25th Paterson Symposium--is there a future for radiosensitivity testing?
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Animals↗
Biomedical subjects
C M West
Publications and source records attributed to C M West.
Photocytotoxic efficacy of sulphonated species of aluminium phthalocyanine against cell monolayers, multicellular spheroids and in vivo tumours.
The problem of relying solely on in vitro data to predict photosensitiser efficacy was demonstrated by examining the uptake and the ability to mediate photocytotoxicity of mono-, di-, tri- and tetra-sulphonated species of chloroaluminium phthalocyanine (AlS1-4Pc) in monolayer cultures of murine Colo 26 cells and in both monolayer and spheroid cultures of human WiDr cells. Cells treated in vitro, whether in monolayer or as spheroids, with the less sulphonated derivatives, AlS1Pc and AlS2Pc, were more susceptible to photocytotoxicity than those treated with AlS3Pc, cells treated with AlS4Pc were even less susceptibile to the cytotoxic effects of light irradiation. Generally these results mirrored the cellular uptake in vitro. When WiDr spheroids were increased in size from 250 microns to 500 microns there was a reduction in uptake of AlS1Pc and AlS2Pc which was reflected by the decreased sensitivity of the larger spheroids to the effects of light irradiation. AlS1Pc had no effect against Colo 26 cells growing as s.c. tumours in syngeneic BALB/c mice; whereas AlS3Pc, AlS2Pc and AlS4Pc produced significant reductions in tumour weights 5 days post laser light irradiation. Of these, AlS2Pc had the most dramatic effect on the colony forming efficiency of tumour cells recovered 24 h after PDT. While, despite their effects on tumour size, AlS3Pc and AlS4Pc scarcely affected the subsequent viability of cells from dissociated tumours. Thus the in vitro efficacy of the sulphonated species of phthalocyanines is not necessarily predictive of their in vivo effectiveness.
Animals↗
Lack of a relationship between colony-forming efficiency and surviving fraction at 2 Gy.
The relationship between in vitro radiation sensitivity and colony-forming efficiency has been examined for primary human tumors of the cervix, colorectum, and lymph gland. Tumors were cultured using the Courtenay-Mills soft agar clonogenic assay and radiosensitivity was assessed as surviving fraction at 2 Gy. There was no correlation between colony-forming efficiency and surviving fraction at 2 Gy, giving confidence to the use of surviving fraction at 2 Gy as a predictor of clinical outcome.
Cell Survival↗
Additive interaction between tamoxifen and rifampicin in human biliary tract carcinoma cells.
A novel two-drug combination of tamoxifen and rifampicin has been investigated for the treatment of biliary tract-associated malignancies. Their effects, alone and in combination, on human tumour cells were studied using two pancreatic carcinoma cell lines. MIA PaCa-2 and AsPC-1, and a gall bladder carcinoma cell line, G-415. Inhibition of growth was used as an endpoint. The cells differed in sensitivity to equimolar doses of the two drugs given as single agents. Combined doses of rifampicin and tamoxifen resulted in growth inhibition to an extent that was suggestive of additive or synergistic drug effects. More detailed analysis was carried out, involving the production of an 'envelope of additivity' for each cell line. An additive effect of drug combination was shown to occur in all three cell lines, thus providing a basis for use of this novel two-drug combination in the treatment of biliary tract-associated malignancies.
Biliary Tract Neoplasms↗
Formation of the Dictyostelium spore coat.
The spore coat forms as a rigid extracellular wall around each spore cell during culmination. Coats purified from germinated spores contain multiple protein species and an approximately equal mass of polysaccharide, consisting mostly of cellulose and a galactose/N-acetylgalactosamine polysaccharide (GPS). All but the cellulose are prepackaged during prespore cell differentiation in a regulated secretory compartment, the prespore vesicle. The morphology of this compartment resembles an anastomosing, tubular network rather than a spherical vesicle. The molecules of the prespore vesicles are not uniformly mixed but are segregated into partially overlapping domains. Although lysosomal enzymes have been found in the prespore vesicle, this compartment does not function as a lysosome because it is not acidic, and a common antigen associated with acid hydrolases is found in another, acidic vesicle population. All the prespore vesicle profiles disappear at the time of appearance of their contents outside of the cell; this constitutes an early stage in spore coat formation, which can be detected both by microscopy and flow cytometry. As an electron-dense layer, the future outer layer of the coat, condenses, cellulose can be found and is located immediately beneath this outer layer. Certain proteins and the GPS become associated with either the outer or inner layers surrounding this middle cellulose layer. Assembly of the inner and outer layers occurs in part from a pool of glycoproteins that is shared between spores, and unincorporated molecules loosely reside in the interspore matrix, a location from which they can be easily washed away. When the glycosylation of several major protein species is disrupted by mutation, the coat is assembled, but differences are found in its porosity and the extractibility of certain proteins. In addition, the retention or loss of proteolytic fragments in the mutants indicates regions of spore coat proteins that are required for association with the coat. Comparative examination of the macrocyst demonstrates that patterns of molecular distributions are not conserved between the macrocyst and spore coats. Thus spore coat assembly is characterized by highly specific intermolecular interactions, leading to saturable associations of individual glycoproteins with specific layers and the exclusion of excess copies to the interspore space.
Chromatography, High Pressure Liquid↗
Spore coat is altered in modB glycosylation mutants of Dictyostelium discoideum.
The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation-defective glycoprotein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which are specific because they are distinctive from changes observed in another glycosylation mutant which affects a different epitope.
Dictyostelium↗
Radiosensitivity testing of primary cervical carcinoma: evaluation of intra- and inter-tumour heterogeneity.
Biopsies from 89 patients with cervical carcinoma were studied using a clonogenic assay to obtain values for the surviving fraction at 2 Gy (SF2). Heterogeneity in intrinsic radiosensitivity was investigated by independently processing multiple biopsies from 18 tumors. No significant differences between intra-tumour SF2 values were demonstrated (p = 0.30). The results have shown that intra-tumour heterogeneity is not a limitation to radiosensitivity testing using the Courtenay-Mills assay. A wide range of values (0.13-0.97) for SF2 was obtained with a mean value of 0.47 +/- 0.18 (+/- 1 S.D., CV = 38%) for 52 squamous cell carcinomas and 0.59 +/- 0.27 for four adenocarcinomas. There were statistically significant differences between the individual tumours (p less than 0.001). From the analysis-of-variance of all the SF2 results it appears to be the surviving fractions below about 0.40 and those above about 0.7 which show significant differences in radiosensitivity between pairs of tumours (p = 0.05). Also 36% of the values of SF2 show significant differences from the mean SF2 of all tumours. The storage of tumour cell suspensions in liquid nitrogen improved the colony-forming efficiency (CFE) but it did not alter the radiosensitivity.
Analysis of Variance↗
A comparison of the sensitivity to photodynamic treatment of endothelial and tumour cells in different proliferative states.
Bovine aorta endothelial and human colon adenocarcinoma (WiDr) cells were exposed to haematoporphyrin derivative plus light. Cell survival was determined using a [3H]thymidine incorporation and a clonogenic assay for both cell types. The endothelial cells were more sensitive to photodynamic treatment. This could be explained in terms of increased drug uptake into the endothelial cells despite there being no difference in cell volume between the cells. For both tumour and endothelial cells, exponentially growing cells were more sensitive to photodynamic treatment than plateau-phase cells. This was associated with a reduction in drug uptake into plateau-phase endothelial cells. However, there was no difference in uptake into exponentially growing and plateau-phase WiDr cells.
Animals↗
The spore coat of a fucosylation mutant in Dictyostelium discoideum.
Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.
Antibodies, Monoclonal↗
Size-dependent resistance of human tumour spheroids to photodynamic treatment.
Spheroids derived from the human colon adenocarcinoma cell line, WiDr, were exposed to 10 micrograms ml-1 Photofrin II and irradiated with light (700 nm, 50 mW cm-2). Compared with exponentially growing monolayer cultures, cells in spheroids of 100, 250 and 500 microns diameter were respectively 1.8, 2.5 and 22-fold less sensitive. The small resistance of plateau-phase cultures (1.3-fold) was insufficient to account for this marked spheroid size-dependent resistance. For monolayer cultures and for spheroids of 100 and 250 microns diameter, the results were the same whether irradiations were carried out pre- or post-trypsinisation. However, there was a difference for the largest spheroid size: when irradiations were carried out pre-trypsinisation, spheroids were more resistant than when irradiations were given post-trypsinisation. Drug extraction studies showed that there was no difference in the average drug uptake between cultures of exponentially growing or plateau-phase cells, and 100 microns diameter spheroids while 250 and 500 microns diameter spheroids took up proportionally 0.5 and 0.4 as much drug. Cell contact effects, drug heterogeneity between cells, hypoxia and problems in drug penetration are suggested as possible reasons for the resistance of large spheroids to photodynamic treatment.
Adenocarcinoma↗
A glycosylation mutation affects cell fate in chimeras of Dictyostelium discoideum.
Prestalk and prespore cells form a simple pattern in the pseudoplasmodium of the cellular slime mold Dictyostelium discoideum. Prestalk cells are distinguished from prespore cells by a low level of expression of a glycoantigen on their surfaces and by reduced intercellular cohesion. We examined the possible significance of these differences, using the modB mutation, which eliminates this glycoantigen genetically, leading to reduced intercellular cohesion, modB mutant cells were allowed to develop together with normal cells to form chimeric slugs. Mutant cells labeled by feeding with fluorescent bacteria were highly enriched in the prestalk cell zone at the anterior end of the slug. In contrast, normal cells, if in a minority, were concentrated in the rear part of the prespore cell zone. Immunoblot analysis and cell-by-cell double-label immunofluorescence of these mixtures showed that mutant cells underproduced several prespore cell markers. Mutant cells tended not to form spores in chimeras unless they exceeded a threshold proportion of ca. 30%. However, mutant cells showed no tendency to produce excess prestalk cells when allowed to develop alone. These findings are most simply explained by postulating that reduced glycoantigen expression and intercellular adhesion encourage a more anterior cell localization, which in turn causes differentiation into a prestalk cell. Since normal prestalk cells also show reduced glycoantigen expression and intercellular adhesion, this suggests that a similar mechanism may contribute to pattern formation during normal development.
Antibodies, Monoclonal↗
Evaluation of surviving fraction at 2 Gy as a potential prognostic factor for the radiotherapy of carcinoma of the cervix.
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Cell Survival↗
The photodynamic effects of photofrin II, hematoporphyrin derivative, hematoporphyrin, and tetrasodium-mesotetra(4-sulfonatophenyl)porphine in vitro: clonogenic cell survival and drug uptake studies.
The human colon adenocarcinoma cell line, WiDr, was exposed to Photofrin II, hematoporphyrin derivative (HPD), hematoporphyrin (HP) or tetrasodium-meso-tetra(4-sulfonatophyenyl)porphine (TPPS4) followed by irradiation with light. Clonogenicity was determined and the resultant survival curves compared and shown to be qualitatively similar in shape. However, for equal amounts of drug in the medium, there were large differences in photosensitizing efficiency with Photofrin II approximately 5, 25 and 50 fold more effective than HPD, HP and TTPS4, respectively. For the same power used, all drugs were less efficient photosensitizers under red light (600-1100 nm) than under white light (300-110 nm). For all drugs this could be explained in terms of changes in light absorption over the two wavelength ranges. Differences in clonogenic cell survival could not be explained in terms of differences in singlet oxygen production (from published values). A reduction in drug uptake into the cells was sufficient to explain the differences between Photofrin II, HPD and HP, while TPPS4 was 5-fold less effective compared to other drugs than would be expected from drug uptake measurements. Two methods for measuring drug uptake were compared and shown to give different results for Photofrin II. Measurements of drug fluorescence in 0.1 N NaOH yielded 5-fold lower values than when measurements were in 1 N HCl following heat treatment to monomerise aggregated drug. Clearly the reliability of the method used in determining drug uptake must be carefully ascertained.
Adenocarcinoma↗
Flow cytometric analysis of intracellular hematoporphyrin derivative in human tumor cells and multicellular spheroids.
Flow cytometry (FCM) has been used to investigate the intracellular fluorescence of hematoporphyrin derivative (HPD) in monolayer and spheroid cultures of WiDr cells. For exponentially-growing monolayer cultures mean cellular fluorescence was directly proportion to the external HPD levels in the range 5-100 micrograms ml-1 (r = 0.99). Heterogeneity of cellular fluorescence was quantified by determining the ratio of the fluorescence value below which were observed values for 98% of the cell population compared to the fluorescence value for 2%. In exponentially-growing cultures, decreasing levels of HPD in the medium led to an increase in the 98:2% ratio, i.e. an increase in heterogeneity of intracellular drug levels. The growth of cells as multicellular spheroids confers a spheroid-size-dependent resistance to photodynamic treatment. With increasing spheroid size (100, 250, 500, 750 and 1000 microns diam.) there was a decrease in mean intracellular HPD levels and a large linear increase in the 98:2% ratio (r = 0.94).
Adenocarcinoma↗
EDTA treatment alters protein glycosylation in the cellular slime mold Dictyostelium discoideum.
We have found that treatment of cells with EDTA resulted in the accumulation of lower molecular weight forms of two cell-type-specific glycoproteins. These new glycoproteins lacked a developmentally regulated glycoantigen defined by monoclonal antibody 54.2. Since EDTA dissociated the cells, the possible involvement of cell separation was tested by immobilizing cells in soft agarose. Glycoantigen expression on these proteins was found to be dependent on cAMP and high oxygen tension but not on cell contact, and was reversibly sensitive to EDTA regardless of the state of cell association. The EDTA effect was mimicked by other soluble, but not particulate, membrane impermeable chelators, could be competed by Zn2+ better than Mg2+, and appeared to involve an intracellular mechanism. Studies with [14C]EDTA showed that EDTA equilibrated with a cellular compartment in a temperature-dependent, Zn2+-insensitive fashion with half-time kinetics of loading and unloading of 30-40 min. If the compartment was assumed to be labeled with the same concentration of EDTA as was present extracellularly, calculations showed that its volume was circa 2% of the total cell volume. This compartment probably consists of intracellular vesicles based on the similar labeling of this compartment with a bulk fluid phase marker, inulin. The data suggest that this step in glycosylation, which was found to be delayed 1 or more hours subsequent to protein synthesis, involves an intracellular, transition metal ion-dependent process which can be modulated by chelators entering the cell through the endocytic pathway.
Cations, Divalent↗
The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum.
In this report we examine the accumulation of glycoconjugates in the extracellular medium and insoluble matrices surrounding developing cells of the cellular slime mold Dictyostelium discoideum. Conditions were employed which permitted advanced development (slug stage and beyond) in suspension culture. Under these conditions, up to one-third of the total culture protein appeared as non-sedimentable, extracellular material over the course of 48 h of incubation. Most of the secreted molecules expressed carbohydrate antigens (glycoantigens) as detected by Western blotting, using a panel of six monoclonal antibodies. Since the glycoantigens are secreted, immunoelectron microscopy was used to localize the glycoantigens in the extracellular matrices surrounding normally developing cells, including the slime sheath, stalk tube, inner spore coat, outer spore coat, and intercellular fluid between spores. Each glycoantigen had a characteristic distribution, and each extracellular matrix space contained a unique combination of glycoantigens. Thus, although each of these matrices (except inter-spore fluid) contains cellulose as a primary component, they could be distinguished on the basis of their glycoantigen and, by inference, glycoprotein compositions. Furthermore, there were differences between anterior and posterior regions of both slime sheaths and stalk tubes. These observations show that secretion as detected in suspension culture occurs under normal conditions as a part of the process of depositing extracellular matrices around the cells. The distributions show that the cell aggregate positionally regulates the expression and deposition of secretory glycoproteins; the resultant patterns of expression of unique protein-linked carbohydrate structures imply a functional role in matrix organization and possibly cell activity which can now be explored.
Antibodies, Monoclonal↗
Growth phase related variation in the radiation sensitivity of human colon adenocarcinoma cells.
Changes in radiation sensitivity with length of time in culture are described for an early passage human colon adenocarcinoma cell line (WiDr). The cells were most radioresistant at the end of lag-phase, 2 days after subculture (Do = 1.7 Gy; n = 10). Radiation sensitivity then increased with time reaching a maximum during plateau-phase, between days 8 and 10 (Do = 1.0 Gy; n = 13). The oxygen enhancement ratio remained constant across the different growth phases of the culture. Cell volume decreased with time in culture as did the proportion of S- and G2M-phase cells. Flow cytometric analyses revealed an increase in the proportion of G1 type cells with a plateau between days 8 and 12 of around 75%. The cell age response measured from synchronized cells following 8 Gy showed that WiDr cells were most radiation resistant in mid S-phase with maximal sensitivity during G1. These cells did not show repair of potentially-lethal radiation damage but were efficient in the repair of sub-lethal damage. The ability to repair sub-lethal damage did not change with culture age.
Adenocarcinoma↗
Cell survival characteristics of a human colon adenocarcinoma cell line after photodynamic treatment: a comparison of Photofrin II and TPPS.
A comparison has been made of the in vitro light-activated drug cytotoxicities of two different porphyrin compounds, Photofrin II and TPPS4. An early passage human colon adenocarcinoma cell line, WiDr, has been exposed to either drug for 24 h, the excess drug washed from the cells and the cells irradiated with light using quartz-tungsten-halogen lamps. Neither light nor drug alone under the experimental conditions employed was toxic to WiDr cells. Together, considerable cytotoxicity could be seen and the shapes of the cell survival curves following exposure to either drug then irradiation with light, were similar. For equal amounts of drug in the medium, Photofrin II was a more efficient photosensitizer of WiDr cells than TPPS4, and differences in cellular uptake could only partly explain this. When the experimental procedure was changed by reducing the temperature of irradiation, a reduction in photosensitizing efficiency could be demonstrated. This was more pronounced for Photofrin II, and was seen as a change in the slope of the final portion of the survival curve; and as a change in the shoulder for TPPS4. Two different batches of the two drugs were compared and shown to give slightly different results for Photofrin II (change in shoulder) but not to differ for TPPS4.
Adenocarcinoma↗