibition of the cellular recovery. The recovery from potentially lethal gamma-irradiation damage cytotoxicity in plateau-phase cells was also inhibited by NU1025. Investigation of DNA strand breakage and repair in gamma-irradiated cells by alkaline elution demonstrated that NU1025 caused a marked retardation of DNA repair. A structurally different PARP inhibitor, NU1064 (2-methylbenzimidazole-4-carboxamide), also potentiated the cytotoxicity of MTIC, to a similar extent to NU1025. NU1064 potentiated a sublethal concentration of a DNA methylating agent in a concentration-dependent manner. Collectively, these data suggest that the most suitable cytotoxic agents for use in combination with PARP inhibitors are methylating agents, bleomycin and ionizing radiation, but not anti-metabolites.

The use of colour difference vectors in diagnosing congenital colour vision deficiencies with the Farnsworth-Munsell 100-hue test.

Colour difference vector analysis provides useful and meaningful information in scoring the Farnsworth-Munsell (FM) 100-hue test. However, the FM 100-hue test is limited in its ability to diagnose type and severity of congenital colour vision defect. Type classification for all subjects is incorrect in 21% of cases, and for deuteranomals the misclassification rate is 38%. Visual inspection of the plots yields a similar misclassification rate and classification of plots with few errors (under 180) is generally less reliable. The FM 100-hue test has a limited ability to separate dichromats from anomalous trichromats. A test protocol based on joint D15 and FM 100-hue tests should pass 36% of anomalous trichromats and 26% of all colour defectives yet fail all dichromatic observers. We conclude that administering the FM 100-hue test is of less value than a combination of D15 panels (Standard D15 and L'Anthony's desaturated D15) in the clinical diagnosis of congenital colour defective observers. Our results for the FM 100-hue panel are similar to those reported previously by other investigators.

Task variables and visual discomfort associated with the use of VDT's.

We investigated task variables that may influence the incidence of visual discomfort amongst subjects who routinely spent a proportion of their day working with visual display terminals (VDT's). A diary was kept by subjects over five consecutive working days, in which they recorded visual symptoms, the visual tasks undertaken, the amount of time spent on workbreaks, work pressure, work interest, and personal moderating factors which may have influenced the symptoms recorded. The symptoms recorded by subjects were then treated as dependent variables in multiple regression analyses with the diary findings (task-related factors) as independent variables. The incidence of some types of symptoms was significantly associated with specific categories of work tasks, personal moderating influences, work pressure, work interest, time of day, and day of week.

Quantitative scoring methods for D15 panel tests in the diagnosis of congenital color vision deficiencies.

Farnsworth's standard D15 and L'Anthony's desaturated D15 panel tests were administered to 99 congenital red-green color defective subjects. The results were analyzed in three ways: (1) by summing the color differences between adjacent caps according to Bowman, (2) by averaging color difference vectors (CDV) according to Vingrys and King-Smith, and (3) by visually inspecting and counting the crossings. The Bowman measure was highly correlated with one of the CDV measures but provides less information regarding a cap arrangement. The desaturated D15 test can be expected to misclassify 5% of dichromats by type (protan/deutan) compared to a type misclassification rate of less than 0.1% for the D15 panel test. The correct diagnostic rates for type were 45% for the standard D15 test and 58% for the desaturated D15 test. However, the improvement in correct diagnostic rate for the latter test was accompanied by an increase in the misdiagnostic rate from 2 to 10%. The main value of the desaturated D15 test in congenital color vision diagnosis would seem to be in providing classification of those subjects who pass the standard D15 test. Quantitative scoring of the tests provides a good estimate of severity of defect. Visual inspection provides a similar diagnostic rate to CDV analysis, but has a lower type misdiagnostic rate at the desaturated D15 test and is more likely to be correct when the two methods disagree. We suggest that quantitative scoring techniques are of limited benefit for the clinical diagnosis of congenital color vision defects but that they are of use in clinical trials or for the monitoring of changes in color vision over time.(ABSTRACT TRUNCATED AT 250 WORDS)

Spatial summation in young and elderly observers.

We measured detection thresholds for targets over a range of sizes at both photopic and scotopic luminance levels in young and elderly observers, and used these data to estimate spatial summation areas 10 degrees in the retinal periphery. There were differences in detection thresholds between the young and old groups at photopic and scotopic luminances, but no differences in spatial summation areas at either background luminance level.

Detection of change of size by young and old observers.

Thresholds for detecting change of angular size were determined for a range of angular separations in young and elderly observers. We found no differences in thresholds between these subjects groups. Thresholds for detection of change of visual angle increased with angular separation in a power function relationship. Angular size change judgements based on successive views of a target are apparently not significantly affected by age.

Peripheral visual acuity and age.

We measured the peripheral visual acuity of a group of eleven older subjects (mean age 54.2 years) and a group of seventeen young subjects (mean age 22.1 years). Peripheral visual acuity was measured using Konig bars with critical detail subtending 2.4 and 4.8 minutes of arc. The older group showed a substantial reduction of peripheral visual acuity.

Peripheral visual acuity with monovision and other contact lens corrections for presbyopia.

We have conducted two experiments to investigate the effect of monovision and other contact lens corrections for presbyopia upon peripheral visual acuity. In the first study, we measured binocular peripheral visual acuity using Landolt rings with seven subjects wearing a monovision correction. The Landolt rings were presented at eccentricities of 10, 20, 40, and 70 degrees on each side of the subject, with near additions of +1.50 D, +2.50 D, and no addition. We found no significant effect of monovision correction on peripheral visual acuity. In the second experiment we measured the peripheral visual acuity of 11 presbyopic subjects wearing distance contact lenses with lookover spectacles, soft progressive bifocal contact lenses, soft concentric bifocal contact lenses, monovision contact lenses, modified monovision contact lenses, and hard bifocal contact lenses using Koenig bar targets. There were no significant differences in peripheral visual acuity between any of the contact lens corrections for presbyopia.

Contrast sensitivity with contact lens corrections for presbyopia.

We measured contrast sensitivity at three distances (330 mm, 660 mm and 4 m) with six contact lens and two multifocal spectacle corrections for presbyopia. The two spectacle corrections were D-segment bifocals and trifocals and the contact lens corrections were distance contact lens with lookover spectacles, soft progressive bifocals, soft concentric bifocals, monovision, modified monovision, and hard crescent segment bifocals. The spectacle corrections in general gave better results for the contrast sensitivity function (CSF), than did the contact lens corrections. Distance contact lenses with lookover spectacles performed best of the contact lens corrections used. However, the differences in CSF between the various contact lens corrections were small and not statistically significant.

Sensitivity to changes in size and velocity in young and elderly observers.

Thresholds for detection of change of size and for detection of change of velocity were measured in young and elderly observers. No differences in thresholds between these subject groups were found at the fovea or in more peripheral retina. The results indicate a degree of functional redundancy in the peripheral retina of elderly subjects, even though there is evidence of reduced numbers of functional photoreceptors in the periphery.

A method for quantitative scoring of the Farnsworth Panel D-15.

The Farnsworth Dichotomous test or Panel D-15 is used extensively for the evaluation of colour discrimination in congenital and acquired colour vision defects. This qualitative assessment of colour vision defect type and severity is based on the hue confusions which are represented diagrammatically on the Panel D-15 score sheet. This paper presents a new proposal for quantitatively scoring the Panel D-15 based on those hue confusions made by the subject. Such a quantitative score can be used to establish relationships with other visual functions or experimental conditions. The application of the scoring technique to clinical results is illustrated.

Recognition of the aircraft navigation light color code.

Navigation lights are a set of color-coded signals intended to indicate the presence, orientation, and relative direction of aircraft at night, and thereby reduce the possibility of midair collisions. It is known that some people with defective color vision have difficulty with quite simple codes. Accordingly, the International Civil Aviation Organization (ICAO) has recommended -- and most countries apply -- that applicants for pilot's licences demonstrate the ability to recognise colored light signals. Pilots who fail to meet this requirement are restricted from flying at night. But is the navigation light signal system effective? This paper concludes that the navigation light system at night can serve as a crude screening method to categorize intruder aircraft into "potential threat" and "no threat" categories. An experiment is described which shows that observers with normal color vision can determine intruder aircraft orientation and relative direction from the navigation light code with a moderately high degree of reliability. The reliability of judgement is, however, decreased by the higher-intensity presence lights also displayed by aircraft.