Response of fibrosarcoma cell subpopulations to small doses of radiation delivered in situ

The survival of cells from 2 tumor subpopulations after γ-ray doses ranging from 1 to 19 Gy was determined using a lung colony assay. Methylcholanthrene-induced fibrosarcomas grown in the hind legs of C3H/Kam pathogen-free mice were irradiated in situ when the tumors were 8–10 mm in diameter. Single cell suspensions prepared from excised tumors were separated on a linear density gradient, and the clonogenicity of predominantly oxic Band 2 (density 1.08 g/cm²) and predominantly bypoxic Band 4 (density 1.14 gm/cm³) cells was measured. The surviving fraction of cells after doses of 1, 2, and 3 Gy was estimated from that measured after total doses of 5 Gy = 5 × 1 Gy, 10 Gy = 5 × 2 Gy, and 15 Gy = 5 × 3 Gy, under the assumption of equal effect per fraction (checked by estimating survival at 3 Gy after different numbers of fractions). Very little curvature was evident in the survival curves of Band 2 and Band 4 cells (β/α = .013–.034 Gy^?1). The initial segment of the survival curve of the predominantly oxic Band 2 cells was steeper (₁D₀ = 3.6 Gy) than that of the predominantly hypoxic Band 4 cells (₁D₀ = 5.2 Gy); both remained linear over a large range, to doses in excess of 3 Gy. These results imply that these tumor subpopulations will be insensitive, in their response to multifractionated regimens, to changes in size of dose per fraction in the range 0 to 3 Gy, a trait shared by two acutely responding normal tissues (marine testis and jejunum).     

Increased tumour response of a murine fibrosarcoma to low temperature hyperthermia and low dose rate brachytherapy

The present animal tumour study was carried out to determine the effectiveness of low temperature hyperthermia combined with low dose rate radiation based on the cell culture studies of our laboratory and others that demonstrated a significant radiosensitization obtained by low temperature hyperthermia and low dose rate radiation. Well-oxygenated murine fibrosarcoma Meth-A tumours growing in Balb/c mice were treated with heat (41d`C tumour temperature) by immersion of the tumour-bearing leg in a waterbath concurrently with low dose rate radiation. Radiation was delivered using <sup>192</sup>Ir interstitial implantation at absolute dose rates of 0.416–0.542 Gy/h. The effect of heat alone on tumour growth and normal tissue was minimal. Tumour growth delay following 30 Gy radiation was 4.9 days. Significant delay in tumour growth was observed with the addition of low temperature hyperthermia delivered concurrently. Enhancement in radiation response was seen with increasing duration of heat treatment; tumour growth delays were 9.5 days following 4h heat (41d`C) treatment and 16 days following 6 h treatment. Three sessions of fractionated hyperthermia 4 h/day during the course of low dose-rate radiation significantly delayed tumour growth to 18.6 days. The results indicate that fractionated heat treatment in conjunction with low dose rate radiation has potential for improving tumour response without adversely affecting normal tissue reaction. This in vivo study represents an extension of the cell culture data and provides further radiobiological basis for the combined use of low temperature hyperthermia and low dose rate radiation.     

Response of MBT-2 bladder carcinoma-induced osteolysis to various agents.

Tumor-bone interactions were experimentally studied using a bladder tumor in mice (MBT-2). The method consisted of subcutaneously inoculating tumor cells over the calvaria in nude mice after the periosteum was disrupted. This resulted in a local tumor that caused fragmentation of the bone. Bone destruction was found to increase in proportion to the number of osteoclasts in the earlier phase. The osteoclasts decreased in number when the tumors had grown large enough to envelop the residual bone. However, bone destruction continued and seemed to be mediated by the tumor cells by a mechanism that did not involve the osteoclasts. The effects of several agents were investigated in this model. High doses of calcitonin and cyclosporine reduced the bone resorption, and these agents may be effective in the early phase of bone destruction. A bisphosphonate derivative (AHBuBP) inhibited bone resorption markedly in the early and late phases of bone destruction. Autoradiography using carbon 14 (14C)-labeled AHBuBP showed that the isotope was concentrated at the surface of the bone adjacent to the MBT-2 tumors. These results suggest that bisphosphonates may make bone less susceptible to the actions of osteoclasts and tumor cells.     

Response of glioblastoma cell lines to low dose rate irradiation

Glioblastoma U251 and U87 cells irradiated with single fraction high dose rate radiation (1.1 Gy/min) were relatively insensitive to inactivation of colony forming ability, similar to other glioblastoma cell lines. Initial rates of cell kill with continuous low dose rate irradiation (0.075 Gy/hr to 0.49 Gy/hr) were low, but at times greater than 20 hours and with dose rates of 0.25 Gy/hr or higher, the rate of cell kill increased. Population doubling times for these cell lines were about 24 hours, suggesting that cell cycle redistribution may be responsible for the increased sensitivity. DNA histograms obtained by flow cytometry support this hypothesis, with cells accumulating in the G2 and M phases of the cell cycle. These results suggest that low dose rate irradiation may be effective in treating glioblastomas. Optimization of time intervals between radiation treatments as well as dose rates used for glioblastoma patients may be influenced by these findings, resulting in better integration of continuous low-dose-rate irradiation (radioactive antibodies and implants) and high-dose-rate irradiation (fractionated external beam) into therapeutic programs.     

Differences in cellular proteins between high and low metastatic potential sublines from the methylcholanthrene-induced rat fibrosarcoma cell line.

The purpose of the present study was to determine with two-dimensional electrophoresis whether two sublines (high and low metastatic potential to lymph nodes) of rat fibrosarcoma induced by methylcholanthrene have differences in cellular proteins. The high metastatic potential subline had a specific protein in the protein map of the whole cell lysate. Its isoelectric point was 6.4 and the molecular weight was 18,000. This specific protein appeared in maps of the precipitate of 1,000 g centrifugation and in the supernatant from treatment with 0.5% Nonidet P-40 (NP-40). But this specific protein was not stained by five lectins, concanavalin A, wheat germ agglutinin, Ulex europeus agglutinin-1, Dolichis biflorus agglutinin, and Ricinus communis agglutinin. Apparently, this high metastatic potential subline has a specific protein in the plasma membrane which has no glycochain.     

Response to pulsed dose rate and low dose rate irradiation with and without mild hyperthermia using human breast carcinoma cell lines.

The purpose of this study was to establish whether a pulsed dose rate (PDR) treatment of 1.5 Gy given every 3 h in combination with 41 degrees C mild hyperthermia or a continuous low dose rate (LDR) treatment with mild hyperthermia could radiosensitize two isogenic human breast carcinoma cell lines in comparison to pulsed dose rate or low dose rate irradiation alone. The radiation resistant cell line was derived from the parental cell line and was transfected to over-express DNA polymerase beta. The end-points assessed were the survival of the cells using the clonogenic assay, the amount of residual DSB(s) using the comet assay and gene expression of polymerase beta using RT-PCR. Results showed that the PDR and LDR treatments combined with mild hyperthermia caused significant radiosensitization when compared to PDR and LDR irradiation alone in terms of the clonogenic and comet assays with both cell lines. RT-PCR results showed that polymerase beta levels of expression were not elevated in response to these treatments, implying that this polymerase may not be involved in sub-lethal damage repair or thermal radiosensitization. These results suggest a potential clinical advantage when combining LDR or PDR with hyperthermia, since they indicate that hyperthermia is an effective radiosensitizer.     

Differences of host immune response and of sensitivity to chemotherapeutic agents between two clones of rat fibrosarcoma with different metastatic potential

Differences of host immune response and of chemosensitivity between two clones derived from a methylcholanthrene-induced rat fibrosarcoma and exhibiting different tumorigenic and metastatic potentials were examined. Clone G, which has no metastatic potential, was more sensitive to natural killer cytotoxicity by syngeneic rat spleen cells than clone A, which is highly metastatic. The colony formation of clone G was strongly inhibited by the supernatant obtained from coculture of lymphocytes with either of these two clones. In contrast, the colony formation of clone A was not inhibited by these supernatants. Inhibition of the colony formation of clone G was dependent on the dilution of the supernatant and the incubation time of the lymphocytes and tumor cells used to generate the supernatant. The supernatant from cocultures of lymphocytes and clone G inhibited the colony formation of clone G more strongly than the supernatant from cocultures of lymphocytes and clone A. There was no difference between these two clones in chemosensitivity to seven of the ten chemotherapeutic drugs tested.     

Immunoprophylaxis and immunotherapy for a murine fibrosarcoma with C. granulosum and C. parvum

Prophylactic and therapeutic effectiveness of killed C. granulosum and C. parvum bacteria was investigated against a methylcholanthrene-induced fibrosarcoma in syngeneic C₃Hf/Bu mice. Subcutaneous or intravenous treatment of mice with 0.25 mg of these bacteria greatly reduced the number of tumor nodules (metastases, colonies) in the lung generated by 10⁵ or 10⁶ fibrosarcoma cells inoculated intravenously 7 days later. The treatment also prolonged the survival of tumor-cell recipients, and to some mice afforded complete protection against tumor growth. Number of lung metastases and survival of the recipients were more affected by intravenous than by subcutaneous treatment with the bacteria. Given intravenously to mice 3 days after intravenous inoculation of 2×10⁵ fibrosarcoma cells, these non-specific immunostimulants significantly reduced the number of pulmonary colonies and prolonged survival of mice. In contrast, subcutaneous application of the bacteria was only slightly effective. Both C. granulosum and C. parvum administered intravenously to mice 3 days following subcutaneous injection of 4×10⁵ fibrosarcoma cells did not affect the development of subcutaneous tumors. However, when the tumors had grown to 25-17 mm in diameter 67% and 80% of them underwent complete and lasting regressions in mice treated with C. granulosum and C. parvum, respectively. C. granulosum and C. parvum were approximately equally effective against both intravenously and subcutaneously injected tumor cells.     

A modelling study of the potential influence of low dose hypersensitivity on radiation treatment planning.

BACKGROUND AND PURPOSE: Low dose hyper-radiosensitivity (HRS) has been observed in both normal tissues and tumours. This modelling study explores the possible impact of HRS on radiation treatment planning. PATIENTS AND METHODS: The interplay between volume-effect and HRS was studied in an idealized comparison of partial versus whole organ irradiation. In the further studies, CT scans of three previously scanned patients were used to estimate normal tissue complication probability (NTCP) for the kidneys after a conformal and a conventional treatment plan with and without consideration of HRS. RESULTS: Idealized treatment plans were compared as pairs of a conventional and a conformal plan both treating the same target volume to the same dose per fraction. Contour maps of the difference in NTCP between paired plans showed a strong dependence on the magnitude of both the volume effect and the HRS effect. For more clinically realistic treatment plans with NTCP calculated for the kidney, the balance between the sparing due to the LQ effect and the increased sensitivity due to the HRS effect was dependent on both the dose distribution and the fractionation. CONCLUSIONS: HRS may potentially affect radiotherapy treatment planning and the relative importance of HRS is larger in a tissue or organ with a pronounced volume effect. If HRS is expressed in some normal tissues or organs, this could offset much of the sparing predicted by the LQ formalism. However, in some clinical situations the NTCP calculated with correction for HRS may still be lower than the NTCP calculated from the uncorrected physical doses.     

Response to X-radiation and cytotoxic drugs of clonal subpopulations of different ploidy and metastatic potential isolated from RIF-1 mouse sarcoma

Clonal subpopulations of different ploidy values and metastatic capacities, isolated from the RIF-1 mouse sarcoma, have been tested for in vitro X-radiation sensitivity, for in vitro sensitivity to adriamycin and for in vitro and in vivo sensitivity to melphalan and CCNU. Following X-radiation, no consistent differences in the survival curve characteristics (Do and n) of diploid, tetraploid and octoploid cells were observed. In addition no relationship between radiation response and metastatic capacity was observed. For drug response, no marked differences were found in the dose response curves of RIF-1 clones treated in vitro with adriamycin. However, a wide variation in the responses of RIF-1 clones to in vitro melphalan treatment was observed which was independent of both ploidy and metastatic capacity. Although the responses of RIF-1 clones to in vitro CCNU treatment were similarly independent of metastatic capacity, a clear relationship between CCNU sensitivity and ploidy was observed. Thus, all diploid RIF-1 clones were markedly more sensitive to CCNU treatment than either tetraploid or octoploid RIF-1 clones. For both melphalan and CCNU treatment the relative sensitivities in vitro correlated with in vivo sensitivities as assayed by clonogenic cell survival.     

Effect of Corynebacterium parvum on the response to irradiation of a C3H fibrosarcoma.

A methylcholanthrene-induced fibrosarcoma of C3H mice has been used as 5th- to 6th-generation syngeneic transplant in an investigation of the role of Corynebacterium parvum as an adjuvant to the therapeutic effect of local irradiation of established tumors. The most effective route for administration of C. parvum in this tumor system was i.v., and the greatest effect of the C. parvum-mediated tumor graft rejection was observed for tumor growing intracutaneously or s.c. An intermediate level of effectiveness was obtained for tumor growing i.m. The combined C. parvum and local irradiation studies were performed using tumors growing in the leg muscle and measuring 8 mm in diameter at the time of local irradiation. Several routes of administration of C. parvum, dose levels of C. parvum, and time relationships between administration of C. parvum and irradiation were investigated. The outstanding finding was that very low doses of radiation were quite effective when administered to tumors growing in animals pretreated with C. parvum. This was true for radiation administered as a single or fractionated dose (10 equal doses spread over 18 days). For single-dose irradiation the effect was relatively dose independent over the range of 200 to 3000 rads. Some indication was obtained that local irradiation may impair the tumor graft rejection reaction. No evidence was obtained of an enhanced growth of tumor. Regression of tumor following irradiation was not modified by pretreatment with C. parvum. The mice that were unsuccessfully treated by radiation and C. parvum had a lower incidence of metastatic disease in the lung than did the mice treated unsuccessfully with radiation alone.     

Response of cells of human origin, normal and malignant, to acute and low dose rate irradiation

Dose response curves were obtained for normal human fibroblasts and for several cell lines derived from human tumors, including melanomas and an osteosarcoma. Most of the tumor lines are similar in radiosensitivity to the normal fibroblasts, except for the melanoma lines, which are significantly more resistant. The two melanoma lines differ, one being much more radioresistant than the other. Potentially lethal damage repair (PLDR) has been studied in these cell lines as well. The extent of PLDR does not appear to correlate with radioresistance; for example, the most resistant melanoma line shows very little repair of PLD. In addition, the normal fibroblasts repair PLD at least as well as any of the tumor derived lines, which casts doubts on the wisdom of introducing into clinical practice inhibitors of PLD until a clear differential between normal tissues and tumors has been demonstrated in vivo. Low dose-rate studies with normal human fibroblasts indicate a smaller dose-rate effect than for most established cell lines of rodent origin. Indeed, in the human cells studied, the effect of sublethal damage repair is quantitatively similar to the repair of potentially lethal damage. Dose response curves for acute and protracted exposures have been obtained for cells derived from patients with cancer-prone syndromes including ataxia telangiectasia (AT) and Bloom's syndrome. Both cell lines are much more radiosensitive than normal human fibroblasts; the AT cells show a dose-rate effect, while Bloom's syndrome cells do not.     

低剂量丝裂霉素C降低胸部癌症患者放射损伤作用研究

目的观察低剂量丝裂霉素C(MMC)对胸部癌症患者放疗中的降低放射损伤作用。方法100例胸部癌症患者中，食管癌54例、肺癌46例，随机分为研究组和对照组各50例，研究组于常规放疗过程中同时用低剂量MMC0．002mg／kg，每周1次，共5～7次；对照组只接受常规放疗。结果100例全部完成治疗，研究组、对照组急性放射性食管炎发生率分别为30％，48％(x^2=3．897，P=0．048)；研究组、对照组急性放射性肺炎发生率分别为4％，16％(x^2=4．001，P=0．045)；血液毒性反应研究组、对照组分别为50％，48％(x^2=0．208，P=0．648)。研究组有效率(CR PK)(84％)明显高于对照组(68％)(x^2=4．089，P=0．043)。结论低剂量MMC配合胸部癌症放疗，明显减轻了急性放射性食管炎及肺炎的发生，无明显的血液毒性反应，同时提高了肿瘤的放疗效果。     

Response of C3H-AvyfB female mice to a low oncogenic, milk-borne mammary tumor virus.

When low oncogenic milk-borne mammary tumor virus (MTV) of strain DD was tested in C3H-AvyfB mice, as compared with the similar strain C3HfB, mammary tumors occurred with a higher incidence and at lower age in C3H-AvyfBfDD (with DD-MTV) than in C3HfBfDD (with DD-MTV), as observed in virgin females. Since the effect of breeding tended to mask any difference between the breeders, mammary tumor incidences were similar with a slightly higher age at tumor occurrence in the C3H-AvyfBfDD females. Thus the C3H-AvyfB virgins (with the Avy gene) were almost as susceptible to mammary tumors with the less potent DD-MTV as were C3HfB virgins with the more virulent C3H-MTV of strain C3H.     

Cytotoxic effect in vivo of selected chemotherapeutic agents on synchronized murine fibrosarcoma cells.

The cytotoxic effects in vivo of single doses of either adriamycin (ADM), 1-beta-D-arabinofuranosylcytosine (Ara-C), bleomycin (BLM), cis-diamminedichloroplatinum (II) (cis-DDP), or cyclophosphamide (CY) on murine fibrosarcoma (FSa) cell populations were determined. Tumour cells were separated and synchronized by centrifugal elutriation. Viable tumour cells from selected elutriator fractions were then injected i.v. into whole-body-irradiated mice. Twenty minutes later selected doses of ADM, Ara-C, BLM, cis-DDP or CY were administered to selected groups of these animals. Fourteen days later the mice were killed. Killing of injected tumour cells by each of the chemotherapeutic agents was evidenced by a reduction in the lung cells by each of the chemotherapeutic agents was evidenced by a reduction in the lung colonies per cell injected in treated animals. Under these conditions the response of FSa cells in vivo to the 5 drugs tested differed both qualitatively and quantitatively. Ara-C was S-phase-specific in toxicity. ADM, BLM, and cis-DDP were preferentially toxic to S, G2+M and G1 cells respectively. CY, a drug requiring bioactivation to form alkylating metabolites, was found to be equally toxic to G1 and G2+M enriched populations, but less effective in killing cell populations enriched with early-S cells.     

Enhancement of radiation sensitivity in iododeoxyuridine labelled cells exposed to low energy x-rays

In order to investigate if low-energy x-rays induce Auger cascades by photoelectric absorption in iodine present in DNA, CHO cells were labelled with iododeoxyuridine (IUdR) for 72 hours. Following labelling, the cells were either irradiated with low-energy x-rays (75 kV, 4 mm Al) or 137Cs-gamma-rays. The radiation response was measured using clonogenic survival, and the survival parameters were analyzed according to the linear quadratic model. The dose modifying factors were determined as the ratios of the alpha-coefficients. The IUdR labelled cells were found to be about 3.2 times as sensitive as the control cells when irradiated with low-energy x-rays. For 137Cs-gamma the ratio was about 1.5. The standard deviations were estimated by Gauss' approximation to be about 0.5 for both irradiation conditions.