The effect of ultrasound on the cytotoxicity of adriamycin

The effect of continuous wave ultrasound exposures on the cytotoxicity of adriamycin has been studied. It has been found that 2.6 MHz, 2.3 Wcm-2 (spatial average) ultrasound can enhance the cell killing potential of adriamycin both in suspensions of single V79 chinese hamster fibroblast cells and in spheroids formed from these cells. The ratio of the slopes of the survival curves for single cell suspensions is 1.5. For spheroids, the growth delay is increased by 1.3 days by simultaneous ultrasound exposure. Flow cytometric studies of the intracellular concentration of adriamycin following ultrasound exposure reveals that this is increased when compared with that measured when the cells are only exposed to adriamycin. Evidence is presented to suggest that this is a non-thermal effect of ultrasound.     

Cure, regression and cell survival: a comparison of common radiobiological endpoints using an in vitro tumour model.

Multicell spheroids of Chinese hamster V79-171 cells grown in suspension culture display many of the characteristics of solid tumours in vivo, and can be used as an in vitro tumour model. Two populations of spheroids differing in age and radiosensitivity were exposed to single doses of gamma-radiation and their response assayed by several techniques: (1) spheroids were reduced to single cells by trypsinization at various times post-irradiation, and viability of the single cells determined by colony formation; (2) entire spheroids were placed in petri dishes and observed for cellular outgrowth; (3)spheroid volume and cell content were monitored as a function of time after irradiation. It was found that spheroid volume changes could not be correlated with either the amount of radiation given or with the relative radiosensitivity. In contrast, the number of cells per spheroid, or cellularity, decreased exponentially with exposure dose at sufficiently long times after irradiation. Radiosensitivity was then quantified by calculating the per cent decrease of cellularity per rad. "Cure" of spheroids as defined by lack of outgrowth when entire spheroids were placed in petri dishes, correlated well with single cell survival, and was achieved 50 per cent of the time after 2,630 rads for the smaller spheroids and 3,750 rads for the larger ones. Since these spheroids contained an average of similar 7,600 and similar 30,700 cells respectively, comparison of cure data with single cell survival data showed that cures were achieved only when every cell was killed. This result may have significant therapeutic implications, since cells of the most radioresistant population of the spheroid, the chronically hypoxic internal cells, were capable of proliferation even when the spheroid was not reduced to single cells after irradiation.     

Radiation response of multicellular spheroids initiated from five human melanoma xenograft lines. Relationship to the radioresponsiveness in vivo

Multicellular spheroids, initiated from five human melanoma xenograft lines (E.E., E.F., G.E., M.F., V.N.) and grown in liquid-overlay culture, were characterised with regard to radiation response. The principal aim of the work was to search for possible correlations with the radioresponsiveness in vivo of the parent xenografts. The spheroids were 100 +/- 5 microns in diameter at irradiation and did not contain radiobiologically hypoxic cells. Single-cell survival measured in soft agar, specific growth delay and spheroid cure were used as end-points. The cellular radiosensitivity was the same whether a melanoma was grown as spheroids or as xenografts. An intercellular contact effect was found for spheroids of the G.E. melanoma but not for spheroids of the E.E., E.F., M.F. and V.N. melanomas, in agreement with previous observations from studies of the corresponding xenografts in vivo. A positive correlation was found between the radiation response of the spheroids, measured as cell survival after 6 Gy or as specific growth delay after 6 Gy, and the radiation response of the parent tumours, measured as specific growth delay after 15 Gy. There was no correlation between the SCD50 (the dose required to cure 50% of the spheroids) and the radioresponsiveness in vivo. It is concluded that differences in radioresponsiveness in vivo among tumours may be identified from studies of the corresponding multicellular spheroids grown in liquid-overlay culture.     

Linear quadratic model of radiocurability on multicellular spheroids of human lung adenocarcinoma LCT1 and mouse fibrosarcoma FSA.

The LCT1 cells derived from a human lung adenocarcinoma and the FSA cells from a mouse fibrosarcoma were found to form spheroids. The cure-dose relationship of spheroids and the survival curves of their component cells were analysed by using a linear-quadratic model for cell survival and a Poisson distribution for cure. The analysis resulted in three conclusions: (1) the double minus logarithm of cure probability was linearly related to radiation dose, (2) the critical cell number was constant at any given cure probability, and (3) cellular radiosensitivity was also constant. The experiments seem to meet these conditions for each of two kinds of spheroids. Control doses (50%) were 20 Gy for LCT1 spheroids and 21 Gy for FSA spheroids, both 400 microns in diameter. The analysis showed that the lower cellular radiosensitivity and the higher number of clonogenic cells made LCT1 spheroids more radioresistant than FSA spheroids and that the higher critical number of 130 cells made the LCT1 spheroids more sensitive than the FSA spheroids with 18 such cells. The overall radiocurability of spheroids was a result of these three opposing effects, indicating that the critical cell number can be one important factor in determining the radiocurability of multicellular systems.     

Identification of genes differentially expressed in V79 cells grown as multicell spheroids

Purpose : Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids. Materials and methods : Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay. Results : Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calciumbinding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide. Conclusions : Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.     

Identification of genes differentially expressed in V79 cells grown as multicell spheroids

PURPOSE: Growth of Chinese hamster V79 cells as multicell spheroids leads to an increase in resistance to killing by ionizing radiation and etoposide. Differential display was used to identify changes in gene expression that occur when cells are grown as spheroids. MATERIALS AND METHODS: Differential display was performed using exponentially growing Chinese hamster V79 cells and the outer cell layer of V79 spheroids. Using six different pairs of primers, 20 altered bands were selected. Eight genes, confirmed using reverse Northerns, showed a match in a GenBank search. Antibodies against a calcium-binding protein, mts1, confirmed differential expression of this protein. Intracellular free calcium levels were measured using fluo-3 fluorescence, and the effect of a calcium-binding agent on etoposide resistance was examined using the comet assay. RESULTS: Genes upregulated in the outer cell layer of spheroids relative to monolayers included: (1) mts1 (S100A4), a calcium binding protein implicated in proliferation, metastasis, cell adhesion, and angiogenesis; (2) cytochrome c oxidase II; (3) B-ind1, a mediator of Rac-1 signaling; (4) TRAM, an endoplasmic reticulum protein. Genes downregulated in spheroids were: (5) phosphoglycerate kinase; (6) ARL-3, a ras-related GTP binding protein; (7) MHC class III complement 4A; and (8) 2,4-dienoyl-CoA. Immunohistochemistry confirmed overexpression of mts1 and another calcium-binding protein, calreticulin, in V79 outer spheroid cells relative to monolayers. C6 rat glioma and SiHa human cervical carcinoma cells that demonstrate a contact effect also showed upregulation of mts1 or calreticulin, while WiDr colon carcinoma cells that lack contact resistance showed no change in expression of either calcium binding protein. Intracellular free calcium levels were found to be almost two times lower in the outer cells of V79 spheroids compared to monolayers. V79 monolayer and outer spheroid cells treated with the calcium chelating agent BAPTA-AM showed a similar level of DNA damage by etoposide. CONCLUSIONS: Expression of genes involved in calcium binding, signaling and metabolism are differentially expressed when V79 cells are grown as spheroids. Differences in the levels of intracellular calcium may underlie the contact effect.     

Cure, cell killing, growth delay and fragmentation of X-irradiated human melanoma HMV-I multicellular spheroids

Human melanoma, HMV-I, multicellular spheroids were irradiated and cure was determined by the absence of cellular outgrowth. Their cellular radiosensitivity was measured by the colony-forming ability of cells dispersed from the spheroid. Analysis of radiocurability of spheroids in terms of their cellular radiosensitivity predicted three necessary conditions: a linearity of dose versus the double-minus logarithm of curability; constancy of a critical cell number; and constancy of cellular radiosensitivity. These conditions were found to exist in the observed data for each of three size classes of spheroids. Analysis suggests that cellular radiosensitivity in multicellular spheroids with diameters of 250 and 400 microns was different from that of monolayers, and that the increase of spheroid-control doses was found to be a function of cellular radiosensitivity, total cell number per spheroid and a critical cell number. The critical cell number increased from 0.8 in a 150 microns spheroid to 4 in a 250 microns spheroid and to 57 in 400 microns spheroid. This number is a unique characteristic of multicellular systems and is one important factor in determining their radiocurability. X-ray-induced growth delay of spheroid size was increased with increasing dose. At high doses a sharp increase in delay time was seen, sometimes accompanying fragmentation of spheroids at late postirradiation times. The clonogenic activity of these fragments may serve as a model of exfoliation, the first step of radiation-induced metastasis.     

MR tracking of transplanted cells with "positive contrast" using manganese oxide nanoparticles

Rat glioma cells were labeled using electroporation with either manganese oxide (MnO) or superparamagnetic iron oxide (SPIO) nanoparticles. The viability and proliferation of SPIO-labeled cells (1.9 mg Fe/ml) or cells electroporated with a low dose of MnO (100 microg Mn/ml) was not significantly different from unlabeled cells; a higher MnO dose (785 microg Mn/ml) was found to be toxic. The cellular ion content was 0.1-0.3 pg Mn/cell and 4.4 pg Fe/cell, respectively, with cellular relaxivities of 2.5-4.8 s(-1) (R(1)) and 45-84 s(-1) (R(2)) for MnO-labeled cells. Labeled cells (SPIO and low-dose MnO) were each transplanted in contralateral brain hemispheres of rats and imaged in vivo at 9.4T. While SPIO-labeled cells produced a strong "negative contrast" due to the increase in R(2), MnO-labeled cells produced "positive contrast" with an increased R(1). Simultaneous imaging of both transplants with opposite contrast offers a method for MR "double labeling" of different cell populations.     

31P NMR spectroscopy of 9L cell line in culture: differential effects of high temperature on anchored cells and spheroids

The differential effects of high temperature on 9L rat gliosarcoma cells cultured in two distinctive forms, namely, anchored monolayer cells and multicellular spheroids, were investigated using 31P nuclear magnetic resonance spectroscopy. The critical temperatures resulting in irreversible changes in cellular energetics were found to be significantly different: 45 degrees C for anchored cells and 43 degrees C for multicellular spheroids, respectively. Phosphomonoester levels in multicellular spheroids were found to be consistently higher than those in anchored monolayer cells regardless of the temperature to which they were exposed. The study demonstrates that tissue derived from a single cell line may exhibit different metabolic properties depending on its growth pattern.     

Dose dependence of the growth rate of multicellular tumour spheroids after irradiation

The present study investigated differences in the growth rate of multicellular tumour spheroids of the MCF-7 line of human breast cancer before and after their irradiation. Growth of the spheroids was analysed according to a model based on a Gompertz function. In this model, normalization to a common initial volume is achieved in a way that enables meaningful comparisons to be made between the results obtained for each spheroid. For irradiated spheroids the model includes an additional term to take account of sterilized cells. We found that the growth rate observed before irradiation is not fully recovered by irradiated spheroids and that growth recovery reduces with higher irradiation doses. Surviving fractions obtained at doses below 3 Gy are comparable with those found in clonogenic assays on spheroids of the same cellular line. At larger doses, discrepancies between the different studies are considerable.     

Multicellular tumour spheroids in radiation biology.

PURPOSE: Multicellular tumour spheroids are being used with increasing frequency in various aspects of tumour biology, including studies dealing with radiation biology. This review attempts to outline recent studies using these three-dimensional systems in radiation biology with particular reference made to papers testing radiotherapeutic protocols with spheroids. DEFINITIONS: Multicellular tumour spheroids are three-dimensional structures composed of cancer cells. They are formed from monolayer tumour cells when these are grown by various in vitro methods (e.g. liquid-overlay, spinner flask and gyratory rotation systems). Because of the cellular organization in spheroids, they have been often shown to recreate in vivo tumours much more closely than two-dimensional in vitro models. CONCLUSIONS: Because of their particular architectural characteristics, multicellular spheroids are demonstrated to be extremely useful in testing radiotherapeutic protocols, including dose rate and fractionation, radioimmunotherapy and the effects of combined treatments (e.g. radiation and anti-neoplastic drugs). Further studies should seek not only to continue testing these protocols, but also to investigate the more fundamental questions of radiation-induced apoptotic cell death, cell-cycle events, cell-cell interactions and cell adhesion phenomena.     

Repair of radiation induced damage in two human tumour cell lines grown as spheroids and monolayers

Growth curves of two human tumour cell lines grown as multicellular tumour spheroid (MTS) were used to obtain survival estimates by back-extrapolation after split and single dose irradiation. Neuroblastoma (NB-100) and squamous cell carcinoma (HN-1) single cells from monolayer culture were assessed for repair of sublethal and potentially lethal damage. The extent of repair was calculated on an iso-effect basis. When grown as spheroids squamous cell carcinoma cells showed a higher capacity to repair sublethal damage than neuroblastoma cells. Repair of potentially lethal damage did not contribute to this higher capacity of HN-1 cells, since this cell line was found to be deficient for this type of repair. Using the recovery ratio to estimate the beta-component of the survival curves, it was found that differences in repair capacity were determined by the alpha-component of the equation. Our results show the feasibility of back-extrapolating multicellular tumour spheroid growth curves to obtain survival estimates that can be applied to establish sublethal damage repair capacity.     

Biophysical aspects of the integrated combination of cytostatic drugs with radiotherapy. Part 2: Dose-effect relationships and 31P NMR spectroscopy of L 1210 cells (monolayers) and 9L glioma cells (monolayers and tumor spheroids) treated with activated isophosphamide, adriamycin, epirubicin and 6 MeV electrons

Dose-effect relations have been evaluated by the treatment of cell cultures (9L glioma cells of rat as monolayers and tumor spheroids, L 1210 cells of mice) with activated isophosphamide, adriamycin, epirubicin and 6 MeV electrons. The magnitude of synergistic effects obtained by combined treatment modalities is strictly pH-dependent, but even for tumor spheroids it appears that there exists an optimum time-interval between drug administration and consecutive irradiation. The determination of the intracellular pH value with the help of pH sensor microelectrodes and 31P NMR spectroscopy indicates that 31P spectroscopy only provides the global pH of the complete culture (average value), whereas the local pH can only be determined by sensors. The ATP-concentration before and after irradiation depends significantly on the glucose supply of the culture medium.     

Study of tea polyphenol as a reversal agent for carcinoma cell lines' multidrug resistance (study of TP as a MDR reversal agent)

The aim of this study was to examine MDR1 expression product P-glycoprotein (Pgp) and study the effect and mechanism of tea polyphenol (TP) in reversion of multidrug resistance (MDR) in carcinoma cell lines. Immunocytochemical method was used for qualitative detection of Pgp. A comparative study of cytotoxicity and multidrug resistance reversion effect was made by MTT assay for tea polyphenol and quinidine in MCF-7 and MCF-7/Adr cell lines. The multidrug resistance reversion effect and mechanism were studied by measuring the uptake of 99mTc-tetrofosmin in the carcinoma cell lines. (1) The Pgp overexpression in MCF-7/Adr cells was found to be strong positive, while the Pgp expression of MCF-7 was negative. (2) Although both tea polyphenol and quinidine could not remarkably change the toxicity of adriamycin to MCF-7, they could improve the sensitivity of MCF-7/Adr to adriamycin. The reversion index of tea polyphenol and quinidine was 3 and 10 respectively. (3) The cellular uptake of 99mTc-tetrofosmin was remarkably lower in MCF-7/Adr than in MCF-7. The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited a 4, 13, 16 fold increase in the presence of 200, 400 and 500 microg/ml of tea polyphenol respectively. The uptake of 99mTc-tetrofosmin in MCF-7/Adr exhibited only a 4-fold increase in the presence of 200 microM of quinidine. Immunocytochemistry can detect P-glycoprotein expression level qualitatively. Tea polyphenol is not only an anti-tumor agent, but also a multidrug resistant modulator similar to quinidine. The multidrug resistance reversion mechanism of tea polyphenol seems to be its inhibition of the activity of P-glycoprotein. Tea polyphenol has the advantage of very low toxicity in tumor treatment.     

Comparison of uptake of99mTc-MIBI,99mTc-tetrofosmin and99mTc-012 into human breast cancer cell lines

Technetium-99m hexakis-2-methoxyisobutylisonitrile (MIBI),^99mTc-tetrofosmin and^99mTc-Q12 were all introduced for myocardial imaging but found additional applications as they are taken up by different tumours, enabling imaging of these lesions in patients. The aim of this study was to compare the uptake characteristics of these compounds in vitro in the human adenocarcinoma breast cell lines MCF-7 and ZR-75. It was shown that^99mTc-MIBI had the highest cellular uptake (15.9%±0.5% dose/mg protein after 60 min in MCF-7, and 14.2%±0.4% dose/mg protein in ZR-75), followed by^99mTc-tetrofosmin (6.8%±0.6% dose/mg protein in MCF-7, and 8.2%±0.2% dose/mg protein in ZR-75) and^99mTc-Q12 (3,2%±0. I% dose/mg protein in MCF-7, and 3.5%±0.3% dose/mg protein in ZR-75 cells). For all three compounds tenfold differences in specific activity did not influence total cell-associated radioactivity. Uptake of^99mTc-MIBI and^99mTc-tetrofosmin was obviously lower at 4° C than at 37° C, whereas^99mTc-Q12 uptake showed only slight temperature dependence. When uptake was compared in cells grown to different cell densities (1 mg/ml cellular protein versus 0.3 mg/ml), no differences in uptake were detected when uptake was corrected for the amount of cellular protein present in the dishes. Furthermore, for all compounds it was shown that cellular radioactivity decreased rapidly after washing. Apart from the differences in cellular uptake of the three compounds after 60 min, no differences in residual cellular radioactivity after washing were found between the different compounds when expressed as a percentage of their 60-min uptake, suggesting that the efflux process of the radiolabelled compounds was similar. The differences in cell-associated activity after 60 min were thus presumably caused by differences in uptake. It was concluded that of the Tc-labelled compounds tested,^99mTc-MIBI had the highest cellular retention in both human breast tumour cell lines. However, for imaging in vivo not only radioactivity in the target organ is important, but also the ratio of radioactivity in the target versus that in the background. Therefore, further studies in vivo need to be performed to investigate which compound is the optimal imaging agent     

Response of chronic hypoxic cells to low dose-rate irradiation

PURPOSE: Compare the sensitivity of human cells in vitro to low dose-rate irradiation in air and in moderate hypoxia (4% O2). MATERIALS AND METHODS: Continuous low dose-rate beta-irradiation at a dose rate of 0.015 or 0.062 Gy/h was given to human T-47D breast cancer cells by incorporation of [3H] -labelled valine into cellular protein. Acute irradiation at a dose rate of 0.4 Gy/min was performed using [137Cs]gamma-irradiation. Cells were cultivated in an atmosphere with 4% O2 using an INVIVO2 hypoxia cabinet. RESULTS: When grown in ambient air with continuous irradiation, T-47D cells were able to continue growth for at least 23 weeks at a dose-rate of 0.015 Gy/h with a surviving fraction stabilized at around 60%. When the dose rate was increased to 0.062 Gy/h the cell culture died out after about 23 days (corresponding to about 22 Gy). When grown in an atmosphere with 4% O2 we surprisingly found that the continuously irradiated T-47D cells (0.015 Gy/h) were severely inhibited in their growth, and cell death became extensive after about 3 weeks while un-irradiated cells continued growth seemingly unaffected by this low oxygenation. Peri cellular oxygenation varied between 4% and below 0.1% over an ordinary passage due to diffusion-limitations through the 2 mm deep medium. Online O2-recordings over a whole passage showed that oxygen was more depleted in the irradiated compared to the un-irradiated cultures indicating increased respiration during irradiation. While cells growing attached to the bottom were inhibited and inactivated during irradiation it was found that cells attached high up in the neck region, i.e., having only a shallow layer of medium above them, survived and formed colonies. When cells cultivated in 4% O2 for 7 weeks were irradiated with acute doses of 137Cs gamma-rays, the radiosensitivity was the same as for cells cultivated in ambient air. CONCLUSION: Continuous irradiation with 0.015 Gy/h for several weeks results in a stronger inhibition for T-47D cells grown in an atmosphere with 4% as compared to 20% O2. The data indicate that this may be due to increased oxygen consumption resulting in more severe hypoxia in [3H]-incorporating compared to control (un-irradiated) cells.     

新生小鼠心肌细胞培养模型的建立及其在毒性评价研究中的应用

目的建立简单实用和重复性好的新生小鼠心肌细胞体外培养方法,构建体外心肌细胞模型用于外源化合物的心肌毒性评价。方法取当天出生的小鼠心脏,用胰蛋白酶、Ⅱ型胶原酶和分散酶消化心室肌组织,用差速贴壁和化学方法纯化心肌细胞,用此细胞模型评价镰刀菌毒素丁烯酸内酯（BUT）的心肌毒性作用。结果新生小鼠心肌细胞培养3d后,大部分细胞开始搏动,随培养时间的延长,心肌细胞胞体逐渐增大。经α-actin抗体免疫组织化学鉴定,心肌细胞的纯度为96%。持续培养90d后,心肌细胞仍保持较好的搏动频率。经BUT染毒后,心肌细胞存活率随BUT浓度的增加而逐渐下降,对心肌细胞的损伤在形态上主要表现为细胞水肿、空泡样变性、肌纤维断裂等。结论该实验建立的新生小鼠心肌细胞体外培养方法,单细胞收获率和心肌细胞纯度高,心肌细胞搏动时间长。BUT对心肌细胞有较强的毒性作用并呈明显的量-效关系。     

Evidence for an adaptive response to radiation damage in plant cells conditioned with X-rays or incorporated tritium

Allium cepa root-tip cells were first exposed to low 'conditioning' doses of ionizing radiation: to X-rays (0.06 or 0.26 Gy) or to incorporated tritium (1.8 x 10(4) or 7.2 x 10(4) Bq/ml; specific activity: 740.0 GBq/mmol) and subsequently given a 'challenge' dose of 1.5 Gy of X-rays. A reduction in X-ray-induced chromosomal damage was brought about by prior exposure to 0.26 Gy of X-rays, while cells receiving the lower conditioning dose (0.06 Gy of X-rays) did not show any significant reduction. In cells grown in the presence of [3H]TdR on the other hand, the adaptive response was evident after both doses given. The results are essentially in agreement with those published by Wolff's group for human lymphocytes in showing that plant cells in vivo can become 'adapted' by exposure to low-level irradiation so that they become more resistant to the clastogenic effects of X-rays delivered subsequently.     

Etoposide sensitivity and topoisomerase II activity in Chinese hamster V79 monolayers and small spheroids.

Chinese hamster V79 cells grown in suspension culture as spheroids are more resistant than monolayers to killing and mutation by ionizing radiation. A change in DNA conformation appears to accompany this increase in radiation resistance. We were therefore interested in whether the activity of topoisomerase II, a nuclear enzyme involved in DNA conformational changes and possibly in DNA repair, might differ in monolayers and small spheroids. One-day-old spheroid cells were more resistant than monolayer cells to the toxic effects of etoposide, a topoisomerase II inhibitor. Fewer strand breaks were induced by etoposide in spheroid DNA than monolayer DNA, as measured by the DNA precipitation and alkali unwinding assays, although identical amounts of damage were produced in monolayers and spheroids by the topoisomerase I inhibitor camptothecin, and the cell cycle specific agent, 5-fluorouracil. There was no evidence of a subpopulation of spheroid cells which were more resistant to etoposide, and no change in the rate of incorporation or DNA chain elongation in spheroids compared to monolayers. Topoisomerase II activity in 1-day-old spheroids, measured by decatenation of trypanosome kinetoplast DNA, was reduced to 68% of the monolayer value; in 3-4-day-old spheroids the level was 32.5%. These results indicate that topoisomerase II activity and sensitivity to a topoisomerase II inhibitor are reduced in 1-day-old spheroid cells. We suggest that the decrease in the activity of this enzyme may be linked to the change in DNA conformation in spheroids and the decrease in their radiation sensitivity.     

Sublethal damage repair and radiosensitivity of human squamous cell carcinoma cells grown with different culture techniques

In this study, cells of a human squamous cell carcinoma line, HN-1, were grown in monolayers and as multicellular tumour spheroids (MTS). Repair of radiation-induced damage was studied by irradiation with single and split doses of X rays (4-8 Gy). It was shown that the amount of sublethal damage that was repaired in this dose range was equal in cells growing in monolayers and as MTS. The radiosensitivity of spheroids, as expressed by spheroid "cure" dose, increased with increasing MTS diameter. It is postulated that, in MTS with no signs of hypoxia, radioresistance diminishes when MTS increase in diameter.