Direct evidence that apoptosis enhances tumor responses to fractionated radiotherapy

Currently, the contribution of cellular apoptotic sensitivity to tumor response after radiation therapy remains controversial. To address this issue, the survival of Rat-1 fibroblasts containing a 4-hydroxytamoxifen-regulated c-Myc allele, c-MycER (T. D. Littlewood et al., Nucleic Acids Res., 23: 1686-1690, 1995), after single and fractionated doses of radiation was investigated. This model system allows pharmacological regulation of apoptosis sensitivity in the same cells in vitro and as xenograft tumors derived from these cells in vivo (G. I. Evan et al., Cell, 69: 119-128, 1992; R. M. Alarcon et al., Cancer Res., 56: 4315-4319, 1996). Activating c-MycER in vitro resulted in marked sensitization of Rat-1 fibroblasts to the effects of both single-dose and fractionated irradiation as measured by the induction of apoptosis and clonogenic survival. Overexpression of the antiapoptosis protein Bcl-2 suppressed the induction of apoptosis and increased clonogenic survival in cells with activated c-Myc after single-dose and fractionated radiation. Systemic time-release implant delivery of 4-hydroxytamoxifen to severe combined immunodeficient mice bearing Rat-1-MycER tumors over the course of either single-dose (10 Gy) or fractionated (five fractions of 2 Gy) radiotherapy resulted in prolonged tumor growth delay relative to identical tumors from mice that received placebo implants. Furthermore, tumors derived from Rat-1-MycER cells that overexpressed Bcl-2 exhibited shorter tumor growth delays relative to similarly treated Rat-1-MycER tumors. The length of tumor growth delay after single-dose or fractionated radiotherapy strongly correlated with the extent of radiation-induced apoptosis in the xenograft tumors as measured by terminal deoxynucleotidyl transferase-mediated nick end labeling. These in vivo results provide direct evidence that increasing the sensitivity of tumor cells to die by apoptosis increases the efficacy of fractionated radiotherapy by reducing tumor cell clonogenic survival.     

Peripheral blood lymphocyte decrease and micronucleus yields during radiotherapy

PURPOSE: To investigate the relationship between lymphocyte decrease and cytogenetic response in individual patients undergoing radiotherapy. MATERIALS AND METHODS: Peripheral blood lymphocytes obtained from 35 patients with pelvic and from 14 with head and neck tumours before and during radiotherapy were PHA-stimulated in vitro and the cultures prepared for the cytokinesis-block micronucleus test. For some patients only, the micronucleus test was carried out after 2 Gy in vitro X-irradiation, and 3-aminobenzamide (2 mM) was used to calculate the 3AB-index. RESULTS: The initially observed individual variation in the decrease of lymphocytes disappeared with increasing number of radiation exposures, reaching a stable level at about 500/microl lymphocytes in the blood. The slope of the relationship between the reciprocal of the lymphocyte decrease ratio and equivalent dose indicates the radiosensitivity of the lymphocyte pool in individual patients. The micronucleus test performed on lymphocytes obtained from patients during radiotherapy (in vivo) provided a lower cytogenetic response than the in vitro micronucleus yield for the same dose, so it was possible to calculate the cytogenetic recovery factor k. A correlation was found between the 3AB-index calculated before radiotherapy, and the recovery factor k. CONCLUSIONS: The 3AB-index, the micronucleus frequency after 2 Gy in vitro X-irradiation, and the cytogenetic recovery factor are proposed as predictive of individual response to radiotherapy.     

Comparison of bond strengths of three denture base resins to treated nickel-chromium-beryllium alloy

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Studies involving fibroblast survival have demonstrated the theoretical feasibility of a predictive assay of radiation sensitivity, but such an assay is still far from clinical application. Using pulsed-field gel electrophoresis (PFGE), we have quantified the initial "apparent" number of DNA double-strand breaks (dsb) induced by the radiation as an alternative measure of sensitivity in 2 different normal cell types from the same patients, epidermal skin cells and lymphocytes. We found significant inter-individual variation in the measured dsb (1-5 dsb/Gy/DNA unit). We also found a linear correlation between molecular damage in lymphocytes and skin samples from the same patient (slope = 0.83; r = 0.694; p = 0.0001). These results suggest that the initial number of dsb could be used as an indicator of the in vivo response to radiation.     

Tuberculosis and HIV infection. Clinical characteristics and diagnosis

The in vitro radiosensitivity of dermal fibroblasts has been found to vary between individuals, and a number of studies have also shown that this parameter correlates with radiation-induced late injuries in clinical radiotherapy. In addition, certain genetic disorders are known to effect radiosensitivity, e.g. normal tissues of patients homozygous or heterozygous for the ataxia teleangiectasia gene show unusual sensitivity to radiation both in vivo and in vitro. Thus, it has been assumed that there is a genetically determined component resulting in a certain intrinsic cellular radiation response in an individual. To study this possible relationship between different cells of a specific patient, we established eight pairs of dermal and tumor fibroblast cultures. The donor patients had either adenocarcinoma of the uterus or squamous cell carcinoma (SCC) of the head and neck. The radiosensitivity of these strains was determined by a 96-well plate clonogenic assay, previously used by us for radiosensitivity testing of cancer cells. From a paired comparison, the values for the cell fraction surviving 2.0 Gy (SF2), of both fibroblast strains, were found to be on the same level in five out of eight cases. In patient 6, the SF2 of tumor fibroblasts was significantly higher than that of dermal fibroblasts (P=0.0014). In two additional cases the tendency was the same, but not statistically significant. As groups, the two types of fibroblasts did not differ from each other, mean SF2 values of 0.24+/-0.07 and 0.21+/-0.05, respectively. The SF2 of tumor fibroblasts from SCC patients proved to be significantly higher than that of the adenocarcinoma patients (P=0.030). These preliminary results indicate that the in vitro radiosensitivity of tumor fibroblasts correlates with normal cell sensitivity in many cases, but not in all. The radiosensitivity of tumor fibroblasts also seems to follow the level of in vitro radiosensitivity determined for the corresponding histological type of tumor cells. Further studies are needed to determine more closely the relationship between the radiosensitivities of tumor cells and tumor fibroblasts, thus evaluating the possibility of testing radiosensitivity from tumor fibroblasts in order to estimate tumor response.     

Radiosensitivity of new and established human melanoma cell lines: comparison of [3H]thymidine incorporation and soft agar clonogenic assays

Seven new low-passage melanoma lines were developed in this laboratory from clinical melanoma specimens and characterised for chromosome complement, DNA ploidy and S-phase content. The radiosensitivity of these lines was compared with that of eight established melanoma cell lines, FME, MM-96, SK-MEL-5, SK-MEL-28, SK-MEL-2, MALME-3M, M19-MEL and LOX-IMVI, using a 96-well microculture assay technique. Dose-response curves were determined using a 5-day incubation period and 6-h terminal [3H]thymidine-labelling period. Radiation (60Co source) was carried out under a lead wedge to provide a radiation dose range of 0-10 Gy, or by irradiating part of the plate (radiation dose 0 or 2 Gy). Data for a range of cell densities in a single 96-well plate were combined into a single regression equation incorporating linear quadratic terms for radiation dose and cell density. SF2 values were defined as the amount of thymidine incorporated following a radiation dose of 2 Gy, expressed as a fraction of that of unirradiated cells, and varied from 0.36 to 0.93. The reproducibility in repeat assays, as defined by the standard error of determinations at different passage numbers, was +/- 0.04. The newly developed lines exhibited a similar range of radiosensitivity to that of the established lines, and melanin content did not correlate with resistance. For nine of the lines, radiation parameters were also determined using a modified Courtenay clonogenic soft agar assay technique, and the results compared with the thymidine incorporation results, and a significant linear correlation was found between SF2 and SF2' (r = 0.89). The linear (alpha) and quadratic (beta) terms of the best-fit linear quadratic dose-response curves, were significantly correlated between the two assays. It is concluded for this series of human melanoma lines that proliferation assays in 96-well plates provide radiosensitivity parameters comparable to those using clonogenic assays.     

An altered intracellular distribution of the autoantigen La/SS-B when translated from a La mRNA isoform

This study reports the successful growth suppression of a rat glioblastoma model (RT-2) both in vitro and in vivo by the insertion of p21(WAF1/CIP1), a negative cell cycle regulatory gene, into the tumor cells. Greater than 95% of the tumor cells expressed p21 protein after being infected with pCL based p21 retrovirus at 4x M.O.I. (multiplicity of infection). The p21-infected cells showed a 91% reduction in colony forming efficiency and a 66% reduction in growth rate. More prominent p21 staining was found in cells exhibiting histologic evidence of senescence. Intracranial implantation of the infected cells showed complete disappearance of the p21-infected cells at day 10 and long-term survival of the animals compared to controls. Injection of pCLp21 virus into tumor established in situ showed tumor necrosis and gene expression. In a clonogenic radiation survival assay, a 93% reduction of surviving colonies of p21-infected cells was seen in comparison to vector-infected control cells and to p53-infected cells after exposure to 8 Gy (800 rads).     

The ratio of initial/residual DNA damage predicts intrinsic radiosensitivity in seven cervix carcinoma cell lines

The single-cell gel electrophoresis (comet) assay was used to measure radiation-produced DNA double-strand breaks (dsbs) in a series of seven cervical tumour cell lines (ME180, HT3, C33A, C41, SiHa, MS751 and CaSki). The proportion of DNA dsbs was measured immediately after radiation treatment (initial damage) and 16 h later after incubation at 37 degrees C (residual damage). Linear dose-response curves were seen for initial (slopes 0.23-0.66) and residual (slopes 0.16-0.87) DNA dsbs. Neither of the slopes of the linear regression analysis on the initial and on the residual DNA dsbs dose-response curves (range 0-80 Gy) correlated with SF2 (surviving fraction at 2 Gy) measured after high- (HDR) or low-dose-rate (LDR) irradiation. An association was evident between SF2 after HDR and LDR irradiation and the ratio of the absolute level of initial and residual damage after a single dose of 60 Gy. However, a significant correlation was found between HDR (r= -0.78, P = 0.04) and LDR (r = -0.86, P = 0.03) SF2 values and the ratio of the slopes of the initial and residual DNA dsbs dose-response curves (range 0.47-0.99), representing the fraction of DNA damage remaining. These results indicate that the neutral comet assay can be used to predict radiosensitivity of cervical tumour cell lines by assessing the ratio of initial and residual DNA dsbs.     

Reduction of radiation-induced chromosome aberration and apoptosis by dithiothreitol

We have examined in vitro and in vivo radioprotective effects of a well-known thiol-containing compound, dithiothreitol (DTT). The treatment of both 0.5 and 1 mM of DTT significantly increased clonogenic survival of gamma-ray irradiated Chinese hamster (V79-4) cells. In order to investigate the possible radioprotective mechanism of DTT, we measured gamma-ray induced chromosome aberration by micronucleus assay. In the presence of 0.5 mM or 1 mM DTT, the frequencies of micronuclei were greatly reduced in all dose range examined (1.5-8 Gy). Slightly higher reduction in micronucleus formation was observed in 1 mM DTT-treated cells than in 0.5 mM DTT-treated cells. In addition, incubation with both 0.5 and 1 mM of DTT prior to gamma-ray irradiation reduced nucleosomal DNA fragmentation at about same extent, this result suggests that treatment of DTT at concentrations of 0.5 and 1 mM reduced radiation-induced apoptosis. In vivo experiments, we also observed that DTT treatment reduced the incidence of apoptotic cells in mouse small intestine crypts. In irradiated control group 4.4 +/- 0.5 apoptotic cells per crypt were observed. In DTT-administered and irradiated mice, only 2.1 +/- 0.4 apoptotic cells per crypt was observed. In vitro and in vivo data obtained in this study showed that DTT reduced radiation-induced damages and it seems that the possible radioprotective mechanisms of action of DTT are prevention of chromosome aberration.     

Evidence into practice, experimentation and quasi experimentation: are the methods up to the task?

PURPOSE: To assess the suitability of the cytokinesis block micronucleus assay as a biological dosimeter following in-vivo radiation using cancer patients undergoing radiotherapy. METHODS: Blood from 4 healthy donors was irradiated in vitro with gamma-rays and the dose response of induced micronuclei in binucleate lymphocytes following cytokinesis block was determined. Micronucleus frequency was ascertained before and at intervals during radiotherapy treatment in 6 patients with various tumors in the pelvic region. Equivalent whole body doses (physical doses) at these times were calculated from radiation treatment plans and cumulative dose volume histograms. RESULTS: Linear dose response relationships were found for induced micronucleus frequency in lymphocytes resulting from both in-vitro and in-vivo irradiation. Doses resulting from in-vivo irradiation (biological doses) were estimated by substitution of micronucleus frequency observed in radiotherapy patients into the dose response curve from in-vitro irradiation of blood. The relationship between the biologically estimated dose (BD) and the calculated equivalent whole body dose (PD) was BD = 0.868 (+/- 0.043)PD + 0.117 (+/- 0.075). CONCLUSION: The micronucleus assay appears to offer a reliable and consistent method for equivalent whole body radiation dose estimation, although our findings should be confirmed using lymphocytes from radiotherapy patients with tumors at anatomical sites other than the pelvis. Except at doses lower than about ).4 Gy, the method yields dose estimates acceptably close to "true" physically determined doses. The assay can be performed relatively rapidly and can be used as a "first line" biological dosimeter in situations where accidental exposure to relatively high radiation doses has occurred.     

Comparison between the in vitro intrinsic radiation sensitivity of human soft tissue sarcoma and breast cancer cell lines

The purpose of this study is to evaluate the radiation sensitivity of human soft tissue sarcoma cell lines in vitro and to compare with that of human breast carcinoma and glioblastoma cell lines. The intrinsic radiation sensitivity parameters of seven human soft tissue sarcomas and eight breast carcinoma cell lines were investigated in vitro by clonogenic assays for single-dose irradiation under aerobic conditions on cells in exponential phase of growth. The results for sarcoma cell lines showed that the mean surviving fraction at 2 Gy (SF2) was 0.39 (SD +/- 0.09) with a range of 0.24 to 0.53, and the average mean inactivation dose (MID) was 1.92 (SD +/- 0.35) range from 1.36 Gy to 2.49 Gy. These values were not different from that of breast cell lines examined concurrently and using the same experimental methods (mean SF2 0.38, SD +/- 0.09; MID 1.9 Gy, SD +/- 0.37). However radiobiological parameters of nine karyotyped human malignant glioma cell lines determined earlier in this laboratory were significantly higher (mean SF2 0.50 +/- 0.14; mean MID 2.61 +/- 0.60). In conclusion, the data presented here do not support the view that cells of sarcomas show unusual radiation resistance. To the extent that the in vitro determined cellular radiation sensitivity reflects the tumor response in vivo, the success rate for radiation applied against sarcoma and breast carcinoma of comparable size could be similar.     

Discrimination of human tumor radioresponsiveness using low-dose rate irradiation

PURPOSE: Evaluation of the theoretical and practical value of using low-dose rate (LDR) irradiation to increase the resolution of radiosensitivity testing of primary human tumors using clonogenic assays. METHODS AND MATERIALS: Fourteen human tumor cell lines were assessed for surviving fraction at 2-8 Gy (SF2-SF8) using low-dose rate irradiation and a clonogenic assay. Further data were collected from the literature for 64 low-dose rate irradiation survival curves from human tumor cell lines. The data were grouped into five different radioresponsiveness categories (A-E). An analysis was made of the ability of the graded survival levels to discriminate between the different radioresponse groups and compared with previous analyses for high-dose rate SF2. Fifteen human cervical carcinoma specimens were analysed for SF2 and SF3.5 following high- and low-dose rate irradiation. RESULTS: Low-dose rate irradiation increased the spread of tumor cell line radiosensitivity data and the ability to discriminate between radioresponse groups was greater at low than at high-dose rates. Using low-dose rate irradiation on primary tumor specimens and a soft agar clonogenic assay decreased the success rate in obtaining data. The latter dropped from 70% for high-dose rate SF2 to 51% for low-dose rate SF3.5. CONCLUSIONS: The work on cell lines illustrates that low-dose rate irradiation does improve the ability of clonogenic radiosensitivity measurements to discriminate between tumors of different radioresponsiveness groups. However, using low-dose rate irradiation on primary human tumors with a soft agar clonogenic assay was not practical because of reducing the success rate for obtaining data for radiosensitivity measurements.     

Angiotensin II mediates cell hypertrophy in vascular smooth muscle cultures from hypertensive Ren-2 transgenic rats by an amiloride- and furosemide-sensitive mechanism

Matrix metalloproteinases (MMP) are enzymes responsible for extracellular matrix degradation, a critical component influencing the growth and metastatic potential of cancer. The purpose of this study was to determine the in vitro effects of MMP inhibition on human pancreatic cancer cells and to document its effect on cancer growth in vivo. The effect of MMP inhibition was determined using the MMP inhibitor BB-94 and a moderately differentiated pancreatic cancer cell line (HPAC). In vitro, a dose response curve was generated over 5 days utilizing the MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. In vivo, using an established orthotopic model for pancreatic cancer (LD100 = 80 days), 22 nude mice with orthotopic tumors (30 were implanted) received either BB-94 or vehicle beginning 4 days prior to implantation and continuing to death or sacrifice on Day 70. Mice were weighted weekly. At death/sacrifice, tumors were weighted, volume determined, and metastases/ distant spread documented. In vitro, BB-94 had little effect on HPAC proliferation at 40 ng/ml but achieved progressively greater to near complete inhibition at doses up to 4000 ng/ml while maintaining cell viability. In vivo, BB-94 significantly increased length of survival (69 +/- 0.1 days vs. 56 +/- 3.1 days) and necropsy weight (25.7 +/- 1.67 g vs. 19.8 +/- 1.14 g) while decreasing metastatic rate (1 vs. 20) and tumor size (0.14 +/- 0.02 g vs. 0.65 +/- 0.1 g). MMP inhibition limits HPAC proliferation in a dose-dependent fashion without direct cytotoxic effects in vitro. Mice harboring orthotopic tumors treated with BB-94 demonstrated significant reductions in tumor weight, volume, and metastases which corresponded to increased animal weight and prolonged survival.     

Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34+ hematopoietic progenitor cells

PURPOSE: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. METHODS AND MATERIALS: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34+ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34+ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. RESULTS: The radiobiological parameters (D[0] and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34+ cells compared to optimal two-factor combinations. The D[0] values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n=1 for all tested GF(s). DNA repair capacity of CD34+ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34+ cells was similar. CONCLUSION: Our data indicate that increased survival of irradiated CB CD34+ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.     

Sensitivity and specificity of mu-capture ELISA for detection of enterovirus IgM

The effect of Taxol on the radiation sensitivity of human squamous carcinoma of the head and neck region was determined in vitro, using clonogenic assays and multicellular tumor spheroids (MTS). Radiosensitivity parameters were determined by alpha and beta for clonogenic assays, and by the residual/control volume ratios at 2 Gy (RSV2) and the dose inducing 50% decrease in MTS number (SCD50) for spheroids. In HTB43 and CAL27 colonies, the combination was antagonist. In spheroids, Taxol induced a decrease of RSV2 and SCD50 in HTB43 and CAL27 MTS and their combinations with radiation were synergistic and additive, respectively. Therefore, the different results obtained by clonogenic assays and MTS may suggest higher drug incorporation through the multiple cell layers of the spheroids than in monolayers.     

Lack of correlation of human fibroblast radiosensitivity in vitro with early skin reactions in patients undergoing radiotherapy

Fibroblasts from breast cancer patients were obtained as outgrowths in vitro from punch biopsies and their radiosensitivity tested in early passages. Skin erythema reactions in the same patients were also measured, as degree of redness using reflectance spectrophotometry. Measurements were taken before and during a 4-week radiotherapy treatment with electrons to the thoracic wall. Of 59 biopsies studied, radiosensitivity and erythema were concurrently studied in 32. In 24, evaluable data from both clinic and laboratory were obtained. A population growth assay in 96-well plates, using absorption of sulphur rhodamine B as the stain for cell numbers, showed good agreement with the colony-formation assay. Plating efficiencies and growth rates in the colony assay were higher using human serum in place of foetal calf serum. Cell survival curves with human serum were mostly exponential with little shoulder. The parameters of survival at 2 Gy (SF2) and the dose required to give 10% survival (D10) were used in the correlations with clinical data; these were 0.25 +/- 0.09 and 3.03 +/- 0.50 Gy, respectively. There was a strong correlation between these two survival curve parameters (r = 0.98). Skin redness was found to linearly increase with time during radiotherapy. The slope of the increase differed markedly from patient to patient, with a range of a factor approx. 10. No correlation was found between SF2 and erythema response in the 24 evaluable patients (r = 0.13, p > 0.5). A similar lack of correlation was found using D10 as the radiosensitivity parameter (r = 0.12, p > 0.5). These data indicate that fibroblast radiosensitivity measured in vitro cannot be used to predict erythema reactions to radiotherapy in breast cancer patients.     

The effect of radiation on tumour growth delay, cell survival and cure of the animal using a single tumour system

The response of a murine tumour to single doses of X rays has been measured using three different assays--animal cure, cell survival in vitro after irradiation in vivo, and tumour growth delay. The dose to cure 50% of the animals, the TCD50, was 79.0 Gy. This was not affected by clamping the tumours to render all the cells hypoxic at the time of irradiation, implying that most of the cells in the tumour were hypoxic already. The enhancement ratio for the hypoxic cell sensitizer Ro-07-0582 was 2.1. The cell survival assay gave an enhancement ratio of 1.6 and an hypoxic fraction of 5%. The discrepancy in the estimates of the hypoxic fraction can be explained by the ability of the naturally hypoxic cells, but not the oxic ones, to recover from potentially lethal damage in vivo. Neither the cell survival assay nor the growth delay assay agreed with the TCD50 assay as to the effect of the hypoxic cell sensitizer, even allowing for recovery from potentially lethal damage. It is doubtful whether the measured survival curve would predict the measured TCD50.     

Expression of manganese superoxide dismutase reduces tumor control radiation dose: gene-radiotherapy

This study investigated the in vitro and in vivo radiation response of tumor cells transfected with human manganese superoxide dismutase (MnSOD) cDNA. A major objective was to test the potential tumor suppressive effect of MnSOD in vivo. Tumor cells studied were an in vitro line derived from a murine spontaneous fibrosarcoma, FSa-II, which expressed an undetectable MnSOD activity. These cells were transfected with pSV2-NEO plasmid (NEO line) or cotransfected with MnSOD plasmid plus pSV2-NEO plasmid (SOD lines) as described previously. The cell lines used were SOD-L and SOD-H, which expressed, respectively, low and high MnSOD activities after transfection, and NEO and parental FSa-II controls. Both SOD-L and SOD-H cell lines were slightly more resistant to ionizing radiation than were the two control cell lines when irradiated in vitro in the presence of oxygen. The dose-modifying factors calculated at the survival level of 0.01 were 1.13 and 1.15 for the SOD-L and SOD-H cells, respectively. To investigate potential tumor suppressive effects, animal tumors of 4 mm diameter were irradiated in vivo under hypoxic conditions, and the radiation dose to control one-half of the irradiated tumors (TCD50) was determined for each tumor. The TCD50S obtained on the basis of the tumor control rate in 120 days after irradiation were substantially lower for the SOD-H and SOD-L tumors compared to the NEO tumors. They were 22.9, 28.6, and 47.5 Gy for SOD-H, SOD-L and NEO tumors, respectively. To analyze these data, survival curves were obtained for hypoxic cells by irradiating NEO and SOD-H tumors under hypoxic conditions in vivo and assaying in vitro. Analysis of these curves suggests that the decrease in the TCD50S of SOD tumors is attributable to the reduced tumorigenicity in these tumors. The hypoxic cell survival curves also showed that SOD did not protect cells from radiation in the absence of oxygen. Electron microscopy showed no morphological differences between these cells. These results suggest that the fraction of tumorigenic cells could be reduced by expression of MnSOD, resulting in a substantial decrease in the TCD50.     

Tumor cell spheroids as a model for evaluation of metabolic changes after irradiation

Tumor cell spheroids provide a good model to evaluate the relationship between tumor volume and the number of viable cells in the volume with the uptake of metabolic tracers before and after therapy. They represent the only in vitro model that allows the determination of the activity per unit volume, a parameter which is relevant for interpretation of PET studies. The purpose of this study was to evaluate this model with respect to the uptake of 14C-FDG, 3H-methionine and 3H-thymidine with and without exposure to irradiation. METHODS: Spheroids of the human adenocarcinoma cell line SW 707 were incubated in media containing 14C-FDG, 3H-methionine or 3H-thymidine for 1 hr at 1, 4, 8, 24 and 48 hr after exposure to a single radiation dose of 6 Gy together with control spheroids. Tracer uptake after incubation was expressed in cpm/ spheroid, cpm/1000 viable cells and cpm/0.01 mm3. In addition, the proliferative capacity of control and irradiated spheroids was determined using the clonogenic assay. RESULTS: Spheroid uptake of FDG decreased with time after irradiation, while the uptake per 1000 viable cells was increased significantly. The activity per unit volume remained unchanged in comparison to control spheroids. Methionine uptake per spheroid was unchanged after irradiation because of the high increase in uptake per 1000 viable cells. Uptake per unit volume also remained unchanged in comparison to controls. Thymidine uptake per 1000 viable cells did not change after irradiation but showed significant differences in uptake per spheroid and per unit volume compared to controls. The percentage of thymidine incorporated into the TCA-precipitable fraction containing DNA was 50% in controls and decreased to 12% at 24 hr after irradiation. The suppressed clonogenic capacity early after therapy recovered with the increase in thymidine uptake and with the increase in thymidine incorporation into DNA. CONCLUSION: The results show that the activity determined within a certain tumor volume is a balance between the increased tracer uptake by surviving cells after therapy and the lack of tracer uptake by dead cells, which still contribute to the tumor volume. Thus, the resulting unchanged activity per unit volume within the spheroid, as found for FDG and methionine, may not fully reflect therapy-induced metabolic changes in tumors.     

Enhancement of fractionated-dose irradiation by retinoic acid plus interferon

PURPOSE: To evaluate the relative cytotoxicity of fractionated-dose radiation in the presence and absence of 13-cis-retinoic acid (RA) plus alpha-2a-interferon (IFN), as a function of overall treatment time. METHODS AND MATERIALS: Studies were performed with the human squamous cell carcinoma line FaDu, in vitro. Attached exponential phase cells were treated with RA + IFN for 8-10 h and then exposed to single graded doses of radiation, or 1 to 6 doses of radiation at 2 Gy per dose, or to 5 doses of radiation at 2 Gy/dose with a time interval of 4-24 h between treatments. Following irradiation, the cells were incubated with drugs present throughout colony formation, and the fraction of survivors in the presence and absence of the combined drugs was calculated. RESULTS: For single graded-dose irradiation, the surviving fraction ratio at 2 Gy in the absence vs. presence of drugs was 1.27 +/- 0.19 in 3 repeat experiments. Following administration of 6 doses of radiation at 2 Gy/fraction with a 5-h time interval between treatments and, after correcting for cell proliferation between treatments, the surviving fractions differed by a factor of 3.25, again indicating an average difference in survival of 1.26 after each of the 6 2-Gy/fractions. Treatment with 5 2-Gy doses of irradiation with 24 vs. 4 h elapsing between doses, resulted in a 3-fold greater decrease in survival in the presence of drugs vs. no drug. The relatively greater cell kill due to 24 vs. 4 h between treatments was due to drug inhibition of cell proliferation between the more prolonged treatments. CONCLUSIONS: The results of this study indicate that retinoic acid plus interferon both sensitizes and inhibits cell proliferation during treatment. These results suggest that this combination of radiation and drugs, when used concurrently, may be effective for inhibiting tumor cell proliferation or accelerated repopulation during clinical fractionated radiotherapy.     

Variation in radiosensitivity due to cell age and split-dose recovery in polykaryons induced by cytochalasin

We have investigated the properties of an in vitro cell survival assay that uses as its endpoint the ability to form polyploid cells (polykaryons) in the presence of cytochalasin B (CB). The criterion for survival is that a polykaryon-forming unit (PFU) must reach the arbitrary DNA content of at least 16C. The age-dependence of PFU sensitivity to 137Cs irradiation was determined using V79-379A cells synchronized at mitosis. Cells assayed as PFUs demonstrated much less variation in radiosensitivity with age than did clonogens, but the changes in curve shape were qualitatively similar. In both assays mitotic cells yielded an exponential survival curve while that obtained at 5 h (mid-late S) had a marked quadratic component. Owing to the small overall variation in PFU survival with age, at doses greater than about 25 Gy the surviving fraction at 5 h was lower than in mitosis. In both V79-379A and HeLa S3 cells, PFUs demonstrated a capacity for split-dose recovery and yielded recovery ratios at 2.6 at 50 Gy in V79 and 1.5 at 20 Gy in HeLa. Since these ratios were much lower than in clonogens at the same dose, we suggest that this is consistent with an association that we have previously demonstrated between PFU response and the clonogenic initial slope. In an attempt to clarify the DNA lesions to which PFUs may be sensitive, we determined PFU response following exposure to 254-nm UV irradiation. In contrast with ionizing radiation, PFU response to UV was very similar to that of clonogens. This suggests that following UV exposure the absence of cytokinesis in polykaryons may confer less protection than in the case of ionizing radiation, possibly due to fundamental differences in the spectrum of DNA lesions produced.