The radiation dose-rate effect in two human neuroblastoma cell lines

The current use of targeted radiotherapy in the treatment of neuroblastoma has generated a requirement for further information on the radiobiology of these cells. Here we report on studies of the dose-rate effect in two human neuroblastoma cell lines (HX138 and HX142) and the recovery that they demonstrate in split-dose experiments. The sensitivity of the two cell lines to high dose-rate irradiation was confirmed. Surviving fractions at 2 Gy were 0.083 for HX138 and 0.11 for HX142. There was little evidence of a dose-rate effect above 2 cGy min-1 but significant sparing was seen at lower dose rates. Substantial recovery was seen in split-dose experiments on both cell lines, to an extent that was consistent with the linear quadratic equation. The data were used to derive values for the beta parameter of the linear-quadratic equation; the values for the neuroblastomas were higher than for any of the other human tumour cell lines that we have investigated to date. Thus, despite their high sensitivity to ionising radiation HX138 and HX142 do exhibit substantial levels of cellular recovery, suggesting that they may have a significant capacity for repair of radiation-induced lesions.     

Radiosensitivity, recovery and dose-rate effect in three human glioma cell lines

The results of radiotherapy in the treatment of high-grade gliomas are disappointing. In this study three recently established cell lines from high-grade human gliomas have been found to exhibit a sensitivity that is at the resistant end of the spectrum of radiosensitivities seen in human tumour cells generally. The results support the view that inherent cellular radioresistance may be an important cause of failure in this disease. All three cell lines showed an increase in survival when the radiation dose rate was reduced. In split-dose experiments, recovery was found to increase with dose in a manner consistent with the predictions of the linear-quadratic equation.     

Lonidamine can enhance the cytotoxic effect of cisplatin in human tumour cells and rodent cells

Lonidamine combined with cisplatin treatment was tested in human glioma (U87MG) human squamous cell carcinoma (UMSCCl) and Chinese hamster (HA1) cells. The order of sensitivity to cisplatin was HA1 greater than U87MG greater than UMSCCl. Lonidamine caused little sensitization in U87MG cells, moderate sensitization in HA1 cells and large sensitization in UMSCCl cells. The degree of sensitization was correlated to the effect of lonidamine on cell growth. The degree of sensitization to cisplatin by lonidamine treatment was dependent on lonidamine exposure concentration, time and treatment sequence. Lonidamine shows potential for enhancement of cisplatin chemotherapy but this appears to be cell type specific and should be tested for cells derived from sites of clinical interest for such drug therapy.     

The effect of sodium butyrate on the growth characteristics of human cervix tumour cells.

Sodium butyrate has been shown to affect cell proliferation, and, at concentrations above approximately 0.5 mM, to cause cell death in some tumour cell lines. When combined with cytotoxic drugs increase in chemosensitivity has been observed. We are presently carrying out a study of the combined effects of sodium butyrate and cytotoxic drugs on cultured cervix tumour cells. To provide a baseline for this study we have carried out a systematic investigation of the effects of sodium butyrate alone on the growth characteristics of cervix tumour cells cultured as multicell spheroids. This has shown that concentrations of n-butyrate of 0.005 mM to 0.50 mM decrease cell proliferation without inducing cell death, the effect increasing with increasing concentration. Butyrate concentrations greater than 0.50 mM cause cell death after a period of 5 to 15 days exposure, dependent on concentration. Concentrations of 0.010 mM and above cause fragmentation of, and increased cell shedding from, multicell spheroids, suggesting an effect on the cell surface. Concentrations of butyrate greater than 0.10 mM cause a considerable increase in the synthesis of cytokeratin, as shown by reaction with cytokeratin antibody. Correlated with this is a marked increase in cell size, concentrations of butyrate of 2.0 or 3.0 mM leading to an approximate doubling of cell diameter, followed by cell disintegration. The effects of butyrate less than 0.25 mM are readily reversible. At concentrations greater than 0.25 mM the effects are reversible up to a limit of about 7 to 20 days depending on concentration, even when cytokeratin synthesis has been induced.     

Spontaneous human lymphocyte-mediated cytotoxicity againts tumour target cells. I. The effect of malignant disease

Spontaneous human lymphocyte-mediated cytotoxicity (SLMC) against tumour-cell targets was examined in a series of patients with localized or malignant disease, both treated and untreated, and patients with untreated chronic lymphocytic leukemia (CLL). The level of SLMC was assessed by means of two previously established assay systems; the xenogeneic assay involving the mouse mastocytoma line P815, and the allogeneic assay in which the human chronic myelogenous leukemia-derived line, K562, was used. The assay systems involve the use of Ficoll-Isopaque-separated, iron-plus-magnetism-purified lymphocytes in an overnight ⁵¹chromium release assay, and reflect the cytotoxic ability of human non-T, complement receptor-, F_c receptor-positive lymphocytes. In the present paper, lymphocytes from all normal donors tested showed significant activity in the SLMC assay, with some variation from day to day. This variation was markedly reduced when different normal donors were tested on the same day and under identical experimental conditions. In contrast, lymphocytes from many patients with malignant disease had decreased SLM activity, and this decrease was highly significant in patients with treated or untreated metastatic disease, or untreated CLL. This was also the case when the data were expressed relative to the number of cytotoxic cells in the normal control population, or in comparison to the relative SLMC activity of lymphocytes from patients with other conditions. Markedly decreased SLMC was observed in some patients in spite of normal T and B lymphocyte proportions, or the presence of the ability to mount a vigorous delayed hypersensitivity reaction to PPD. A comparison of the xenogeneic and allogeneic assays showed that the same information with respect to whether SLMC was normal or abnormal was obtained with both assays in the majority of cases. The significance of the data is discussed with respect to the possible role of SLMC in vivo and the relevance of SLMC to the assessment of specific cell-mediated cytotoxicity in malignant disease.     

The dose-rate effect revisited: radiobiological considerations of importance in radiotherapy

A wide range of dose-rates have been used in radiation biology and radiation therapy, extending from a few cGy per day to hundreds of Gy in a fraction of a second. The dose-rate range of importance in radiotherapy extends from about 0.1 Gy/hr to several Gy/min. In this range, the fraction of cells killed by a given dose decreases as the dose-rate is reduced, principally because of the repair of sub-lethal damage. In some cell lines, an inverse dose-rate effect is observed where, over a narrow range of dose-rates, the effectiveness of a given dose increases with decreasing dose-rate if cells move through the cycle and are arrested in G2, which is a radiosensitive phase. In recent years data have accumulated for cells of human origin. About 40 data sets have been analyzed for values of the survival curve parameters and the rate of repair of sub-lethal damage. These data have been used to address three questions of relevance to radiotherapy. (1) The proposal to use pulsed rather than continuous irradiation in interstitial brachytherapy. (2) The equivalence of high dose-rate and low dose-rate intracavitary treatments for carcinoma of the cervix. (3) An analysis of equivalent doses for a range of dose-rates in interstitial implants.     

The effect of antiserum fractions on Ehrlich ascites tumour cells

Hyperimmune heterologous serum produced in sheep against mouse Ehrlich ascites tumour cells was absorbed with normal mouse tissue and fractionated by DEAE column chromatography into IgG1 and IgG2 fractions. In vitro cytotoxicity test showed that sheep anti-Ehrlich ascites tumour IgGl fraction was cytotoxic to ⁵¹Cr labelled tumour cells whereas IgG2 had no cytotoxic effect. Pretreatment of the tumour cells with the non-cytotoxic IgG2 fraction slightly inhibited the cytotoxic action of IgG1 in vitro.     

Radioimmunotargeting of human tumour cells in immunocompetent animals

A tumour model system is reported that for many purposes may be an alternative to xenografted nude mice. The model allows immunotargeting of human tumour cells in immunocompetent animals. The target cells are contained in i.p. diffusion chambers (DC) with micropore membrane walls that are permeable to molecules, including the cell specific monoclonal antibodies (MoAb), but impermeable to cells. Thus, the tumour cells are protected from the host immunocompetent cells. In the work here presented the model was tested in immunocompetent mice and pigs, with tumour cells and antibody preparations that had demonstrated specific targeting in the nude mouse xenograft model. Hence, the DC were filled with cells from the human cell lines Hep-2 (expressing placental alkaline phosphatase, PLALP), or OHS (a sarcoma cell line), and the MoAb preparations injected i.v. were a 125I-labelled Fab fragment of the PLALP specific antibody H7, or a 125I-labelled F(ab')2 fragment of the sarcoma specific antibody TP-1. Specific targeting of the human tumour cells was demonstrated in both mice and pigs. The target: blood ratios were comparable in the two species, reaching a maximum of about 15 after 24 h with the Fab preparation, and a ratio of 25 after 72 h with the F(ab')2. The target uptake relative to injected dose was lower in pigs than in mice, but the difference between the two species was smaller than expected, presumably due to a slower antibody clearance in the pigs than in the mice. An artificial cell targeting system like this has several advantages in the search for solutions to many of the fundamental problems experienced in immunotargeting. Firstly, parallel binding experiments can be carried out in vitro with the same target. Because in vitro results are only influenced by the diffusion into the DC and the immunological binding characteristics of the antibodies, targeting differences between antibody preparations due to these factors can then be distinguished from differences due to pharmacokinetical properties. Secondly, the animals can be implanted with any type and number of target cells, or with antigen negative control cells. Thirdly, and perhaps most important, the system opens a possibility for evaluation of the murine MoAb in xenogenic species, and this may predict the clinical targeting potential better than experiments on mice.     

The effect of a somatostatin analogue on the release of hormones from human midgut carcinoid tumour cells.

The use of a somatostatin analogue (SMS 201-995) has greatly facilitated the treatment of patients with the midgut carcinoid syndrome. Clinical studies have shown that SMS reduces the peripheral levels of tumour-produced serotonin (5-HT) and tachykinins, e.g. neuropeptide K (NPK), basally and after pentagastrin provocation. Some studies have indicated an inhibitory effect of SMS on tumour cell growth as well. In the present study we have investigated the effects of SMS on four different human midgut carcinoid tumours maintained in long term culture. Media levels of 5-HT and NPK-LI in tumour cell cultures decreased rapidly during incubation with SMS (10(-8)-10(-10) M) in all four tumours studied without evidence for tachyphylaxis (up to 6 weeks observation period). SMS treatment (10(-8) M) during 4 days reduced the media concentrations of 5-HT by 56%, while the intracellular contents of 5-HT were decreased by 27% indicating dual inhibitory effects on synthesis and secretion of 5-HT from tumour cells. The DNA contents of cultures were not affected by SMS (10(-8) M or 10(-10) M) treatment for 4 or 14 days. When tumour cell cultures were challenged with isoprenaline (IP) (10(-6) M) no reduction of the IP induced release of 5-HT could be detected after pretreatment of tumour cell cultures with SMS (10(-8) M) for 1 h, 4 h or 4 days. These studies provide evidence for a direct action of the somatostatin analogue on midgut carcinoid tumour cells, reducing both synthesis and secretion of hormones from tumour cells. This effect appears not to be related to inhibition of tumour cell growth. The inhibition of 5-HT secretion from tumour cells by SMS seems to operate via a second messenger system different from the one mediating the beta-adrenoceptor stimulated release of 5-HT.     

Clonogenic cell survival in cryopreserved human tumour cells.

Cells from 3 human tumours have been grown in soft agar contained in Millipore diffusion chambers and implanted i.p. in mice. Clonal growth was obtained from fresh biopsy samples, from cryopreserved tissue, and from xenografts of the tissues in immune-suppressed mice. The radiosensitivities of a melanoma and an ovarian carcinoma were evaluated by in vitro irradiation before assay for colony formation. Xenografting did not modify the radiosensitivity of the melanoma. Cells from another tumour were exposed to Adriamycin or cyclophosphamide whilst contained within i.p. diffusion chambers; the sensitivity was similar for cryopreserved and xenografted cells. The results encourage further attempts to quantify the sensitivity of human tumour cells by these methods.     

The inhibition of cellular recovery in human tumour cells by inhibitors of topoisomerase

A human bladder carcinoma cell line was irradiated at high and low dose rates and exposed to camptothecin and VP16, inhibitors of topoisomerase I and topoisomerase II respectively. Although camptothecin substantially modified the cytotoxic effects of high dose rate irradiation, abolished low dose rate sparing and inhibited the repair of sublethal and potentially lethal damage, VP16 had no effect on the survival curves even at highly cytotoxic doses. Thus, it is argued that there is a role for topoisomerase I but not topoisomerase II in the repair of DNA damage induced by ionising radiation.     

Chromosomal DNA replication pattern in human tumour cells in vitro

The present paper deals with the chromosomal DNA replication pattern in human solid tumour cells in vitro. This was studied at the terminal stages of the S-period. All the cell lines of female origin showed a late replicating chromosome in group XX6-12. In cell lines of male origin one of the chromosomes of group 21-22Y was later replicating than the rest of the members of the group. The DNA replication pattern of the autosomes and the sex chromosomes was similar to that of the cultured human leucocytes. The results of the present study show that the DNA replication pattern of the chromosome in neoplastic cells is basically unchanged despite the changes in the chromosome number and morphology. Therefore the abnormal behaviour of the neoplastic cells cannot be related to the changes in the pattern of the chromosomal DNA replication.     

The ESTRO Breur lecture. Cellular sensitivity to low dose-rate irradiation focuses the problem of tumour radioresistance

This paper emphasises the radiobiology of human tumour cells irradiated at the relatively low dose rate of 1 Gy/h (i.e.1-2 cGy/min), described here as the "Regaud dose rate". Continuous irradiation at this dose rate is approximately isoeffective with fractionated radiotherapy using 2 Gy/fractions. At the Regaud dose rate, cell survival curves are approximately exponential and they appear to extrapolate the initial slope of the high dose rate survival curve. Little recovery occurs after such treatments since it has largely taken place during irradiation. At the Regaud dose rate human tumour cell lines show a wide range of radiosensitivities, differing by a factor of around 7. This may well be the most clinically-relevant way of describing the radiosensitivity of tumour cells. Current models of radiation cell killing envisage a component of damage that increases linearly with dose. It is this component that dominates the slope of the Regaud survival curve. It may be produced by DNA damage due to clusters of ionisation events, or perhaps by damage to hypersensitive parts of the genome. The steepness of this component of damage may be modified by exogenous inhibitors of DNA damage repair.     

The effect of adriamycin and 4'-deoxydoxorubicin on cell survival of human lung tumour cells grown in monolayer and as spheroids

Using growth delay and clonogenic cell survival as end points, we have shown that the 3-dimensional structure of human lung tumour spheroids confers a degree of resistance to the anthracyclines adriamycin and 4'-deoxydoxorubicin, relative to cells grown as monolayer. 4'-deoxydoxorubicin induces a longer growth delay and greater clonogenic cell kill than adriamycin in spheroids, although it is no more cytotoxic in monolayer (exponential and plateau phase). There is a log linear relationship between clonogenic cell survival and duration of adriamycin exposure in monolayers, and biphasic curve with a lesser degree of cell kill for disaggregated spheroid cells. Using fluorescent microscopy we have demonstrated, qualitatively, that the more lipophilic analogue partitions into the spheroid more rapidly and to a greater degree than adriamycin. It is possible that adriamycin penetration is a relatively important aspect of spheroid drug resistance, which may be related to intraspheroidal pH gradients, and that we have partially overcome this by using a lipophilic analogue.     

The effect of hydralazine on blood flow and misonidazole toxicity in human tumour xenografts

The effect of post-irradiation hypoxia induced by 5 or 30 mg/kg hydralazine has been studied in three human tumour xenografts (two rectocolic adenocarcinomas and one melanoma) treated with two doses of misonidazole similar to those used in patients (0.1 and 0.2 mg/g). Only a small sensitization was detected using an in vitro colony assay. These results are in marked contrast to the results obtained with rodent tumours. This difference between human tumour xenografts and rodent tumours might be explained by differences in the reduction of tumour blood flow after hydralazine administration (5 and/or 10 mg/kg). Using the laser Doppler technique, the tumour blood flow reduction was 33% and 25% of the control for NA11 and HRT18 tumours, respectively. In contrast, hydralazine induced a 60-70% reduction in blood flow in the murine SCCVII tumour. Using the fluorescent marker Hoechst 33342, the reduction in perfusion was again more pronounced in the murine tumour as compared to the Na11 and HRT18 xenografts. The differences between human tumour xenografts and rodent tumours are not linked to the mouse strain used (nude versus C3H) nor to a tumour bed effect.     

Radiation sterilisation of cultured human brain tumour cells for clinical immune tumour therapy

The aim is to investigate the radiosensitivity of noninfected cultured human glioma cells to ascertain that intracutaneously administered cells are viable enough to produce interferon-gamma but not able to proliferate. Cell cultures were established from five patients undergoing brain tumour surgery. By karyotyping, we found four malignant (three glioblastoma multiforme (GBM), one giant cell glioma) and one normal. The cells were irradiated with (137)Cs-gamma rays at absorbed dose levels of 0, 20, 40, 60, 80, 100 and 120 Gy. The fraction of viable cells was examined by MTT incorporation assay. The average of the data obtained from three GBM cell cultures was fitted to an exponential model. The parameters were: extrapolation number n=0.85+/-0.10, mean lethal dose D(0)=12.4+/-3.2 Gy and an additional uncertainty parameter deltaS=0.14+/-0.03. By setting deltaS=0, the corresponding values of the parameters were n=0.86+/-0.16 and D(0)=30.0+/-8.1 Gy. The rate of proliferation was examined by (3)H-thymidine incorporation. The average of the proliferation data obtained from three GBM cell cultures was fitted to an exponential model yielding n=0.943+/-0.005 and D(0)=5.8+/-0.5 Gy for deltaS=0.057+/-0.005, and by setting deltaS=0, n=1.00+/-0.02 and D(0)=8.4+/-1.6 Gy. No outgrowth of plated cells was observed after 4 weeks at an absorbed dose of 100 Gy. This absorbed dose is recommended for irradiation of 2 x 10(6) glioma cells used for clinical immunisation.     

Radiation-induced DNA double-strand break rejoining in human tumour cells.

Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t1/2) of rejoining ranged from 18.0 +/- 1.4 to 36.4 +/- 3.2 min (fast rejoining process) and from 1.5 +/- 0.2 to 5.1 +/- 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t1/2 of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell.     

Genetic basis of drug sensitivity in human testis tumour cells

Testicular germ cell tumours (TGCT) are cured in over 80% of patients by using combination chemotherapy. However, the mechanism regulating this sensitivity has not been defined. Because cells derived from patients with DNA repair syndromes are similar to TCGT in their sensitivity to certain DNA-damaging agents and some of the genes involved have been cloned by functional complementation, the purpose of our study was to determine whether drug sensitivity in TGCT also has a genetic basis. Three testis tumour cell lines (cisplatin-sensitive) and 3 bladder cancer cell lines (cisplatin-resistant) were fused with a cisplatin-sensitive cell line (D98 or CI). The authenticity of the hybrids was confirmed by karyotyping and PCR analysis of locus-specific sites, and sensitivities to cisplatin were measured by colony forming assays. The hybrids between sensitive cell lines were more resistant to cisplatin than the parental cells, indicating that functional complementation had occurred. The hybrids between the cisplatin-resistant and sensitive cells were intermediate in their cisplatin sensitivity, indicating that resistance is incompletely dominant. We conclude that cisplatin sensitivity has a genetic basis in TGCT and that resistance to cisplatin can be conferred by somatic cell fusion. Our data indicate that gene(s) controlling sensitivity to chemotherapy in TGCT might be identified by expression cloning. © 1996 Wiley-Liss, Inc.     

Cytotoxic activity of topotecan in human tumour cell lines and primary cultures of human tumour cells from patients.

The cytotoxic activity and cross-resistance pattern of the novel topoisomerase I inhibitor topotecan (Topo) were investigated in ten cell lines, representing different mechanisms of cytotoxic drug resistance, and in 218 fresh human tumour samples using the fluorometric microculture cytotoxicity assay (FMCA). Resistance to Topo in the cell lines was associated with expression of the multidrug resistance-associated protein (MRP), whereas the cell lines with P-glycoprotein (P-gp), topoisomerase II and glutathione-associated resistance did not show decreased sensitivity to the drug. Topo was more active in haematological than in solid tumour samples, but substantial activity was observed in carcinomas of the ovary and breast, sarcoma and childhood solid tumours. Cross-resistance to standard drugs representing different mechanisms of action was generally low in patient cells. The effect of Topo was better after longer exposure, but this time-dependent effect was largely abolished when adjustment for in vitro exposure was made. Topo showed activity both in proliferative and non-proliferative cell systems. The results indicate that Topo is insensitive to major mechanisms of resistance except for MRP. Proliferation does not seem to be necessary for the effect of Topo, and no superiority for protracted dosing schedules was observed. The results also suggest that, for example, leukaemias, lymphomas, sarcomas and childhood solid tumours may be suitable targets for future phase II trials.     

Mesothelial cells stimulate the anchorage-independent growth of human ovarian tumour cells

Results are presented which show that mesothelial cells (MC) from ovarian cancer patients can both stimulate and inhibit the clonogenic growth of ovarian tumour cells (TC) in a dose-dependent fashion. TC lines from both non-ovarian and ovarian tumours were variable in their response to MC. Colony formation was rarely induced when the TC population was non-clonogenic and a bladder cell line showed inhibition of colony formation in the presence of MC. Primary tumour cultures from ovarian cancer patients also showed a variable response to MC. Fibroblasts from malignant, benign and non-neoplastic sources were significantly less effective in stimulating the clonogenic growth of responsive cell lines. Conditioned medium was a poor substitute for the presence of intact viable cells, and distance between feeder cell and TC was an important factor in determining the magnitude of response. A significant relationship between the feeder effect of MC and their proliferation in soft agar was observed when epidermal growth factor was used in the medium. The relevance of the findings in the context of the pattern of spread of ovarian cancer is discussed.