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Biomedical subjects

A C Begg

Publications and source records attributed to A C Begg.

At least 37 records · Page 2Linked to original sources
PubMed1997

Detection of radiation-induced chromosome aberrations using fluorescence in situ hybridization in drug-induced premature chromosome condensations of tumour cell lines with different radiosensitivities.

A potential assay for radiosensitivity of human tumours is that of radiation-induced chromosome damage determined on metaphase spreads of human solid tumours. It is often difficult, however, to obtain enough metaphases for cytogenetic analysis after radiation. A possible solution would be to use the technique of premature chromosome condensation (PCC), enabling the study of interphase cells. The induction of PCCs using mitotic inducer cells is technically difficult, however, and the frequency of induction relatively low. We have attempted to use another approach, to induce PCCs using the phosphatase inhibitors okadaic acid and calyculin A. Both inhibitors were found to induce PCCs in several human tumour cell lines, with calyculin A producing the higher incidence. Determination of radiation-induced chromosome aberrations using fluorescence in situ hybridization on these chemically induced PCCs showed a clear difference between a radiosensitive (SCC61) and a radioresistant (A549) tumour cell line, with more aberrations in the sensitive line. Owing to incomplete condensation compared with that in standard metaphases, accurate classification of aberration types was not possible. Despite this limitation, the present data indicate that this relatively quick and simple method may be useful for determining chromosome aberrations in interphase cells and potentially in human solid tumours for predictive assay purposes.

Animals↗
PubMed1996

Enrichment of tumor cells for cell kinetic analysis in human tumor biopsies using cytokeratin gating.

PURPOSE: To determine the feasibility of using cytokeratin antibodies to distinguish normal and malignant cells in human tumors using flow cytometry. The goal was ultimately to increase the accuracy of cell kinetic measurements on human tumor biopsies. MATERIAL AND METHODS: A panel of four antibodies was screened on a series of 48 tumors from two centres; 22 head and neck tumors (Amsterdam) and 26 esophagus carcinomas (Leuven). First, screening was carried out by immunohistochemistry on frozen sections to test intensity of staining and the fraction of cytokeratin-positive tumor cells. The antibody showing the most positive staining was then used for flow cytometry on the same tumor. RESULTS: The two broadest spectrum antibodies (AE1/AE3, E3/C4) showed overall the best results with immunohistochemical staining, being positive in over 95% of tumors. Good cell suspensions for DNA flow cytometry could be made from frozen material by a mechanical method, whereas enzymatic methods with trypsin or collagenase were judged failures in almost all cases. From fresh material, both collagenase and trypsin produced good suspensions for flow cytometry, although the fraction of tumor cells, judged by proportion aneuploid cells, was markedly higher for trypsin. Using the best cytokeratin antibody for each tumor, two parameter flow cytometry was done (cytokeratin versus DNA content). Enrichment of tumor cells was then tested by measuring the fraction of aneuploid cells (the presumed malignant population) of cytokeratin-positive cells versus all cells. An enrichment factor ranging between 0 (no enrichment) and 1 (perfect enrichment, tumor cells only) was then calculated. The average enrichment was 0.60 for head and neck tumors and 0.59 for esophagus tumors. CONCLUSIONS: We conclude that this method can substantially enrich the proportion of tumor cells in biopsies from carcinomas. Application of this method could significantly enhance accuracy of tumor cell kinetic measurements.

Aneuploidy↗
PubMed1996

Radiation-induced differentiation of human skin fibroblasts: relationship with cell survival and collagen production.

The aim of this study was to determine whether significant inter-individual differences exist between skin fibroblast strains obtained from radiotherapy patients in both radiation-induced differentiation and collagen production in vitro, for use as potential parameters for a predictive assay for fibrosis following radiotherapy in patients. Morphological cell differentiation was determined 7 days after irradiation in seven early-passage primary human fibroblast cell strains and correlated with cell survival. Collagen production was measured in two cell strains by flow cytometry and incorporation of 3H-proline. There was a wide variation in the extent of radiation-induced differentiation for the seven cell strains, each showing a dose-related increase. The correlation between induced differentiation and cell survival was poor (r = 0.64) but statistically significant (p < 0.01). Collagen synthesis increased 7 days after irradiation for one cell strain (HF-48), as measured by incorporation of 3H-proline, but not in radiation sensitive AT-1 cells. The collagen I content of the two cell strains was assessed by flow cytometry but no significant differences were observed between the strains tested or with increasing dose. In conclusion, marked variations in radiation-induced fibroblast differentiation were observed between patients, this being an important criterion for a predictive assay.

Aged↗
PubMed1996

Lethality of radiation-induced chromosome aberrations in human tumour cell lines with different radiosensitivities.

In order to find an explanation for the eventual disappearance of all chromosome aberrations in two radiosensitive human tumour cell lines, the type and stability of different aberration types was investigated in more detail. To classify the aberrations into unstable and stable types, three-colour fluorescence in situ hybridization was performed, including a whole-chromosome probe, a pancentromere probe, and a stain for total DNA. This technique enables the appropriate classification of the aberrations principally by the presence (stable) or not (unstable) of a single centromere per chromosome. Unstable-type aberrations were found to disappear within 7 days (several divisions) in the two radiosensitive and the two radioresistant tumour lines investigated. Stable-type aberrations were found to remain at an approximately constant level over the duration of the experiment (14 days; 8-10 divisions) in the two radioresistant lines. In contrast, the majority of these stable-type aberrations had disappeared by 14 days in the two radiosensitive lines. The previous findings of disappearance of total aberrations in radiosensitive cells was therefore not due to a reduced induction of stable-type aberrations, but the complete disappearance of cells with this aberration type. These results could not be explained by differences in apoptosis or G1 blocks. Two possible explanations for these unexpected findings involve non-random induction of unstable-type aberrations, or lethality of stable-type aberrations. The results suggest caution in the use of stable-type aberration numbers as a predictor for radiosensitivity.

Apoptosis↗
PubMed1995

Defective repair of DNA double-strand breaks and chromosome damage in fibroblasts from a radiosensitive leukemia patient.

A radiation-sensitive fibroblast culture (180BR) established from an acute lymphoblastic leukemia patient who died following radiotherapy is defective in the repair of radiation-induced DNA double-strand breaks. The cells also show a reduced capacity to repair interphase chromosome damage visualized by means of premature chromosome condensation and metaphase chromosome aberrations measured by fluorescence in situ hybridization on chromosome 4. This case represents the first example in humans where hypersensitivity to ionizing radiation can be ascribed directly to a defect in DNA and chromosome repair, and the defect may underlie the cancerous phenotype observed.

Cells, Cultured↗
PubMed1995

Can measurements of potential doubling time (Tpot) be compared between laboratories? A quality control study.

The purpose of this study was to investigate the reproducibility of potential doubling time measurements of human tumors in different laboratories and to distinguish which steps in the measurement procedure were subject to the greatest variation. This was achieved by comparing measurements on the same source material in two different laboratories in which three aspects of the technique were separately studied, namely, preparation and staining of the nuclear suspensions, running the samples on the flow cytometer (FCM), and analyzing the two-parameter FCM data. This involved exchange between the two centers of fixed tumor material, stained nuclear suspensions, and FCM data on floppy disks. The analysis step was found to be the least variable step. For DNA synthesis time, Ts, and the labeling index, LI, the coefficients of determination (R2) ranged from 92% to 95.4%. A small systematic bias was observed, with one center measuring approximately 15% higher values for both LI and Ts. Different criteria for window placements were found to be a contributing factor. Variations in the FCM step were approximately equal to those for analysis, with no systematic deviations. Variations for the preparation and staining step were the largest (R2 = 60.5% and 38.1% for LI and Ts, respectively). However, this step was the only one subject to intratumoral variability, which was the largest contributing factor to the variations observed. In addition, however, LI was on average 41% higher in one center compared to the other, resulting in a systematic bias. Differences in the level of green fluorescence of the labeled cells implicated antibody differences as a possible cause. The variations found here for the three procedural aspects were significantly smaller than variations observed between tumors, a requirement for a predictive assay. Suggestions for implementation of quality control procedures include objective (computer-assisted) data analysis on two-parameter histograms and optimization of antibody combinations.

Carcinoma, Squamous Cell↗
PubMed1995

Use of fluorescence in situ hybridization to determine the relationship between chromosome aberrations and cell survival in eight human fibroblast strains.

A predictive assay of normal tissue radiosensitivity could benefit 'treatment tailoring' of radiotherapy for certain categories of tumour. The use of present clonogenic cell survival assays for this purpose would be impractical in routine clinical practice because of the lengthy assay time. Fluorescence in situ hybridization (FISH) using whole chromosome probes on metaphases was investigated as a potential substitute. Eight human fibroblast cell strains with a range of radiosensitivities were tested. For each strain, cell survival curves were determined and correlated with chromosome aberrations detected by FISH performed on metaphase cells collected 52 h after irradiation. A whole chromosome probe for chromosome 4 was used for all cell strains. The results revealed an increase in the percentage of metaphases with aberrant chromosomes (translocations and/or breaks) with increasing radiation dose for all strains. For the more radiosensitive cell strains there were relatively more aberrant metaphases for a given radiation dose when compared with fibroblasts from a normal donor. The relationship between surviving fraction and chromosome aberrations showed some variation between strains, but a linear regression for all data showed a highly statistically significant correlation (r = 0.89, p < 0.0005). These results suggest that an assay of chromosome damage using FISH could substitute for the clonogenic assay to predict the radiation sensitivity of human fibroblasts.

Cell Survival↗
PubMed1994

Characterization of resistance mechanisms to cis-diamminedichloroplatinum(II) in three sublines of the CC531 colon adenocarcinoma cell line in vitro.

Cisplatin resistance was developed in sublines of the CC531 rat colon adenocarcinoma cell line by continued low level drug exposure. Two relatively stable lines were obtained (RL2 and RL4) which were 6- and 20-fold more resistant to cisplatin. In addition, a subline more sensitive than the parent line by a factor of 2 (RLS) was obtained by subculture from a treated tumor. Mechanisms of resistance to cisplatin were investigated in these four lines, with the aim of determining the relative contributions of different resistance mechanisms at various resistance levels. Drug accumulation linearly decreased with increasing drug resistance. A 20-fold resistance was associated with only a 5-fold decrease in accumulation, suggesting that other resistance mechanisms may be involved in the total degree of resistance. Intracellular glutathione, measured fluorometrically, also increased with increasing resistance, varying by a factor of 4 between the most and least resistant lines. Reduction of glutathione levels by buthionine sulfoximine to parent line levels increased sensitivity but the cells remained considerably more resistant than parent cells. Resistant lines cultured in the absence of drug became progressively more sensitive, without accompanying changes in total glutathione levels. DNA-drug adducts, the presumed toxic lesion, were measured immunocytochemically. Initial levels decreased with increasing platinum resistance, although not proportional to resistance (factor of 5 decrease for 20-fold resistance). Drug dose ratios for equal initial adducts were similar to dose ratios for equal drug accumulation, implying that intracellular concentrations solely determine DNA adduction and that differences in glutathione level had little influence on the proportion of drug which eventually formed adducts. After 48 h, a better correlation between remaining adducts and resistance was found (factor 12 less adducts for 20-fold resistance). This implies that repair of adducts was important in determining survival. These data indicate that decreased drug accumulation played a proportionally greater role in the moderately resistant cell line and that adduct repair played a progressively greater role in the highly resistant cell line.

Adenocarcinoma↗
PubMed1994

The effect of radiation on G2 blocks, cyclin B expression and cdc2 expression in human squamous carcinoma cell lines with different radiosensitivities.

The purpose of the present study was to investigate the role of cyclin B and cdc2 in the G2 delay and to test whether the magnitude of the G2 delay correlated with sensitivity to ionizing radiation in two human cell lines. Cell cycle delays were measured by flow cytometry after pulse labeling with bromodeoxyuridine, and expression of cell cycle control genes were measured in Western blots in radiosensitive SCC61 and radioresistant SQ20B cell lines. Flow cytometry data demonstrated that the duration of the G2 arrest was dose dependent in both cell lines, amounting to approximately 1.1 h/Gy. No difference was found between the cell lines in the length of the G2 block. Radiation exposure did not result in a decrease of cyclin B. Cyclin B protein levels in both asynchronous and synchronized populations in fact showed a dose dependent increase, concomitant with the rise in the fraction of cells in G2/M. Similarly, the cdc2 protein levels did not decrease after irradiation. However, it was found that the levels of hyperphosphorylated, and therefore inactive, kinase were significantly higher in irradiated cells than in unirradiated cells. The accumulation of this hyperphosphorylated form correlated with the arrest of cells in the G2 phase. Finally, immunocytochemical staining of cyclin B revealed an increase of this protein in the cytoplasm after irradiation and a decrease in nuclear staining. This differential localization could possibly account for the reduced nuclear phosphorylation of cdc2 kinase leading to the G2 arrest.

Blotting, Western↗
PubMed1994

Improvement of combined modality therapy with cisplatin and radiation using intratumoral drug administration in murine tumors.

The aim of these studies was to increase the therapeutic ratio by achieving higher tumor concentrations of cisplatin during the course of a fractionated irradiation treatment. Specific goals were to test, firstly, whether multiple drug injections could be replaced by a single slow release implant of cisplatin, and secondly, whether the therapeutic potential of the combined treatment could be increased by administering the drug intratumorally. Drug administration routes tested were intraperitoneal (i.p.) of drug in solution, intratumoral (i.t.) of drug in solution, and intratumoral of drug in a slow release formulation. The latter consisted of a hydrogel polymer formulated into rods which were implanted into the center of subcutaneous tumors. For drug alone, both i.t. routes (solution or polymer) produced higher therapeutic gains than i.p. administration, as judged by tumor growth delay for a given weight loss. When combined with radiation, dose response curves were always shifted to lower doses and were steeper than for radiation or drug alone, although isobologram analysis indicated additivity. In a first series, drug enhancement ratios ranged from 1.6 to 2.6, and were highest for the i.t. groups. In a second series, X-ray enhancement ratios ranged from 1.1 to 1.7, with overlap between results from the different routes. Therapeutic ratios, however, were highest for the i.t. groups in both series. Slow release rods produced the highest therapeutic gains in the first series, while i.t. administration of drug in solution was approximately as effective in the second series.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Quantification of patient to patient variation of skin erythema developing as a response to radiotherapy.

A method is described to determine accurately skin redness during a course of radiotherapy using reflectance spectroscopy utilizing information from across the visible spectrum according to the L*a*b* color coordinate system. The method was used to quantify the development of skin erythema during and after electron beam irradiation of the chest wall following mastectomy. A number of factors were identified which could influence the wide variation in response seen between patients. These were: intra- and inter-observer variation; intra- and inter-patient variation and variation in the actual dose delivered. Statistical analysis, including an analysis of variance of inter- and intra-patient variation, revealed that the major factor that accounts for the observed difference between patients is a true inter-patient variation, with a coefficient of variation, corrected for intra-patient variation, of 43%. Within the narrow dose range administered in this study, there was no demonstrable dose-effect relationship, raising questions about the role of cell death in the basal layer of the epidermis in the pathogenesis of radiation induced erythema.

Adult↗
PubMed1994

Pharmacokinetics in melanoma-bearing mice of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a candidate compound for boron neutron capture therapy.

Blood pharmacokinetics and tissue distribution of 5-dihydroxyboryl-6-propyl-2-thiouracil (BPTU), a boron carrier with postulated melanin-seeking properties for boron neutron capture therapy, were determined in C57/BL mice with subcutaneous pigmented or non-pigmented B16 melanomas. Borocaptate sodium (BSH) was used as a boron compound without melanin-seeking properties in a comparative biodistribution study in the same animal tumour models. Administration of single doses showed that BPTU was retained better in the pigmented B16 tumour than in the non-pigmented variant. BPTU was found in large concentrations in kidney and liver. Brain boron was approximately 10-fold lower than tumour boron. On a molar basis, BPTU demonstrated higher affinity for B16 tumours than BSH. Owing to solubility limits, tumour boron concentrations in this mouse study were too low for effective application of BNCT. However, the high tumour-to-blood and tumour-to-normal tissues ratios indicate that, with appropriate formulation, BPTU could be a promising candidate for clinical BNCT.

Animals↗
PubMed1994

Prediction of repopulation rates and radiosensitivity in human tumours.

An accurate prediction of response to radiotherapy would allow treatments to be modified for those individuals where a relatively poor response is expected with conventional treatment. Present assays receiving most attention are those for repopulation, radiosensitivity and hypoxia. This paper discusses our experience with the first two, together with relevant data from other groups. Attention is focussed particularly on correlations with outcome in clinical trials, and the accuracy of present assay methods.

Chromosome Aberrations↗
PubMed1994

A comprehensive PC-based computer model for microdosimetry of BNCT.

A computer model is described that performs microdosimetric calculations of the radiation dose delivered to tumour and normal tissue in boron neutron capture therapy (BNCT) by simulating capture reactions in a predefined three-dimensional space. The role of intracellular boron distributions and cellular dimensions on the radiation dose in clinical and experimental BNCT has been studied using a PC-based computer model. In order to calculate the radiation dose to low boron uptake cells, the extent of irradiation by boron containing adjacent cells (cross fire) is also dealt with. Radiation doses from boron and nitrogen neutron capture are converted to a biological effect by means of relative individual ion track segment efficacies, based on linear energy transfer along the particle track. A good correlation was found after comparing predicted values with previously published experimental data. A number of examples is given to illustrate the program's features.

Boron Neutron Capture Therapy↗
PubMed1994

Use of fluorescence in situ hybridization to measure chromosome aberrations as a predictor of radiosensitivity in human tumour cells.

Fluorescence in situ hybridization (FISH) is a potential assay for determining cellular radiosensitivity based on the detection of chromosome damage. This approach was chosen because of its relative simplicity and short assay time. Two radiosensitive and two radioresistant human tumour cell lines were used. The radiosensitive lines were an ovarian carcinoma line (A1847) and a squamous carcinoma line (SCC61). The radioresistant cells were a lung adenocarcinoma line (A549) and a second squamous line (SQ20B). Whole chromosome-specific probes were used to detect radiation-induced chromosome aberrations in mitotic cells. Available probes were first screened to characterize the intrinsic chromosome aberrations before irradiation and the appropriate probes (minimum fluorescent spots) were selected for each cell line. Maximum radiation-induced aberrations were found 24 h after irradiation. Dose-response curves corrected for target size (proportion of genome probed) differed for all cell lines. The radiosensitive A1847 cell line showed more induced aberrations compared with the radioresistant A549 cell line, in agreement with the survival data. In contrast, the SQ20B cell line showed more induced chromosome aberrations than the more radiosensitive SCC61 cell line, leading to the hypothesis that the SQ20B cells could tolerate more aberrations. Dose-response curves obtained in surviving cells 14 days postirradiation indeed showed elevated levels of chromosome aberrations for SQ20B cells. The difference in chromosome aberrations between 1 and 14 days showed a good correlation with the survival data for all four cell lines. In conclusion, FISH of mitotic cells with whole chromosome probes appears to be a suitable assay to predict radiosensitivity. It seems necessary, however, to determine both induced and remaining chromosome aberrations, since different processing or tolerance of radiation-induced aberrations, including stable types, could lead to different correlations with cell survival.

Cell Survival↗