UVB-activated indole-3-acetic acid induces apoptosis of PC-3 prostate cancer cells

Indole-3-acetic acid (IAA) has recently shown anticancer activity in combination with horseradish peroxidase. The current study demonstrated that IAA irradiated with ultraviolet B (IAA(UVB)) is able to generate free radicals and induce cell death in a time-dependent fashion in PC-3 prostate cancer cells, while PC-3 cells treated with IAA alone exhibited no toxic responses. It was also found through Western blot analysis that the cytotoxic effect of IAA(UVB) resulted from apoptosis. Treatment with IAA(UVB) for 24 hours showed a significant increase in phosphorylated p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, the stress signaling proteins. Furthermore, pro-caspases (-3, -8, and -9) were clearly down-regulated and poly(ADP-ribose) polymerase cleavages were demonstrated in the group treated with IAA(UVB). Flow cytometric analysis also demonstrated the induction of apoptosis by IAA(UVB) in PC-3 cells. In conclusion, this study demonstrated that IAA induced cell death in combination with UVB irradiation by increasing apoptosis in PC-3 cells.     

吲哚-3-乙酸对人外周血淋巴细胞微核和SCE频率的影响

背景与目的:研究吲哚-3-乙酸对人体外周血淋巴细胞微核和SCE频率的影响.材料与方法:应用人体外周血淋巴细胞测定吲哚-3-乙酸诱导微核形成率试验和姊妹染色单体互换率(SCE).结果:各吲哚-3-乙酸处理组与阴性对照组相比,微核形成率和SCE差异均存在显著性.结论:吲哚-3-乙酸对人外周血淋巴细胞的遗传物质具有损伤作用.     

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

Indole-3-acetic acid (IAA), when oxidized by horseradish peroxidase (HRP), is transformed into cytotoxic molecules capable of inducing cell injury. The aim of this study was to test if, by targeting hematopoietic tumors with HRP-conjugated antibodies in association with IAA treatment, there is induction of apoptosis. We used two lineages of hematologic tumors: NB4, derived from acute promyelocytic leukemia (APL) and Granta-519 from mantle cell lymphoma (MCL). We also tested cells from 12 patients with acute myeloid leukemia (AML) and from 10 patients with chronic lymphocytic leukemia (CLL). HRP targeting was performed with anti-CD33 or anti-CD19 antibodies (depending on the origin of the cell), followed by incubation with goat anti-mouse antibody conjugated with HRP. Eight experimental groups were analyzed: control, HRP targeted, HRP targeted and incubated with 1, 5 and 10 mM IAA, and cells not HRP targeted but incubated with 1, 5 and 10 mM IAA. Apoptosis was analyzed by flow cytometry using annexin V-FITC and propidium iodide labeling. Results showed that apoptosis was dependent on the dose of IAA utilized, the duration of exposure to the prodrug and the origin of the neoplasia. Targeting HRP with antibodies was efficient in activating IAA and inducing apoptosis.     

Development of a novel enzyme/prodrug combination for gene therapy of cancer: horseradish peroxidase/indole-3-acetic acid

This paper demonstrates the potential for utilizing the plant enzyme, horseradish peroxidase (HRP), in a gene-directed enzyme prodrug therapy context. Human T24 bladder carcinoma cells transfected with a mammalian expression vector containing the HRP cDNA were selectively sensitized to the nontoxic plant hormone, indole-3-acetic acid (IAA). The HRP/IAA-induced cell kill was effective in normoxic and anoxic conditions. The activated drug is a long-lived species able to cross cell membranes, and cell contact appears not to be required for a bystander effect to take place. These preliminary results suggest that the delivery of the HRP gene to human tumors followed by IAA treatment may provide a novel cancer gene-directed enzyme prodrug therapy approach, with potential to target hypoxic cells.     

Genotoxic activity of plant growth-regulating hormones in Aspergillus nidulans

Three plant growth-regulating hormones, indole-3-acetic acid(IAA), indole-3-butyric acid (IBA), and kinetin (6-furfuryl-aminopurine),were tested for their genetic activity in Asper-gillus nidulansin a plate test. The first two hormones were found to greatlyincrease somatic segregation in the fungus whereas kinetin wasnot effective. Several concentrations of the plant hormoneswere used and it was found that increasing concentrations ofIAA and IBA increased mitotic segregation of the fungus withmost of the segregants being produced by mitotic crossing-over,together with non-disjunctional segregants at a lower level.The metabolic activation technique was also used and it wasshown that when S9 mixture was added to IAA and IBA a further3- to 5-fold increase in the number of segregants was obtained.In the case of kinetin the S9 had no effect.     

Nitric oxide: its production in host-cell-infiltrated EMT6 spheroids and its role in tumour cell killing by flavone-8-acetic acid and 5,6-dimethylxanthenone-4-acetic acid

Summary Flavone-8-acetic acid (FAA) and its more dose-potent analogue 5,6-dimethylxanthenone-4-acetic acid (5,6-MeXAA), appear to exert their antitumour effects through vascular and other host-mediated mechanisms and are known to induce the synthesis of nitric oxide by murine macrophages. We investigated the role of nitric oxide in the cytotoxic effects of these drugs in host-cell-infiltrated spheroids. EMT6 murine mammary adenocarcinoma cells were grown in culture to produce multicellular spheroids in vitro spheroids), which were then inoculated i. p. into mice. After 6 days the spheroids were removed ex vivo spheroids). Exposure to FAA (890 m) and 5,6-MeXAA (80 m) in vitro for 20 h increased nitrite concentrations to 6.7 and 9.7 nmol/spheroid, respectively, as compared with 0.7 nmol/spheroid in the absence of drug. FAA and 5,6-MeXAA did not increase nitrite production in in vitro spheroids in cells obtained by peritoneal lavage. However, mixed cultures of in vitro spheroids and peritoneal cells treated with 5,6-MeXAA produced nitrite (2.5 nmol/spheroid), indicating that interactions between host cells and tumour cells were important for induction. The effets of these drugs on ex vivo spheroids were prevented by co-incubation withN ^G-monomethyl-l-arginine, indicating that nitrite originated from the oxidation ofl-arginine to nitric oxide. Cell sorting of disaggregated spheroids into EMT6 cells andMac-1-positive macrophage populations indicated that both of these cell populations could be induced to synthesise nitric oxide by subsequent incubation with 5,6-MeXAA. Incubation of ex vivo spheroids with FAA and 5,6-MeXAA decreased the clonogenicity of EMT6 cells, and this effect was wholly (FAA) or partially (5,6-MeXAA) reversed by the presence ofN ^G-monomethylarginine (250 m). FAA and 5,6-MeXAA may therefore exert some of their cytotoxic effects on tumour cells through the production of nitric oxide.This work was supported by grants from the New Zealand Lotteries Board, the Cancer Society of New Zealand and the Health Research Council of New Zealand     

靶向HRP/IAA自杀基因系统联合IL-12基因治疗Lewis肺癌的研究

目的:观察人端粒酶逆转录酶(hTERT)启动子驱动辣根过氧化物酶(HRP)/吲哚乙酸(IAA)系统联合白细胞介素-12(IL-12)基因对小鼠Lewis肺癌的疗效。方法:建立Lewis肺癌移植瘤模型,随机分为对照组、HRP组、IL-12组和联合组,分别给予AdCMVGFP、AdhTERTHRP、AdCMVmIL-12单一或联合注射,然后腹腔注射IAA,观察肿瘤生长情况。蛋白质印迹法和ELISA检测瘤组织HRP和IL-12表达;免疫组化检测瘤组织内CD4+、CD8+淋巴细胞浸润情况。结果:与对照组、单一治疗组相比,联合组小鼠肿瘤生长受到显著抑制(联合组vs对照组:P=0.000,9 d;联合组vsHRP组:P=0.005,15 d;联合组vsIL-12组:P=0.046,12 d),生存期显著延长(联合组vs对照组:χ2=9.529,P=0.002;联合组vsHRP组:χ2=9.039,P=0.003;联合组vsIL-12组:χ2=8.595,P=0.003),肿瘤组织广泛坏死,CD4+、CD8+淋巴细胞浸润显著增多。结论:IL-12基因治疗可诱导宿主产生抗肿瘤免疫应答,与靶向HRP/IAA自杀基因系统联合具有...     

Multicellular tumor spheroids in radiotherapy research (review)

Culturing of human tumor cells as multicellular spheroids can be a tool to study radiation responses. The degree of structural and functional differentiation in the primary tumor may be retained in spheroids rather than in conventional monolayer cultures. In the liquid overlay culture technique spheroids can be individually assessed for their responses to treatment, whereas in spinner flasks, large quantities of similarly sized spheroids can be produced. Studying the response of spheroids to irradiation can be performed on single cells obtained after disaggregation of these spheroids, or on intact spheroids, using cure and growth delay as endpoints. Clonogenic cell survival is especially difficult to perform on spheroids of human tumor cells. Modern calculation methods, however, may offer promising correlates between growth curves and single cell survival. Spheroids of human tumor cell lines show tumor type dependent radiation responses, offering an approach for comparison of radiosensitivity of tumor cell lines of different histologic origin. Contact effect, as a modifying factor of radiation response in spheroids, has especially been studied in murine cell lines. The use however, of human tumor cell lines, may offer new insight in this phenomenon. Radiobiologic hypoxia has been observed in spheroids of both murine and human origin. Reoxygenation after irradiation has also been described by radiobiologic parameters. So far, no physiologic reoxygenation processes after radiation treatment have been identified. In view of the clinical relevance of oxygen to radiation responses and treatment outcome, reoxygenation processes should be further elucidated in spheroids of human origin. Repair of potentially lethal damage in spheroids has been reported for only one murine cell line. In an indirect manner it has also been studied in spheroids of human origin. Sublethal damage repair has been studied rather extensively in murine cell line spheroids. However, only recently it has been reported in human tumor spheroids in relation to the clinical curability of the tumors of origin. Use of human tumor cell lines to study radiation responses of spheroids is necessary to determine tumor type dependent differences in several radiation related phenomena, such as reoxygenation, contact effect, and repair processes.     

Use of a tritiated thymidine suicide technique in the study of the cytotoxic drug response of cells located at different depths within multicellular spheroids

A technique using 'tritiated thymidine suicide' has been established as a means of studying the response to cytotoxic drugs of cells at different depths within multicellular tumour spheroids. Because of the characteristic spatial arrangement of cycling cells (mostly in the outer regions) and non-cycling cells (mostly at the inner regions) of spheroids, cells surviving after long term (24 h) exposure of spheroids to high doses of 3HTdR will be those located furthest from the surface. By comparing the drug response of cells from 3HTdR pre-treated and untreated spheroids, the individual response of total cells, cells near to the surface and cells lying deeper within the viable rim of spheroids can therefore be deduced. In this study, large spheroids of about 800 micron in diameter of a mouse mammary cell line, EMT6/Ca/VJAC, and of a human small cell lung cancer cell line, POC, have been used. Using clonogenic assay, the response of these two cell types to adriamycin (ADM), nitrogen mustard (HN2), CCNU and vincristine (VCR) (POC only) were measured. The preliminary part of this study has confirmed that the cells killed are those which incorporate 3HTdR during the DNA synthesis period; the cells killed are mainly located in the outer regions of spheroids i.e. surviving cells are mostly located in the inner part of the viable rim and 3HTdR pretreatment does not sensitise surviving cells to subsequent cytotoxic drug treatment. Results from large EMT6 spheroids agree with our previous findings (obtained using a selective disaggregation method) that cells in the outer regions of spheroids are more sensitive to ADM and HN2 than cells in the inner regions whilst the opposite is true for CCNU. For POC spheroids, cells in the outer region of spheroids are more sensitive to ADM and VCR than cells in the inner region whilst a reverse trend is seen for the response to CCNU. The response to HN2 is similar at all depths. Amongst the factors governing the response of cells in spheroids to cytotoxic drugs, the responses to ADM and VCR are thought to be largely dictated by cell cycle distribution and limited drug penetrability, whilst for HN2 the response may be determined by the factor of cell cycle distribution. For CCNU, we believe that the cellular response is largely dependent upon microenvironmental factors prevailing within spheroids.     

Heat-shock protein 70-dependent dendritic cell activation by 5-aminolevulinic acid-mediated photodynamic treatment of human glioblastoma spheroids in vitro

Background:

T-cell responses contribute to the anti-tumoural effect of photodynamic therapy (PDT). For such responses to occur, dendritic cells (DCs) have to migrate to the tumour, take up tumour antigens and respond to danger signals with maturation, before they engage in T-cell activation. Here, we have studied the effect of 5-aminolevulinic acid (ALA)-mediated PDT on DCs in vitro in a human spheroid model of glioblastoma (GB).

Methods:

Spheroids of the GB cell lines U87 and U251 were treated with ALA/PDT, and effects on attraction, uptake of tumour antigens and maturation of DCs were studied. To block heat-shock protein-70 (HSP-70) on the spheroids, neutralising antibodies were used.

Results:

5-Aminolevulinic acid /PDT-treated GB spheroids attracted DCs that acquired tumour antigens from the spheroids effectively. Moreover, co-culture with ALA/PDT-treated spheroids induced DC maturation as indicated by the upregulation of CD83 and co-stimulatory molecules as well as increased T-cell stimulatory activity of the DCs. Heat-shock protein-70 was upregulated on the spheroids after ALA/PDT treatment. Uptake of tumour antigens and DC maturation induced by the ALA/PDT-treated spheroids were inhibited when HSP-70 was blocked.

Conclusion:

ALA/PDT treatment of glioma spheroids promotes the three initial steps of the afferent phase of adaptive immunity, which is at least partially mediated by HSP-70.     

Evaluation of the antitumoral potential of different nitric oxide-donating non-steroidal anti-inflammatory drugs (NO-NSAIDs) on human urological tumor cell lines

Our work aimed at identifying the antitumoral potential of new nitric oxide (NO)-releasing non-steroidal anti-inflammatory drug (NSAID) derivatives on human prostate and bladder carcinoma cell lines. Among all molecules tested, two sulindac derivatives, NCX 1102 ((Z)-5-fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl] methylene]-1H-indene-3-acetic acid 4-(nitrooxy)butyl ester) and NCX 1105 ((Z)-5-fluoro-2-methyl-1-[[4-(methylsulfinyl)phenyl] methylene]-1H-indene-3-acetic acid 6-(nitrooxymethyl)-2-methylpyrydyl ester hydrochloride), were the most cytotoxic compounds. In contrast to its parent molecule sulindac, cell cycle analysis showed that NCX 1102 led to cell accumulation in the G2-M transition stage in all cell lines, and induced apoptosis in five out of the six cell lines. Thus, NO-NSAIDs may be useful for the elaboration of new therapeutic strategies in the management of bladder and prostate cancer.     

Mathematical model of simultaneous diffusion and binding of antitumor antibodies in multicellular human tumor spheroids

Multicellular tumor spheroids are widely used as in vitro models of poorly vascularized tumor nodules in vivo. The uptake kinetics of tumor-associated antibodies in multicellular tumor spheroids is assumed to be governed by passive diffusion and irreversible binding of the antibodies with binding sites on the cell surface. By further assuming that the spheroids are homogeneous with respect to diffusion and binding, a mathematical model has been developed which permits the extraction of the macroscopic diffusion constant D and the macroscopic binding rate k from empirical studies. The model was applied to uptake kinetics data obtained (a) with a melanoma-associated monoclonal antibody 96.5 (isotype IgG2a)-human multicellular melanoma spheroid system exhibiting strong antibody to cell binding and (b) with the same monoclonal antibody-human multicellular colon adenocarcinoma HT29 spheroid system exhibiting nonspecific binding. The spheroids had approximately 300 microns diameter. The constants D and k were estimated to be 0.45 micron2 s-1 and 2.0 x 10(-3) s-1, respectively, for the system with specific binding. Saturation of binding sites occurred. In the nonspecific binding system, D and k were found to be 0.10 micron2 s-1 and 1.0 x 10(-5) s-1. No saturation of binding sites occurred. D and k were also estimated to be, respectively, 0.52 micron2 s-1 and 6.4 x 10(-5) s-1 for another melanoma-associated monoclonal antibody 140.240 (same isotype as 96.5) in the melanoma spheroid system exhibiting moderate cell binding with the antibody. The mathematical model describes well the system exhibiting nonspecific binding, but requires modifications and further development for the systems exhibiting moderate to strong binding.     

Comparisons between two monoclonal antibodies that bind to the same antigen but have differing affinities: uptake kinetics and 125I-antibody therapy efficacy in multicell spheroids.

It has been predicted that low affinity antibodies (Abs) should penetrate into tumors more readily than high affinity Abs. However, the absolute uptake and residence time of a high affinity Ab may be better. It is, therefore, not clear whether a high affinity Ab would have a therapeutic advantage. This is particularly relevant with 125I radioimmunotherapy, where targeting of every cell is important. This study compared the uptake kinetics and toxicity in multicell spheroids of two murine monoclonal Abs labeled with 125I. 17-1A was produced by immunization with a human colon cancer cell line and has an affinity of 5.15 x 10(7) M-1. 323/A3 was produced by immunization with a human breast cancer cell line and has an affinity of 1.87 x 10(9) M-1. Binding of both Abs to LS174T spheroids was similar at 4 degrees C, but binding of 17-1A was 8-10-fold less than that of 323/A3 at 37 degrees C. Despite this difference, the toxicity of 125I-17-1A in spheroids after 7 days of incubation was similar to that of 125I-323/A3. Autoradiography showed that 17-1A penetrated the spheroids much more deeply and evenly than did 323/A3. It appears that much of the radiation dose to spheroids treated with 125I-323/A3 was wasted because of the uneven Ab distribution. This study demonstrates the potential advantage of using Abs of lower affinity for 125I radioimmunotherapy, because of their more even distribution. It also suggests that a large number of binding sites per cell may be a disadvantage if more 125I is bound than is necessary to kill the cell, because this may slow Ab penetration.     

Integrin expression on colorectal tumor cells growing as monolayers,as multicellular tumor spheroids,or in nude mice

In this study we compared the expression of integrin alpha chains 2, 3, 4, 5, 6, v and the beta chains 1, 3, 4 in 2 colorectal carcinoma cell lines (HRT-18 and CX-2), growing in confluent and subconfluent monolayer cultures, as multicellular tumor spheroids and in nude mice, using the immunofluorescence technique (confocal microscopy) and flow cytometry. The fast-growing cell line HRT-18 expressed, in confluent and subconfluent monolayer cultures, alpha 2, 3 and beta 1 with a continuous membranous staining pattern, whereas alpha v, alpha 6, and beta 4 were expressed continuously membranous in the intermediate and apical part of the cell layer, and clustered at focal contacts at the base of the cells. In spheroids and tumors of nude mice the focal pattern of alpha v, 6 and beta 4 was changed into a diffuse one. Using flow cytometry, the expression of alpha 3 was found to be reduced in spheroids of HRT-18. The slowly-growing cell line CX-2 expressed, under the same conditions in monolayer culture, alpha 6, beta 1 and beta 4, and very weakly alpha 2, 3, 5 and v. Alpha 3 was expressed in spheroids of CX-2 only at the outer rim where the cells proliferate. In contrast, alpha 2 and 5 were expressed mainly in the quiescent, non-proliferating area. Alpha 6 was reduced in spheroids of CX-2. In the nude mouse tumor of CX-2, alpha 5 was expressed only focally and very weakly, alpha 2 was no longer detectable, but alpha v appeared to be enhanced in a focal pattern. These data indicate that integrin expression of tumor cells depends upon the culture system and that integrin expression in multicellular tumor spheroids is more similar to the in vivo situation in nude mouse tumors. © 1995 Wiley-Liss, Inc.     

The use of multicellular spheroids in establishing human sarcoma cell lines in vitro

By a new procedure stable monolayer cultures were derived from spheroids in 8 out of 17 different human sarcomas (16 soft-tissue and I osteogenic sarcoma). Eleven of the sarcomas were obtained from patients undergoing surgery, and 6 from BALB/c nude mice carrying s.c. growing xenografts. The new procedure involves aggregation of single-cell suspensions into spheroids and cultivation of these in agar-coated flasks until the growth rate levels off, at which time the spheroids are transferred to uncoated flasks. Cells proliferating from the rim of adhering spheroids are trypsinized and aggregated to form new spheroids. By 3 to 5 such alternations, monolayer cultures were obtained that have now been subcultured for about 6 months. The cell lines all gave rise to colonies in a clonogenic soft-agar system, and upon s.c. injection into athymic nude mice 3 lines tested formed growing tumors. The histology of spheroids formed from late monolayer passages closely resembled that of the original tumors. That the new procedure is superior to other methods of establishing sarcoma cell lines is indicated by the fact that a stable monolayer culture could be obtained directly from the tumors in only 1/8 cases where the above procedure was successful, and in only 2 instances from soft-agar colonies derived from the tumors.     

Differential mechanisms of radiosensitization by 2-deoxy-D-glucose in the monolayers and multicellular spheroids of a human glioma cell line

In vitro studies using monolayer cultures of human tumor cell lines have shown that 2-DG selectively inhibits energy-dependent DNA repair and cellular recovery processes in cancer cells. However, monolayer cultures differ greatly from the complex environmental conditions generated in solid tumors that develop inhomogeneous hypoxic and necrotic regions. In contrast, multicellular spheroids mimic heterogeneous cellular behavior and the consequent functional characteristics of in vivo solid tumors, and serve as important in vitro model to investigate tumor biology and responses to potential therapeutic agents. The present study compares the radiomodification by 2-DG in monolayer cultures and spheroids of a human glioma cell line (BMG-1) to gain insight into the effects in solid tumors. In spheroids, the glucose consumption (2.1 p mole/cell/h) and lactate production (3.67 p mole/cell/h) was nearly 2-3 fold higher than in monolayer cells (0.83 and 1.43 p mole/cell/h respectively). Presence of 2-DG (5 mM) for 2-4 h inhibited the glucose usage and lactate production by 70% in spheroids, while a 35% reduction was observed in monolayer cells. Under these conditions, 2-DG drastically enhanced the radiation-induced cell death of spheroids (by 2-3 folds); while a 40% increase was observed in monolayer cells. Radiosensitization by 2-DG in monolayer cells was primarily due to an increase in mitotic death (23%) linked to cytogenetic damage (micronuclei), whereas a profound induction of apoptosis (40%) accounted for the sensitization in spheroids. Although the Bcl-2 and Bax levels were significantly higher in spheroids, Bcl-2/Bax ratio was similar in monolayers and spheroids. Comet assay revealed a late onset of DNA breaks in the presence of 2- DG following irradiation only in spheroids, which corroborated well with the late onset of oxidative stress. 2-DG did not induce a significant cell cycle delay in monolayers, while a transient G(2) delay was apparent in spheroids.     

Glucose diffusivity in multicellular tumor spheroids

In order to understand the role of glucose limitations in controlling multicellular tumor spheroid growth, knowledge of the glucose diffusion coefficient is essential. The effective diffusivity of glucose in spheroids of rodent and human tumor cell lines has been determined by measuring the efflux of tritium labeled L-glucose from spheroids with time. When the rapid and irreversible binding of L-glucose in spheroids is properly taken into account, measurements of the efflux of this diffusion tracer from spheroids into label-free medium can be correlated to the diffusion equation in order to obtain the effective glucose diffusivity in spheroids. Such measurements have been made in EMT6/Ro mouse mammary tumor spheroids as well as in spheroids derived from human colon carcinoma cells (HT29, CO112, and WiDr) and from human squamous carcinoma cells (CaSki and A431). EMT6/Ro spheroids have a glucose diffusivity of 1.1 x 10(-6) cm2/s, while glucose diffusion coefficients in the human cell spheroids studied vary from 5.5 x 10(-7) cm2/s to 2.3 x 10(-7) cm2/s. These values are low enough to suggest that significant gradients in glucose concentration may exist in spheroids and tumors. It is thus believed that these glucose diffusivities, as well as their variation with cell line, may have important implications for the role played by glucose in the growth and cellular heterogeneity of spheroids and tumors.     

The radiation response of V79 and human tumour multicellular spheroids--cell survival and growth delay studies

Chinese hamster cells (V79 379A) cells from a human small cell carcinoma of the lung (ME/MAR) and two xenografted human melanomas (HX117 and HX118) have been grown as multicellular spheroids in vitro. The radiation response of these four cell types has been compared when grown as spheroids (200 or 400 micron in diameter) and as single cells from disaggregated spheroids. The radiation sensitivity of the three human lines irradiated as single cells in air, is similar. In comparison, the V79 cells are more radioresistant. Only the V79 and HX118 cells show a spheroid size dependent radiation response. The radiation response of spheroids has been assayed using both cell survival and growth delay. V79, ME/MAR and HX117 cells demonstrate a good correlation between the two endpoints whereas with HX118 there appears to be greater cell kill for a given level of growth delay. This may be because HX118 is efficient in the repair of potentially lethal damage (PLD). The results support the view that extrinsic factors such as three dimensional contact, hypoxia and repair of PLD can be important and together with the intrinsic cell radiosensitivity will determine the radiation response of tumours.