Biomedical subjects
Publications and source records attributed to C M West.
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Dosimetry data are required for many epidemiologic studies of radiation workers. For several reasons, these data may not be available for some workers for some periods of time. An estimate of a worker's dose for such a time period can be made using data from nearby time periods for the worker or using data obtained for other workers during the same time period. This paper reports the results of an evaluation of a procedure for estimating external doses for workers included in retrospective epidemiologic studies. This evaluation was conducted using data for workers who had recorded doses for each year in a 5-y period. The dose for the middle year of the 5-y period was treated as unavailable and the dose was estimated several ways. These estimates were compared with the recorded dose. It was observed that averaging a worker's dose data for the years preceding and following a year for which data hypothetically were not available most often yielded the best estimate of the dose. At one facility studied, the use of nearby data for the same worker was a more reliable estimate for a dose than was the department or facility median or mean dose. However, at another facility, use of the department median or mean dose produced a more accurate estimate than did nearby data for the worker. These results indicate that a single procedure for estimating doses is not best for all facilities, and it is recommended that a test of the type reported in this paper be performed before a decision is made on how to estimate doses. The use of department medians or means to estimate doses consistently produced better estimates than were obtained using facility medians or means.
An assessment has been made of the reproducibility of measuring tumour proliferation using in vivo iododeoxyuridine (IUdR) labelling and flow cytometry. The variation that occurs between different institutions (Paterson Institute for Cancer Research, Manchester and the Gray Laboratory, Northwood), different observers and different runs on the same flow cytometer have been measured on 139 samples from 53 patients with colorectal cancer. The results demonstrate that the IUdR technique for measuring tumour proliferation is reproducible. Correlations were seen between measurements of Tpot obtained by different individuals and on separate machines. However, direct comparisons of the measured parameters showed that there were highly significant differences in the values obtained between institutes and observers (P < 0.001). Despite these variations, there were still significant detectable differences in Tpot measurements between individual tumours (P < 0.001). Analysis of the results obtained by running the same samples on two separate occasions on the same machine showed that the technique was highly reproducible and that the staining procedure was stable. Eighty per cent of the samples were similarly assigned to either above or below the median Tpot value, regardless which observer/laboratory combination was utilised. These data suggest that large clinical trials using Tpot should employ a single centre and a single individual to prepare, run and analyse samples.
Intra-tumoural heterogeneity of proliferation has been assessed by taking multiple biopsies from 30 colorectal cancers. Following in vivo IUDR labelling, dual parameter flow cytometry was used to measure tumour DNA index (DI) and labelling index (LI) and to derive DNA synthesis time (Ts) and potential doubling time (Tpot). Heterogeneity was seen for all parameters under investigation. Overall coefficients of variation (CV) and logarithmic transformation of Ts and Tpot (due to their non-gaussian distributions) indicate that LI (CV 25%) was the most variable parameter. Intra-tumoral heterogeneity in Tpot (lnTpot CV = 22%) was less than inter-individual variation (CV = 63%), suggesting that this variation should not be a limitation to the possible usefulness of this technique as an independent prognostic indicator. Correlations of Tpot values were examined between the shortest, the median and the value for a pooled homogenate sample from a single tumour. Using an homogenate, it was possible to accurately predict classification of tumour Tpot values as being below the median ('fast tumours') in 15 of 19 cases (79%). The data suggest that assaying an homogenate may allow a more rapid analysis of a multiply sampled tumour.
The intrinsic radiosensitivity of cervical carcinoma has been measured using a soft agar clonogenic assay. All patients received radical radiotherapy alone with a minimum of 2 years post-treatment follow-up. Only women with stage I, II and III disease were included in the analysis. Values for cell surviving fraction at 2 Gy (SF2) were obtained for 88 tumours with an assay success rate of 73%. The 53 patients alive and well at the time of analysis had tumours with a mean SF2 that was significantly lower than the value from the 22 patients with locoregional failure (P < 0.01). Patients with radioresistant tumours (SF2 > 0.40, the median) had a significantly lower 3 year survival level than those with sensitive tumours (SF2 < or = 0.40) (P = 0.002). Also the frequency of local recurrence was higher (P = 0.001) whether these were central (P = 0.009) or peripheral (P = 0.046). Cell surviving fraction at 3.5 Gy was obtained for 46 tumours and the 3 year patient survival rate was significantly higher for those with SF3.5 values less than the median (P = 0.043). There was, however, no difference in the level of local recurrence (P = 0.24). The ability to grow in culture was not associated with significantly poorer patient survival (P = 0.56) or failure to control the primary disease (P = 0.17). While high colony forming efficiencies were associated with an increased rate of local recurrence (P = 0.029) they did not predict for overall patient survival (P = 0.32). These data suggest that, for cervical carcinoma treated with radical radiotherapy, intrinsic radiosensitivity is important in determining treatment outcome.
A comparison has been made of high (1.55 Gy min-1) and low dose-rate (0.0098 Gy min-1) irradiation in determining the intrinsic radiosensitivity of peripheral blood lymphocytes from normal individuals. Samples from 19 people were assessed using a limiting dilution assay and used to investigate the variability associated with evaluation of lymphocyte radiosensitivity after both high and low dose-rate irradiation. Repeat experiments on a single sample from one donor stored over a period of 6 months have been used to compare assay variability using the different dose-rates. Multiple samples taken from a single person over a period of 5 months were assayed at low dose-rate to assess intraindividual variation in measured in vitro radiosensitivity. At high dose-rate significant interexperimental variation in the measured parameters was demonstrated and, after allowing for this variability, no significant interindividual differences were found. At low dose-rate, sparing of cell kill was seen for all lymphocytes and led to an increase in the spread of data between individuals such that interindividual differences reached statistical significance for surviving fraction at 4 Gy, alpha (linear fit) and D with p < 0.004 for all parameters.
A study has been made of the intrinsic radiosensitivity of peripheral blood lymphocytes from normal individuals. Cell survival following in vitro high (1.55 Gy min-1) and low (0.0098 Gy min-1) dose-rate irradiation was obtained for single lymphocyte samples from three individuals in six experiments. Despite wide interexperimental variability the ranking of intrinsic radiosensitivity for these individuals was reproducible. Further studies were carried out using low dose-rate irradiation only, on: (1) single lymphocyte samples from 18 people; (2) 14 samples taken on different occasions from one of these donors over a period of 5 months; and (3) a large store of lymphocytes from another individual. The latter was assayed in every experiment and served as an internal standard to which results could be normalized. In comparison with uncorrected values, the normalized results for the 14 samples from the single individual showed a reduction in the spread of data while those for the 18 different donors were unchanged. An analysis of variance was carried out on a larger data set of 38 measurements which included repeat assays on several of the samples (two samples from the multiply-sampled individual and five samples from other donors were assayed twice). In comparison with the uncorrected values, the normalized results showed a reduction in experimental variability such that the coefficient of variation (CV) for surviving fraction at 4 Gy for the multiple samples from a single individual decreased from 41 to 19%. In contrast, there was little change in the variation between 18 individuals with CVs of 56 and 62%, respectively, for uncorrected and normalized results. These data suggest that the use of an internal standard for the determination of the intrinsic radiosensitivity of individuals using peripheral blood lymphocytes should aid the development and evaluation of predictive tests for the radiotherapy of cancer.
The prestalk and prespore cells from the Dictyostelium discoideum multicellular slug stage of development differ in assembly of glycoconjugates. Prespore cells are 2- to 3-fold more active than prestalk cells in the assembly of N-linked glycans and 20-fold more active in their fucosylation. Only prespore cells synthesize an O-linked glycan consisting in part of Fuc alpha-linked to N-acetylglucosamine. Incorporation of fucose, glucosamine, mannose and galactose into large pronase-resistant glycoconjugates was almost exclusively into prespore cells. Such glucosamine-labelled glycoconjugates resist fragmentation by beta-elimination and include a glycoantigen dependent on the modB genetic locus. In contrast, large fucose-labelled glycoconjugates consisted of multiple, small, O-linked oligosaccharides on carrier peptides. The spore coat protein SP96 has several fucosylated O-linked oligosaccharides, one of which correlates with a fucose epitope previously shown to localize in prespore vesicles and the outer layer of the spore coat.
FP21 is a 21-kDa fucoprotein which fractionates with the cytosol after high-speed centrifugation of gently lysed Dictyostelium cells. Less than 0.7% of FP21 is associated with vesicles. In proliferating cells, 4 x 10(5) fucosyl moieties/cell are associated with FP21 as anionic, possibly O-linked oligosaccharides equal in size to 4.8 glucose units. FP21 is underfucosylated in a mutant strain (HL250) that depends on extracellular fucose for synthesis of GDP-fucose. To determine the cellular site of FP21 fucosylation, cytosolic and vesicular preparations from strain HL250 were compared for their ability to transfer fucose from GDP-fucose to FP21. Cytosolic preparations fucosylate endogenous FP21 in a time-, concentration-, and divalent cation-dependent fashion, with a Km for GDP-fucose of 1.4 microM. Activity in normal cell cytosol is dependent on exogenous mutant FP21, demonstrating that FP21 is normally fully fucosylated. Both mutant and normal cytosols are also able to alpha-fucosylate a type 1 glycolipid substrate (8-methoxycarbonyloctyl-Gal beta 1-3 beta GlcNAc), but not related substrates, with Km values for the type 1 glycolipid of 0.99 mM and for GDP-fucose of 1.6 microM. Competitive inhibition between FP21 and the type 1 glycolipid shows that the same enzyme fucosylates both substrates. Intact and permeabilized vesicle preparations from wild-type cells are unable to fucosylate FP21 or the type 1 glycolipid by a divalent cation-dependent mechanism, and thus are devoid of FP21-fucosyltransferase. Since control experiments showed that vesicle leakage is minimal during cytosol preparation, these results indicate that FP21 is synthesized and fucosylated in the cytosolic compartment, by an unusual soluble fucosyltransferase.
Biopsies from 117 patients with cervical carcinoma were studied using a clonogenic assay to assess in vitro growth. Successful colony growth was achieved in 84 tumours (72%) with a mean colony-forming efficiency (CFE), based on total viable nucleated cell counts, of 0.18 +/- 0.49% (+/- 1 standard deviation). There was a wide range of values, from 0.003-4.28%, with a coefficient of variation of 272%. The relationship between clinical features of cervical carcinoma and in vitro colony formation was investigated. No significant association was demonstrated between in vitro growth and either clinical stage (r = 0.02), tumour differentiation (r = -0.08) or patient age (r = -0.12). There was no significant difference in tumour grade between the group of tumours which failed to grow in culture and those which grew well (p = 0.70). In addition, there was no correlation between CFE and the degree of macrophage (r = 0.001), lymphocyte (r = 0.12), or granulocyte (r = 0.08) infiltration. There was no significant difference between CFEs of tumours from patients who had died and from those who were alive and well after a minimum of 2 years' follow-up after radiotherapy (p = 0.51). Ability to form colonies in agar was not associated with a worse prognosis (p = 0.49). Although CFE is an independent biological parameter, our results suggest that, for cervical carcinoma, it is not useful as a univariate prognostic factor.
The murine tail has been used as a model for injury to skin when hyperthermia (HT) and photodynamic therapy (PDT) using haematoporphyrin derivative, are used in combination. Skin injury by either agent alone was quantitated by the probability of tail necrosis as a function of dose of agent. 'Tolerance' doses of each modality were given and changes in skin vascular function were measured by the rate of clearance of 133Xenon. This was promptly inhibited but restored to normal by 7 days. The absolute numbers of hypodermal vessels of different sizes were measured in tail cross-sections and capillary numbers were found to be greatly reduced between 1 and 7 days, and restored to normal by 21-28 days. When a tolerance dose of PDT was followed at 1, 7, 21 and 28 days by test doses of HT, or vice versa, marked enhancements in probability of necrosis were observed when the interval was 1 or 7 days (Enhancement ratio (ER)PDT-HT = 1.5 and ERHT-PDT = 1.8). Prolonging the interval between modalities to 21-28 days spared the tissue (ERHT-PDT/21 DAYS = 1.1; ERPDT-HT/28 DAYS = 1.0). Close temporal apposition of PDT and HT, such as has been advocated to improve tumour control, may also increase injury to normal tissue through vascular effects common to both.
Chinese hamster ovary cells have been exposed to high-voltage electric fields causing electroporation (EP) and the interaction between EP and radiation-induced cell lethality investigated. There was a voltage-dependent decrease in plating efficiency, assessed immediately following EP, and cell viability, assessed at 24 h. A linear decrease was seen for both. These decreases were accompanied by a voltage-dependent increase in cell volume, assessed immediately following EP. A good correlation between increases in cell volume and decreases in plating efficiency was seen (r = -0.91). The application of electric fields immediately prior to, or following, irradiation led to a radiosensitization of the cells. This radiosensitization still occurred when a 6 h interval was left between radiation and EP but was lost when cells were irradiated 24 h prior to EP. When cells were irradiated following EP, the radiosensitization was lost with a 1 h interval between the two treatments. These results suggest that, when studying the combined cellular effects of EP of macromolecules and radiation, care should be taken that sufficient time has elapsed between the two modalities to prevent the radiosensitization of cells.
At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.
The influence of cell heterogeneity on response to photodynamic treatment (PDT) has been investigated using the human colon adenocarcinoma line WiDr, grown as spheroids and exposed to hematoporphyrin derivative. The spheroids show a marked spheroid size-dependent resistance to PDT. Using a flow cytometer, cell sub-populations have been separated, on the basis of drug fluorescence, from single cell suspensions prepared from 500 microm diameter spheroids. Cells low in fluorescence have been shown to be resistant to PDT, have a smaller median cell volume, and be enhanced in G1-type cells. These cells also show reduced low density lipoprotein uptake. The results suggest that spheroid size-dependent resistance to PDT is related to a decreasing growth fraction with increasing spheroid size. Heterogeneity of drug uptake could be a potential limitation to clinical PDT.
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A serum-free medium has been developed to support the clonogenic growth in soft agar of human cervical carcinoma cell lines xenografted into nude mice. Using the Courtenay-Mills assay, colony-forming efficiencies and colony sizes equivalent to those obtained using Ham's F12 plus 15% fetal calf serum can be obtained. Validation of the assay using the developed medium was obtained through establishing linearity between the number of cells plated and colonies formed, and by producing radiation survival curves.
For mouse kidney cells assayed in primary culture, the Do and n values were 1.1 +/- 0.06 Gy and 7 +/- 2 for single cells irradiated in vitro, and 1.3 +/- 0.08 Gy and 25 +/- 11 for in vivo irradiation. The lower radiosensitivity in vivo was shown not to be caused by natural hypoxia, as the average oxygen enhancement ratios were 2.6 +/- 0.3 for in vitro and 2.8 +/- 0.4 for in vivo irradiation. Irradiations of fragments of kidney tubules produced similar survivals as irradiations of kidneys in situ, even for irradiation immediately before the fragments were disaggregated into single cells. The critical point of change in radiosensitivity from in vivo to in vitro values due to this contact effect was the time that the kidney cells were monodispersed.