Biomedical subjects

R M Sutherland

Publications and source records attributed to R M Sutherland.

At least 55 records · Page 3Linked to original sources

PubMed1992

Potentiation of alkylating chemotherapy by dual function nitrofurans in multi-cell spheroids and solid tumors.

The cytotoxicity and chemosensitizing potential of four dual function nitrofurans was determined in human HT-29 multi-cell spheroids and rodent KHT sarcoma solid tumors. Spheroids were treated with a range of doses of the bioreductive drugs for a period of up to 48 h and the extent of cell kill was assessed at various times after treatment. Cytotoxicity was determined using a clonogenic cell-survival assay. The results demonstrated that two of the nitrofurans were even more toxic to spheroid cells than was the potent bioreductive nitroimidazole aziridine RSU 1069. The dose of the nitrofuran which, after a 24-h exposure, led to a survival value between 0.5 and 1.0, then was chosen for subsequent studies aimed at assessing the ability of these agents to potentiate the efficacy of the nitrosourea CCNU. Exposure to this chemotherapeutic agent was for a period of 1 h. The results indicated that all four dual function nitrofurans enhanced the cell killing of the conventional chemotherapeutic agent by factors ranging from 1.1 to 1.7. Subsequent studies evaluated the therapeutic benefit of combining these bioreductive agents and CCNU in KHT sarcoma-bearing C3H/HeJ mice. The nitrofurans were administered i.p. 0.5 h prior to the chemotherapy and tumor response was assessed by measuring the survival of clonogenic KHT cells 22-24 h after treatment. Normal tissue toxicity was determined using a bone marrow stem cell (CFU-GM) assay. Combining these bioreductive agents with CCNU increased the tumor cell kill by factors of 1.2 to 1.7.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Reoxygenation associated radiosensitization after chronic hypoxia: effect of temperature and oxygen tension.

A431 human squamous carcinoma cells reoxygenated after 12 hr hypoxia were found to be more radiosensitive than aerobic cells not previously exposed to hypoxia. Experiments were carried out to investigate the effect of temperature and oxygen tension during the hypoxia and the reoxygenation periods on the sensitization effect. Incubation at 0 degrees C during 12 hr of hypoxia inhibited the sensitization seen after reoxygenation. During the reoxygenation period, reduced temperature did not modify the levels of enhancement but suppressed the decay of the sensitization. If oxygen tension during 12 hr of hypoxia was less than 10 ppm, maximal enhancement by reoxygenation was seen, but there was no enhancement if the oxygen concentration was greater than 1%. In cells subjected to less than 10 ppm oxygen for 12 hr, the level of sensitization by reoxygenation in 2 to 20% oxygen concentration was similar. The results indicate that radiosensitization by reoxygenation may require metabolic change(s) of cells during both the hypoxia and reoxygenation periods. The sensitization may also involve oxygen dependent species of reactions.

Carcinoma, Squamous Cell↗

PubMed1992

Effect of flunarizine on micro-regional distributions of intravascular HbO2 saturations in RIF-1 and KHT sarcomas.

Flunarizine, a calcium channel-blocker, preferentially active on peripheral blood vessels and red blood cells, has previously been shown to increase not only tumor blood flow, but also both chemosensitivity and radiosensitivity. The mechanisms behind these effects are not clear at present, and different tumor lines have been demonstrated to respond quite differently to the same flunarizine treatment. To clarify the underlying mechanisms at the micro-regional level, the present investigation examined the effects of flunarizine on spatially-resolved distributions of intravascular oxyhemoglobin (HbO2) saturations. Tumors were implanted s.c. in the flanks of C3H mice and grown to a volume of 500-1000 mm3. Flunarizine was injected i.p. at 5 mg/kg; animals were anesthetized, and following a 20 min delay, tumors were quick-frozen. HbO2 saturations were determined cryospectrophotometrically at systematically selected sites across each tumor cross-section. Three primary questions were addressed: (a) Does flunarizine have a direct effect on tumor oxygen delivery as measured by intravascular HbO2 saturations? (b) Are changes in oxygen delivery uniformly distributed throughout the tumor volume? (c) Are HbO2 distributions for tumor lines of dissimilar radiobiological hypoxic fraction affected similarly by flunarizine? For KHT tumors, flunarizine substantially increased the % vessels greater than or equal to 10% saturated throughout the tumor volume, with a somewhat larger increase for the interior vessels than for the peripheral vessels. In contrast, for the RIF-1 tumors, HbO2 saturations following flunarizine remained near control levels. These results indicate that fundamental differences in vascular geometry, hemodynamics, or blood rheology may exist between the KHT and RIF-1 tumor lines, and furthermore, that the response of one tumor line to a given blood-flow modifier may be completely unrelated to that of another.

Animals↗

PubMed1992

Cell cycle dependence of epidermal growth factor induced radiosensitization.

The effect of epidermal growth factor on the radiation response of two human squamous carcinoma cell lines, A431 (from vulva) and SiHa (from cervix), was examined. In both lines, cells in S phase were more radioresistant than cells in other cell cycle phases. Epidermal growth factor present after irradiation enhanced the radiation response of A431 cells in different cell cycle phases, whereas no effect was seen for SiHa cells. The enhancement was maximum with 10 ng/ml epidermal growth factor and was associated mainly with a reduction in the shoulder region of the cell survival curve. The ratio between the n values of the control and epidermal growth factor treated total cell population, G1, S, and G2M cells is 2.2, 4.1, 1.7, and 2.2, respectively. Epidermal growth factor reduced plating efficiency by about 50% for A431 cells in different cell cycle phases whereas a slight increase in plating efficiency was seen for SiHa cells. The present results indicate that epidermal growth factor related radiosensitization is dependent on both cell line and cell cycle.

Carcinoma, Squamous Cell↗

PubMed1992

Enhancement of transforming growth factor-alpha synthesis in multicellular tumour spheroids of A431 squamous carcinoma cells.

Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.

Blotting, Northern↗

PubMed1992

Mathematical modelling of microenvironment and growth in EMT6/Ro multicellular tumour spheroids.

In order to determine the role of micromilieu in tumour spheroid growth, a mathematical model was developed to predict EMT6/Ro spheroid growth and microenvironment based upon numerical solution of the diffusion/reaction equation for oxygen, glucose, lactate ion, carbon dioxide, bicarbonate ion, chlorine ion and hydrogen ion along with the equation of electroneutrality. This model takes into account the effects of oxygen concentration, glucose concentration and extracellular pH on cell growth and metabolism. Since independent measurements of EMT6/Ro single cell growth and metabolic rates, spheroid diffusion constants, and spinner flask mass transfer coefficients are available, model predictions using these parameters were compared with published data on EMT6/Ro spheroid growth and micro-environment. The model predictions of reduced spheroid growth due to reduced cell growth rates and cell shedding fit experimental spheroid growth data below 700 microns, but overestimated the spheroid growth rate at larger diameters. Predicted viable rim thicknesses based on predicted near zero glucose concentrations fit published viable rim thickness data for 1000 microns spheroids grown at medium glucose concentrations of 5.5 mM or less. However, the model did not accurately predict the onset of necrosis. Moreover, the model could not predict the observed decreases in oxygen and glucose metabolism seen in spheroids with time, nor could it predict the observed growth plateau. This suggests that other unknown factors, such as inhibitors or cell-cell contact effects, must also be important in affecting spheroid growth and cellular metabolism.

Animals↗

PubMed1991

Epidermal growth factor reduces resistance to doxorubicin.

Epidermal growth factor (EGF) increased the sensitivity to doxorubicin (DOX) of a human squamous carcinoma cell line, A431. The relative enhancement of sensitivity by EGF was greater in 2 DOX-resistant sublines, A431/A5 and A431/A10, established by growing cells from surviving colonies after treatment of A431 cells with DOX. A greater number of EGF receptors (both high- and low-affinity binding sites) was found for resistant A431/A5 and A431/A10 cells than for parental cells. The enhanced drug responsiveness is not directly related to EGF effects on growth, as growth inhibition by EGF appears to be similar among the 3 sublines.

Cell Division↗

PubMed1991

Oxygen regulated 80 kDa protein and glucose regulated 78kDa protein are identical.

Ischemic stress of cells within solid tumors arises from inadequate perfusion of regions of the tumor and results in microenvironments which are hypoxic and deficient in nutrient delivery and waste product removal. Stressed cells within these microenvironments show growth inhibition and synthesize unique sets of proteins referred to as glucose and oxygen regulated proteins (GRPs and ORPs respectively). The commonality of proteins induced by glucose-starvation and hypoxia has not been proven. To this end, ORPs were induced in Chinese hamster ovary cells in the presence of high glucose concentration in the media and ORP 80 isolated from two dimension gels. Eleven tryptic peptides of the 80 kDa ORP were sequenced and found to be identical to GRP 78 sequences. The data demonstrate that GRP 78 and ORP 80 have the same primary amino acid sequence and suggest that glucose-starvation and hypoxia can induce the same cellular responses.

Amino Acid Sequence↗

PubMed1991

Nonperturbing test for cytotoxicity in isolated cells and spheroids, using electron paramagnetic resonance.

Electron paramagnetic resonance imaging (EPRI) and EPR spectroscopy of nitroxides have been applied to detect and quantify cytotoxic effects in mammalian cells in vitro. Images depicting microscopic viable and nonviable areas in the same multicellular spheroid (a model of solid tumors) have been acquired prior to and following the treatment with an antitumor drug, Adriamycin. The loss of viability in the inner region of the viable rim was detected. Such mapping of the time-course changes of the localization of viable and nonviable cells in the same intact biological object is not possible with routinely used methods to measure viability, such as histological examination. The experimental conditions required for high accuracy and sensitivity of the EPRI of spheroids have been evaluated and directions for further development of this approach are indicated.

Animals↗

PubMed1991

Time-dependent effects of anesthetic agents on 31P NMR high-energy phosphates in KHT and RIF-1 fibrosarcomas.

Previous studies have reported significant radiobiological and hemodynamic effects associated with sodium pentobarbital (PB) anesthetization. The present work contrasts the effects of PB with azaperone-ketamine (AZ) in RIF-1 and KHT tumors while animal body core temperature is maintained at 37 degrees C. The primary aims were to evaluate both agents in terms of: (i) duration of anesthetic; (ii) effect on absolute levels of 31P NMR phosphocreatine (PCr) + beta-nucleoside triphosphate (beta-NTP)/inorganic phosphate (Pi) ratios; and (iii) effect on temporal variability of PCr + beta-NTP/Pi ratios. In terms of overall duration, AZ was the clear preference. Although the maintenance of 37 degrees C core temperature significantly reduced overall durations for both anesthetics, AZ animals invariably remained immobile for a minimum of 80 min. For PB, durations were highly unpredictable. With AZ, mean PCr + beta-NTP/Pi ratios were constant over the entire 80 min period for both lines. With PB, PCr + beta-NTP/Pi ratios were lower in relation to AZ for KHT at select timepoints, but highly variable among RIF-1 tumours. Since ratios under PB varied substantially with time for RIF-1 lines, measurements taken with PB are clearly not representative of the control state. Furthermore, in light of the consistent and reproducible results obtained with AZ, this anesthetic is considered a marked improvement over PB for animal studies of this nature.

Anesthetics↗

PubMed1991

A new mouse T-cell receptor alpha chain variable region family.

Sequence analysis of the rearranged T-cell receptor alpha chain gene segments from an influenza reactive T-cell clone T2.5-5 and a hemin chloride reactive T-cell hybrid SJL-HE-1.1 have revealed a previously undescribed V alpha gene family. We have designated this family V alpha 15. Southern hybridization analysis has indicated that this family most probably contains only two members, and that these are conserved in each of six mouse strains representing three previously described V alpha haplotypes: V alpha a, V alpha b, and V alpha c.

Amino Acid Sequence↗

PubMed1991

Epidermal growth factor modification of radioresistance related to cell-cell interactions.

The effect of epidermal growth factor on radiosensitivity of cells in spheroids and its relationship to radioresistance associated with cell-cell interactions was examined. A human squamous carcinoma cell lines, CaSki, was used. Epidermal growth factor present for 48 hr before irradiation reduced the plating efficiency but did not affect the radiosensitivity of cells. However, epidermal growth factor present after irradiation, that is, during the period of colony formation, reduced the plating efficiency and increased the radiosensitivity of cells from spheroids. Both effects were maximum at 10 ng/ml epidermal growth factor. The enhancement in radiation response was not related to epidermal growth factor effects on potentially lethal and sublethal damage repair. In the absence of cell-cell interactions, such as monolayer cultures and spheroids disaggregated for 15 hr before irradiation, radiosensitivity enhancement by epidermal growth factor was associated with reduced shoulder of the cell survival curve. However, in the presence of cell-cell interactions, such as intact spheroids and spheroids disaggregated immediately before irradiation, in addition to reduced shoulder, epidermal growth factor treatment increased the slope compared to that of the monolayer cultures. The results indicate that epidermal growth factor enhances cellular radiosensitivity and modifies effects of cell-cell interactions.

Carcinoma, Squamous Cell↗

PubMed1991

The effect of antigen concentration, antibody valency and size, and tumor architecture on antibody binding in multicell spheroids.

Intact IgG1 and F(ab')2 anti-carcinoembryonic antigen antibodies penetrate human colon adenocarcinoma multicell spheroids much more slowly than Fab fragments and the molecular weight and the binding site valency appear to be the most important factor in determining the rate of penetration. The rate is also influenced considerably by the number of antigen binding sites per cell, with a high antigen concentration slowing penetration appreciably. The tumor cell architecture appears to have a minor effect on antibody penetration when compared to antibody size or antigen concentration.

Adenocarcinoma↗

PubMed1991

The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells.

Chronic hypoxia increases the expression of a set of stress proteins (oxygen regulated proteins or ORPs) which is implicated in the development of drug resistance and radiation sensitivity in tumour cells. Five major ORPs have been documented, and two, ORP 80 and ORP 100, are considered to be identical to the glucose regulated stress proteins GRP78 and GRP94, respectively. We report here that ORP 33 is a form of the heme catabolic enzyme, heme oxygenase, using evidence obtained from northern blotting, two-dimensional polyacrylamide gel electrophoresis and western analysis. Heme oxygenase is believed to be an important component of the cellular response to oxidative stress. The significance of heme oxygenase as a hypoxia-induced stress protein is discussed.

Aerobiosis↗

PubMed1991

Differences in EGF related radiosensitisation of human squamous carcinoma cells with high and low numbers of EGF receptors.

Previous studies have shown that the presence of epidermal growth factor (EGF) after irradiation enhanced the radiosensitivity of CaSki cells. To examine the role of EGF receptor density and related growth response in EGF associated radiosensitisation, four human squamous carcinoma cell lines were used. The total number of EGF receptors for HN5, A431, CaSki, and SiHa cells is 5.2 x 10(6), 1.6 x 10(6), 7.9 x 10(5) and 1.1 x 10(5) respectively, and the dissociation constant (Kd) for low affinity EGF receptors is 11.8, 3.8, 1.7 and 0.8 nM respectively. The Kd for high affinity receptors differs slightly among the four cell lines, 0.09 to 0.21 nM. EGF inhibited the growth of A431, CaSki, and HN5 cells, but stimulated the growth of SiHa cells. Due to the presence of 10 ng ml-1 EGF after irradiation, radiosensitivity enhancement associated with reduced shoulder size of the survival curve was observed. The extent of sensitisation was similar for A431, CaSki, and HN5 cells, with no effect on SiHa cells. At this concentration, EGF present during the clonogenic assay period after irradiation also reduced the plating efficiency (PE) of the monolayer cultures of A431, CaSki, and HN5 cells, but increased that of SiHa cells. The radiation response of mouse 3T3 cells (less than 5,000 receptors) was not sensitised by EGF. A similar level of radiosensitivity enhancement by EGF was observed for parental and conditioned A431 cultures. The conditioned cells were grown in 50 ng ml-1 EGF for 10 weeks and did not demonstrate growth inhibition and PE reduction by treatment with EGF. The EGF receptor numbers and binding affinity of these cells were the same as for the parental cells. The results from the conditioned cells support the hypothesis that EGF related radiosensitisation is EGF receptor density dependent.

Animals↗

PubMed1991

Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance.

In this report, we investigate several examples of hypoxia-induced drug resistance and compare them with P-glycoprotein associated multidrug resistance (MDR). EMT6/Ro cells exposed to drugs in air immediately after hypoxic treatment developed resistance to adriamycin, 5-fluorouracil, and actinomycin D. However, these cells did not develop resistance to colchicine, vincristine or cisplatin. When the cells were returned to a normal oxygen environment, they lost resistance. There was no correlation between the content of adriamycin and the development of adriamycin resistance induced by hypoxia. There was no difference between the efflux of adriamycin from aerobic cells and that from hypoxia-treated cells. The mRNA for P-glycoprotein was not detected in the hypoxia-treated cells. These results suggest that hypoxia-induced drug resistance is different from P-glycoprotein associated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1991

The influence of cell-cell contact on radiosensitivity of human squamous carcinoma cells.

Previous reports have indicated that for a number of cell lines, radiation resistance was greater when cells were grown as small spheroids than when grown as monolayer cultures. Experiments were conducted to study the increased radioresistance relative to recoveries from potentially lethal and sublethal damage (PLD and SLD, respectively) in two human squamous carcinoma cell lines, A431 and CaSki. Exponential-phase monolayer cultures and small spheroids 30-70 microns in diameter were used. No significant difference in the distribution of cells in the stages of the cell cycle was seen between the two cultures. To study PLD repair, cells were irradiated and then allowed to repair, all at 37 degrees C, for 6 h. In split-dose experiments, the two doses were given 6 h apart. After irradiation, the surviving fraction of cells was measured by clonogenic assay. Cells from CaSki spheroids (D0 = 1.25 Gy, Dq = 3.08 Gy) were more radioresistant than cells from the monolayer culture (D0 = 0.92 Gy, Dq = 2.8 Gy). No difference in radiation response was seen between the spheroid and monolayer cultures of A431 cells. The radioresistance of CaSki cells, which was maximum in intact small spheroids and immediately after disaggregation of spheroids, decayed completely by 15 h at 37 degrees C. The decay process was inhibited if cells were incubated at 0 degrees C. Relative PLD and SLD repair capacities for both cell lines were greater for monolayer cultures than for small spheroids. However, no correlation was found between the two cell lines in terms of repair capacity and cellular radiosensitivity.

Carcinoma, Squamous Cell↗

PubMed1990

Assessment of tumor energy and oxygenation status by bioluminescence, nuclear magnetic resonance spectroscopy, and cryospectrophotometry.

The energy and oxygenation status of tumors from two murine sarcoma lines (KHT, RIF-1) and two human ovarian carcinoma xenograft lines (MLS, OWI) were assessed using three independent techniques. Tumor energy metabolism was investigated in vivo by 31P nuclear magnetic resonance spectroscopy. After nuclear magnetic resonance measurements, tumors were frozen in liquid nitrogen to determine the tissue ATP concentration by imaging bioluminescence and to register the intracapillary oxyhemoglobin (HbO2) saturation using the cryospectrophotometric method. There was a positive correlation between the nucleoside triphosphate beta/total resonance ratio or a negative correlation between the Pi/total resonance ratio and the model ATP concentration obtained by bioluminescence, respectively. This was true for small tumors with no extended necrosis irrespective of tumor type. Moreover, a positive correlation was obtained between the HbO2 saturations and the ATP concentration measured with bioluminescence. The results demonstrate the potential of combined studies using noninvasive, integrating methods and high-resolution imaging techniques for characterizing the metabolic milieu in tumors.

Adenosine Triphosphate↗