Protective effect of hypoxia in the ram testis during single and split-dose X-irradiation

Spermatogonial stem-cell survival in the ram was studied after single (6Gy) and split-dose (2 × 3Gy, interval 21–24 h) X-irradiation both under normal and hypoxic conditions. Hypoxia was induced by inflation of an occluder implanted around the testicular artery. The occluders were inflated about 10 min before irradiation and deflated immediately after. Stem-cell survival was measured at 5 or 7 weeks after irradiation by determination of the Repopulation Index (RI) in histological testis sections. The RI-values after fractionated irradiation were only half those after single dose irradiation. Hypoxia had a protective effect on the stem-cell survival. After split-dose irradiation under hypoxic conditions two times more stem cells survived than under normal oxic conditions; the RI-values increased from 34% (oxic) to 68% (hypoxic). This effect of hypoxia was also found after single dose irradiation where the RI-values increased from 68% (oxic) to 84% (hypoxic). The development of the epithelium in repopulated tubules was also studied. Under hypoxia, a significantly higher fraction of tubules with complete epithelium was found after single (38 vs. 4%) as well as after split-dose irradiation (12 vs. 0%).     

Adenovirus-mediated expression of a dominant negative Ku70 fragment radiosensitizes human tumor cells under aerobic and hypoxic conditions

Ku70 is one component of a protein complex, the Ku70/Ku80 heterodimer, which binds to DNA double-strand breaks and activates DNA-dependent protein kinase (DNA-PK), leading to DNA damage repair. Our previous work has confirmed that Ku70 is important for DNA damage repair in that Ku70 deficiency compromises the ability of cells to repair DNA double-strand breaks, increases the radiosensitivity of cells, and enhances radiation-induced apoptosis. Because of the radioresistance of some human cancers, particularly glioblastoma, we examined the use of a radio-gene therapy paradigm to sensitize cells to ionizing radiation. Based on the analysis of the structure-function of Ku70 and the crystal structure of Ku70/Ku80 heterodimer, we designed and identified a candidate dominant negative fragment involving an NH(2)-terminal deletion, and designated it as DNKu70. We generated this mutant construct, stably overexpressed it in Rat-1 cells, and showed that it has a dominant negative effect (i.e., DNKu70 overexpression results in decreased Ku-DNA end-binding activity, and increases radiosensitivity). We then constructed and generated recombinant replication-defective adenovirus, with DNKu70 controlled by the cytomegalovirus promoter, and infected human glioma U-87 MG cells and human colorectal tumor HCT-8 cells. We show that the infected cells significantly express DNKu70 and are greatly radiosensitized under both aerobic and hypoxic conditions. The functional ramification of DNKu70 was further shown in vivo: expression of DNKu70 inhibits radiation-induced DNA-PK catalytic subunit autophosphorylation and prolongs the persistence of gamma-H2AX foci. If radiation-resistant tumor cells could be sensitized by down-regulating the cellular level/activity of Ku/DNA-PK, this approach could be evaluated as an adjuvant to radiation therapy.     

DNA Damage in Human Endothelial Cells after Irradiation in Anoxia

Endothelial cells and fibroblasts have been reported to respond differently to oxidative stress. Both the effects of high oxygen tension and radiation involve the action of free radicals. DNA damage (single strand breaks, SSB, and double strand breaks, DSB) was assayed in human umbilical cord vein (HUV) cells and in Chinese hamster fibroblasts (V79) after irradiation under oxic or anoxic conditions. The cells were exposed to single doses in the range of 2-18 Gy of γ-radiation from ⁶⁰Co. Significantly more DNA damage was induced in the V79 cells than in the HUV cells. As a consequence, a higher oxygen enhancement ratio was obtained for the HUV cells (6.3) as compared to the V79 cells (2.8). The repair of SSB was slower in the HUV cells than in the V79 cells, irrespective of oxic state. For the higher doses, the damage remaining at 60 min after anoxic irradiation, i.e. DSB, was only detected in the V79 cells.     

Increased radiosensitivity with chronic hypoxia in four human tumor cell lines

PURPOSE: It is well known that the radiosensitivity of tumor cells can be significantly reduced under hypoxic conditions. However, most of the reports in the literature refer to an experimental setup in which the supply of oxygen is kept low for a short period of time only. In tumors, chronic hypoxia would seem to be the more typical situation, because of an insufficient vascularization and the limited diffusion of oxygen into the tissue. Under such conditions, certain changes in the proliferation patterns of tumor cells, in which the cell cycle checkpoint protein p53 seems to play a role, have been shown to occur. We therefore decided to study radiosensitivity and cell cycle progression under conditions of chronic hypoxia in several human tumor cell lines differing in their p53 status. METHODS AND MATERIALS: Four human tumor cell lines (melanomas Be11 and MeWo and squamous carcinomas 4197 and 4451) were incubated for 3 h, 24 h, and 72 h under either oxic or hypoxic conditions and subsequently exposed to graded doses of X-rays. In some cases, cells were kept under hypoxia for the same periods of time, but then reoxygenated immediately before irradiation. Cell survival was assessed with the usual colony formation assay, and cell cycle distributions were determined by two-parameter flow cytometry after labeling with bromodeoxyuridine (BrdU). RESULTS: As expected, the oxygen enhancement ratio at 3 h was 2.0 or more in all cases. Differences, however, became evident with longer incubation times. At 24 h, the sensitivity of cells kept under hypoxic conditions both before and during irradiation was practically unchanged with cell lines Be11, 4197, and 4451, but clearly increased with MeWo. This resulted in an oxygen enhancement ratio of only 1.1 for the latter cell line when the sensitivity of aerated cells was used as reference. Cells kept under hypoxia for 24 h and reoxygenated shortly before irradiation, however, also showed an increase in sensitivity, so that the oxygen enhancement ratio based on differences in irradiation atmosphere alone was still around 2.0. At 72 h, the two p53 wild-type cell lines were not available for experiments, because they quickly degenerated under hypoxic conditions. Both mutant cell lines now showed similar results, the sensitivity being increased with irradiation under continued hypoxia as well as after reoxygenation. The oxygen enhancement ratios with reference to aerated cells were 1.3 and 1.5 for MeWo and 4451, respectively. Flow cytometric measurements after labeling with BrdU revealed that in all cell lines, the fraction of active S-phase cells during incubation tended to decrease under hypoxic conditions. Only in the p53 mutant cell lines, however, was this accompanied by an increase of the percentage of S-phase cells that were not actively incorporating BrdU. CONCLUSIONS: It is suggested that these quiescent cells in the S-phase compartment develop because of a general breakdown of cellular energy metabolism. In the p53 mutant cells, this may lead to a cessation of cell cycle progression in all phases alike, because checkpoint control has been lost; p53 wild-type cells, on the other hand, settle down preferentially in G(1) under the same conditions. Independently of the p53 status, however, energy depletion may be the cause of a decreased ability to cope with radiation damage and thus the cause of the observed increase in radiosensitivity. This would become more easily apparent in the p53 mutant cell lines, because they are less sensitive than the p53 wild types to hypoxia as such.     

Effect of etoposide,carmustine, vincristine, 5-fluorouracil,or methotrexate on radiobiologically oxic and hypoxic cells in a C3H mouse mammary carcinoma in situ

Summary The effect of etoposide (VP16), carmustine (BCNU), vincristine (VCR), methotrexate (MTX), and 5-fluorouracil (5-FU) on the oxic and hypoxic cells in a C3H mammary carcinoma in CDF₁ mice was investigated using an in situ local-tumor-control (TCD₅₀) assay. The surviving fraction (SF) was calculated from the size of the radiation dose needed to inactivate the surviving tumor cells in drug-treated tumors relative to untreated controls. Preferential drug cytotoxicity towards oxic and hypoxic cells was evaluated from the difference in the response to irradiation under ambient and clamped conditions, respectively. Three drugs caused a significant (P<0.05) reduction in the survival of hypoxic cells, the SFs being 0.31 (VP-16), 0.13 (BCNU) and 0.16 (VCR). VCR was also toxic towards oxic cells (SF, 0.17), whereas VP16 and BCNU had no significant effect on these cells (SF, 0.5 and 0.76, respectively). Two drugs produced significant killing of cells in the oxic compartment: 5-FU (SF, 0.10) and MTX (SF, 0.22); these two drugs had no effect on hypoxic cells (SF, 0.78 and 1.11, respectively).This work was supported by the Danish Cancer Society and the Jacob and Olga Madsen Foundation     

Repair of DNA breaks after irradiation in misonidazole or oxygen

Biphasic kinetics (half-times 7' and 250') adequately describe the aerobic repair of DNA breaks after irradiation of mammalian cells. The proportions repaired by the two components are affected by conditions prior to, during and after irradiation. DNA of cells irradiated in hypoxia have approximately twice as much of the damage which is repaired by the slow component as cells irradiated in air. If, instead of oxygen, misonidazole is present during hypoxic radiation, the repair resembles the repair of oxic damage more closely than repair of hypoxic damage. The biphasic nature of the repair curves is interpreted to be due to two classes of initial damage, proportions of which can be altered by sensitizers such as O2 and misonidazole.     

Is tumour radiosensitization by misonidazole a general phenomenon?

The response of 14 mouse tumour sub-lines to the radiosensitizing action of a large single dose of misonidazole (MISO) has been assessed by regrowth delay. In 13 of these, significant enhancement of radiation effect occurred under ambient conditions, indicating sensitization of naturally hypoxic cells. The enhancement observed (SER'') varied with the radiation dose, as would be predicted for a mixed oxic/hypoxic cell population. The maximum SER'' in these 13 tumours did not depend on histology or regrowth rate. The 14th tumour, a slow-growing sarcoma, was not sensitized under ambient conditions, but showed marked sensitization when clamped to produce acutely hypoxic cells. This is consistent with no hypoxic cells occurring naturally in a sarcoma with a slow rate of growth. Faster-growing variants of this tumour showed radiosensitization under ambient conditions. The slow-growing carcinoma, RH, however, appears to contain hypoxic cells and did show sensitization. The cytotoxic action of MISO was compared with the radiosensitization by administering it after irradiation in 8 of the tumour lines. In 2 tumours no cytotoxicity was observed. In the rest cytotoxicity was significant, but much smaller than the sensitization observed when MISO was administered before irradiation. These regrowth-delay data have been used to calculate hypoxic fractions in 3 ways. Estimates of hypoxic fraction ranged from less than 0.1% in the slow sarcoma to greater than or equal to 30% in several tumours. There is considerable variation in the estimate, according to the technique used.     

Cytotoxicity of the bioreductive agent RH1 and its lack of interaction with radiation

BACKGROUND AND PURPOSE: RH1 is a new bioreductive agent that was developed as a cytotoxic agent with selectivity for tumour cells expressing high levels of the enzyme DT-diaphorase (DTD). The aim of the present study was to investigate the cytotoxicity of RH1 in relation to cellular levels of reducing enzymes and any interaction of RH1 with ionizing radiation under oxic and hypoxic conditions. PATIENTS AND METHODS: The MB-MDA231 human breast cancer cell line (WT) and WT cells transfected with the NQO1 gene encoding DTD (the D7 cell line) were used to examine the dependency of RH1's cytotoxicity on cellular DTD activity. The role of the 1-electron reducing enzyme P450 reductase was also studied using a P450 reductase-transfected isogenic cell line (R4). A clonogenic assay was used to investigate the cytotoxicity of RH1 with and without irradiation in air and in nitrogen. In all cases drug exposure was for 3 h. RESULTS: DTD levels were around 300-fold higher in D7 compared to WT and R4 cells. RH1 was cytotoxic at nanomolar concentrations to all the cell lines, and was 2-3 times more toxic in the D7 cells with high DTD than in the other two cell lines. Doses of RH1 was around 2-fold more effective in hypoxic than in oxic WT cells, but not by as much in D7 cells. RH1 did not radiosensitise the cells but showed an additive effect when combined with irradiation under oxic and hypoxic conditions. CONCLUSIONS: RH1 shows high clonogenic cytotoxicity to MDA231 cells with high DTD activity but its selectivity based on the presence of DTD is much less than as shown in previous reports. RH1 showed an additive cell killing effect when combined with irradiation under both oxic and hypoxic conditions.     

The role in cancer therapy of inhibiting recovery from PLD induced by radiation or bleomycin

Effects on survival of allowing and inhibiting potentially lethal damage recovery (PLDR) after X ray or bleomycin (bleo) exposure, especially at clinically applicable doses, were examined in plateau phase Chinese hamster ovary (CHO) cells. Dose modifying factors (DMF's) between immediately explanted cells and those trypsinized 24 hours after various doses of X rays were 2.51 +/- 0.12 (mean +/- 2 S.E.) at 70% surviving fraction (SF), 2.03 +/- 0.085 at 40% and 1.71-1.79 at lower SF levels. Thus, at doses used clinically (1-4 Gy), the DMF by PLDR was more than 2.0, suggesting its importance in radiotherapy of cancers. Feeder layers were found to increase the SF, especially for replating immediately after bleo exposure. Among the inhibitors tested, 3'-deoxyguanosine at 3.7 mM and caffeine at 10.3 mM inhibited x and bleo PLDR (DMF of 1.0-1.2) when trypsinized 24 hours after treatment. However, interestingly, 3 aminobenzamide, a specific inhibitor of poly (ADP) ribose synthesis, even at 14.7 or 29.4 mM, was not as effective in suppressing both x and bleo PLDR, suggesting the role of repair processes independent of poly (ADP) ribose levels in PLDR in plateau phase cultures. Possibilities of clinical application of PLDR inhibitors as radio- and chemosensitizers are discussed.     

CB 1954 revisited

We have assessed the antitumour activity of the nitrophenylaziridine CB 1954 in vitro and in vivo. For EMT6 mouse mammary tumour multicellular spheroids under hypoxic conditions in vitro, a 6-h exposure to 40 micrograms/ml reduced the surviving fraction to as low as 10(-3) and the growth delay was 5.4 days. Oxic cells were twofold less sensitive. Phenyl AIC protected oxic and hypoxic cells equally. Under oxic conditions minimal cell killing was seen with HT29 cells, either in multicellular spheroids or in monolayer; a 6-h exposure to 40 micrograms/ml gave a spheroid growth delay of 1.5-1.7 days. No growth delay was seen with single maximum tolerated doses of CB 1954 against HT29 grown as a xenograft in immunosuppressed mice. Only minimal growth delays of 1-2 days were seen with similar doses against the EMT6 tumour and the RIF-1 and KHT sarcomas in mice. Little activity was seen with maximum tolerated doses given once a day for 5 days against EMT6 and RIF-1. No chemosensitization was measurable with CCNU, cyclophosphamide or melphalan in the KHT tumour.     

Enhancement of radiation-induced cell kill by platinum complexes (carboplatin and iproplatin) in V79 cells

Two second generation platinum complexes currently undergoing clinical chemotherapeutic trials, carboplatin (CBDCA) and iproplatin (CHIP), were evaluated for their ability to alter the survival of cultured Chinese hamster V79 cells following irradiation. Two protocols were employed. In the first, the drug was added to preplated cells, some of which were subsequently made hypoxic with nitrogen gas. These hypoxic cells were irradiated following 1 hour exposure to drug and survival was assessed by standard colony forming unit (CFU) methods. Enhancement ratios (ER) of approximately 1.4 were obtained for irradiation under hypoxic conditions, if the cells were exposed to equitoxic doses of CBDCA (500 microM) CHIP (50 microM). In the second series of experiments, cells were treated with 10 Gy in air and then incubated for various times prior to trypsinization and serial dilution of single cell suspensions. Six hours after irradiation, cells treated with X rays alone had recovered to produce a surviving fraction twice that of cells trypsinized immediately after irradiation (not held). Post-irradiation administration of CBDCA (50 microM) or CHIP (20 microM), at a time when free radical-mediated radiosensitization would not be possible, operationally inhibited this recovery from radiation-induced potentially lethal damage (PLD). Inhibition, expressed as recovery inhibition factor (RIF) after 6 hr with drug, was 2.0 for CBDCA and 1.2 for CHIP. These results suggest that the rationale for designing clinical trials to exploit interactions between cisplatin and radiation might also extend to include combined modality therapy using radiation with either of these two platinum complexes.     

Interaction of platinum drugs with clinically relevant x-ray doses in mammalian cells: a comparison of cisplatin, carboplatin, iproplatin, and tetraplatin

Whereas the interaction between radiation and platinum complexes has never been pronounced in radiobiological experiments (to 30 Gy in mammalian cells), there have been reports of interest in this combination in the clinic, where fractionated doses of approximately 2 Gy are used. Our studies on the marked interaction in hypoxia at the 80% survival level (1-2.5 Gy) with cisplatin have been extended to second generation platinum drugs of clinical interest. The studies in the lower radiation dose region have been facilitated by the use of the cell analyzer DMIPS to identify individual cells and follow them microscopically to assess for clonogenic ability. Chinese hamster V79 cells were used, which were exposed to drug for 1 hr prior to irradiation in hypoxia (or air). None of the drugs give an enhancement ratio (ER) greater than 1.3 in the high radiation dose region, whereas all can produce ER80% (ER calculated at iso-survival of 80%) of 2 or higher at low doses in hypoxic cells. The enhancement of radiation kill in oxic V79 cells (ER's to 1.1 at 1-2% S) disappears at low doses (ER80% = 1.0) except for tetraplatin, where a moderate ER80% (to 1.64) was measured. Comparison of the hypoxic interaction on a concentration basis suggests that cisplatin is the best drug at low x-ray doses and low concentrations, but the interaction reaches a plateau at ER80% approximately 2.0. Tetraplatin continues to give better interaction with increasing concentration (up to ER80% = 3.7 at 25 microM). Interaction of radiation with the less toxic drugs, iproplatin and carboplatin, used at around 100 microM can be improved by longer exposure times prior to irradiation. Comparison on the basis of toxicity, for which the plating efficiency was used, suggests that cisplatin gives a better interaction than the three newer drugs for a given level of toxicity in hypoxic V79 cells.     

Superfractionation as a potential hypoxic cell radiosensitizer: prediction of an optimum dose per fraction

Purpose: A dose “window of opportunity” has been identified in an earlier modeling study [1] if the inducible repair variant of the LQ model is adopted instead of the pure LQ model, and if all survival curve parameters are equally modified by the presence or absence of oxygen. In this paper we have extended the calculations to consider survival curve parameters from 15 sets of data obtained for cells tested at low doses using clonogenic assays.

Methods and Materials: A simple computer model has been used to simulate the response of each cell line to various doses per fraction in multifraction schedules, with oxic and hypoxic cells receiving the same fractional dose. We have then used pairs of simulated survival curves to estimate the effective hypoxic protection (OER′) as a function of the dose per fraction.

Results: The resistance of hypoxic cells is reduced by using smaller doses per fraction than 2 Gy in all these fractionated clinical simulations, whether using a simple LQ model, or the more complex LQ/IR model. If there is no inducible repair, the optimum dose is infinitely low. If there is inducible repair, there is an optimum dose per fraction at which hypoxic protection is minimized. This is usually around 0.5 Gy. It depends on the dose needed to induce repair being higher in hypoxia than in oxygen. The OER′ may even go below unity, i.e. hypoxic cells may be more sensitive than oxic cells.

Conclusions: If oxic and hypoxic cells are repeatedly exposed to doses of the same magnitude, as occurs in clinical radiotherapy, the observed hypoxic protection varies with the fractional dose. The OER′ is predicted to diminish at lower doses in all cell lines. The loss of hypoxic resistance with superfractionation is predicted to be proportional to the capacity of the cells to induce repair, i.e. their intrinsic radioresistance at a dose of 2 Gy.     

Effect of hypoxia on 1,3-bis(2-chloroethyl)-1-nitrosourea cytotoxicity in 9L cells

The cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) on 9L rat brain tumor cells were studied under oxic and hypoxic conditions. Acute hypoxia was produced by gassing exponentially growing monolayer cells with 95% nitrogen/5% CO2 for 2 h, after which cells were treated with graded concentrations of BCNU for 1 h. Cell survival was assayed with a colony forming-efficiency assay and the extent of DNA cross-linking was measured with the alkaline elution assay. BCNU was more cytotoxic to hypoxic than to oxic cells. There were far more interstrand cross-links formed in hypoxic than in oxic cells, and the number of cross-links could be measured readily in hypoxic cells at very low concentrations of BCNU. This allowed cell survival and cross-linking to be compared at the same dose levels, a correlation not possible for oxic cells in which cross-links are not measurable at low doses. The total intracellular levels of glutathione were lower in hypoxic cells, but the reduced glutathione levels may not be related to the enhanced cell kill because survival of oxic cells treated with BCNU was not affected when cells were depleted of glutathione with buthionine sulfoximine. These results indicate that the additional cell kill produced in 9L cells under hypoxic conditions is related to increased cross-link formation.     

Toxicity of RSU-1069 for KHT cells treated in vivo or in vitro: evidence for a diffusible toxic product

RSU-1069 is a highly effective hypoxic cell cytotoxin in KHT sarcomas treated in vivo. However, relative to the hypoxic cells, the oxic cells in the tumor appear more sensitive to the drug than would have been predicted on the basis of results with CHO (AA8-4) cells treated in vitro with the drug under oxic and hypoxic conditions. To examine possible reasons for this difference, suspensions of KHT cells were prepared from tumors growing in vivo, and treated with RSU-1069 in vitro under oxic or hypoxic conditions. The sensitivity of the KHT cells was similar to that of AA8-4 cells, regardless of whether the cells were obtained from untreated tumors or from tumors given 15 Gy in vivo just prior to the preparation of the cell suspension. We observed, however, that the sensitivity of both AA8-4 cells and KHT cells to drug treatment under hypoxic conditions increased with the density of the cells in the treated suspension. This result suggests the possibility that a diffusible toxic product may be released from cells. Such a product could contribute to the toxicity of the drug for oxic cells in tumors in situ.     

Radioresponsiveness, sublethal damage repair and stem cell rate in spheroids from three human tumor lines: comparison with xenograft data.

Dose-control curves after fractionated irradiation were generated for small oxic spheroids from the two human glioma cell lines, U87 and A7, as well as the squamous cell carcinoma line FaDu. These data were fitted by the linear quadratic model assuming Poisson statistics. The alpha/beta values of A7, U87, and FaDu spheroids, respectively were 10.3 (8.1-12.9) Gy, 17.8 (15.1-21.1) Gy, and 37.9 (29.1-51.5) Gy. These data were compared with those previously published by Suit et al. (31) and Zietman et al. (40) for 6 mm xenografts of U87 and FaDu after fractionated irradiation and for A7 after single dose irradiation under clamped conditions. A good agreement in the alpha/beta values was observed for U87 and Fadu xenografts and spheroids assuming an oxygen enhancement ratio (OER) of 2.7. In addition, the ranking according to the single doses needed to control 50% of the tumors agreed for xenografts and spheroids from the three cell lines. U87 was the most resistant line in both model systems, followed by A7 and FaDu. However, the absolute values of alpha and beta, obtained from the direct fit to the dose-control data were only about half as high for U87 and FaDu xenografts than for the spheroids. Monte Carlo simulations showed that this discrepancy can be explained by a greater tumor heterogeneity of the xenografts. While the number of critical stem cells or spheroid rescuing units equaled the number of cells per spheroid for the three cell lines, the percentage of tumor rescuing units for Fadu and U87 xenografts was estimated to be below 1%. In a next step, survival curves were generated for exponentially growing cells of the three lines. A7 cells were significantly more radioresistant when plated on tissue plastic than in soft agar. Using the most resistance-promoting colony assay conditions for each cell line, a good agreement was observed for the alpha and SF2Gy values calculated from the colony and spheroid control data. This study shows that the spheroid model can quantitatively predict the repair capacity of sublethal damage as well as the rank order of radiation sensitivity of in vivo tumors.     

Hypoxia and reoxygenation in human melanoma xenografts

Tumor hypoxia and reoxygenation pattern following single dose (10.0 Gy) and fractionated (7 fractions of 2.0 Gy, 1 fraction per day) irradiation were studied in five human melanoma xenograft lines using the paired survival curve method. The hypoxic fractions differed significantly among the melanoma lines; they were found to be 6 +/- 3% (E.E.), 22 +/- 8% (E.F.), 31 +/- 11% (G.E.), 45 +/- 17% (M.F.), and 15 +/- 5% (V.N.). There were no clear correlations between hypoxic fraction and tumor volume-doubling time or vascular density, suggesting that intrinsic cellular characteristics, for example, rate of oxygen consumption and ability to retain clonogenicity under hypoxic stress, also may play an important role for the magnitude of the hypoxic fractions in the melanomas. Reoxygenation was rapid and extensive in all melanoma lines; 12-24 hr after the single dose irradiation or the last fraction of the fractionated irradiation, the hypoxic fractions were similar to those in untreated tumors and stayed at that level up to at least 10 days after irradiation. The hypoxic fractions 1-10 days after irradiation tended to be higher after fractionated than after single dose irradiation, but the differences were not statistically significant. There was a positive correlation between the hypoxic fractions in untreated tumors and the hypoxic fractions after irradiation and reoxygenation, suggesting that it may be possible to predict radiation resistance caused by hypoxia from the hypoxic fractions in tumors before start of radiation therapy. However, hypoxia is probably not a major cause of failure in the radiation therapy of malignant melanoma.     

Trimodality therapy (drug/hyperthermia/radiation) with BCNU or mitomycin C

To develop multimodality treatment combinations with high curative potential in advanced local disease, BCNU (N,N'-bis(2-chloroethyl)-N-nitro-sourea) and mitomycin C were tested with hyperthermia and radiation in the FSaIIC fibrosarcoma system. Growth delay experiments demonstrated that, while neither BCNU nor mitomycin C produced dose modification of the radiation response, and hyperthermia (43 degrees C, 30 min) produced only a moderate dose modification (1.4 +/- 0.2), the combination of BCNU plus hyperthermia resulted in a radiation dose modifying factor (DMF) of 1.9 +/- 0.3, and mitomycin C plus hyperthermia a dose modifying factor of 2.1 +/- 0.4. Tumor cell survival over a range of BCNU doses administered i.p. immediately before hyperthermia resulted in a dose modifying factor of 1.8 +/- 0.2 versus drug alone. With mitomycin C however, giving the drug immediately prior to heating produced a dose modifying factor due to hyperthermia of only 1.2 +/- 0.10. Hoechst 33342 diffusion was used to separate tumor cells into predominately oxic and hypoxic subpopulations. Administration of the single, double and trimodality therapies showed that BCNU was 3.1-fold more toxic to the oxic versus the hypoxic cells whereas mitomycin C was 3.5-fold more toxic to the hypoxic compared to the oxic cells. Hyperthermia was 1.4-fold more toxic to the hypoxic versus the oxic cells whereas 10 Gy of radiation was 2.0-fold more toxic to the oxic compared to the hypoxic cells. The combination of hyperthermia plus radiation increased killing in both Hoechst dye defined subpopulations but relatively more in the hypoxic cells in which killing was 1.8-fold greater than in the oxic cells. When heat was delivered immediately after i.p. administration of the anticancer drugs, hyperthermia increased BCNU killing in the oxic cells by 17.2-fold versus 4.4-fold in the hypoxic cells and increased mitomycin-killing by 2.6-fold in the oxic cells versus 17-fold in the hypoxic cells. Use of the full trimodality treatment, given in the sequence drug (BCNU, 50 mg/kg or mitomycin-C 5 mg/kg)----heat (43 degrees C, 30 min)----radiation (10 Gy) produced a 3 log kill in the oxic cells versus a 2 log kill in the hypoxic cells with BCNU and a 2 log kill in the oxic cells versus a 3 log kill in the hypoxic cells with mitomycin C. These results indicate that the use of selected anticancer drugs with hyperthermia and radiation can produce highly cytotoxic interactions which markedly modify the effect of radiation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Resistance of hypoxic cells to ionizing radiation is influenced by homologous recombination status

PURPOSE: To determine the role of DNA repair in hypoxic radioresistance. METHODS AND MATERIALS: Chinese hamster cell lines with mutations in homologous recombination (XRCC2, XRCC3, BRAC2, RAD51C) or nonhomologous end-joining (DNA-PKcs) genes were irradiated under normoxic (20% oxygen) and hypoxic (<0.1% oxygen) conditions, and the oxygen enhancement ratio (OER) was calculated. In addition, Fanconi anemia fibroblasts (complementation groups C and G) were compared with fibroblasts from nonsyndrome patients. RAD51 foci were studied using immunofluorescence. RESULTS: All hamster cell lines deficient in homologous recombination showed a decrease in OER (1.5-2.0 vs. 2.6-3.0 for wild-types). In contrast, the OER for the DNA-PKcs-deficient line was comparable to wild-type controls. The two Fanconi anemia cell strains also showed a significant reduction in OER. The OER for RAD51 foci formation at late times after irradiation was considerably lower than that for survival in wild-type cells. CONCLUSION: Homologous recombination plays an important role in determining hypoxic cell radiosensitivity. Lower OERs have also been reported in cells deficient in XPF and ERCC1, which, similar to homologous recombination genes, are known to play a role in cross-link repair. Because Fanconi anemia cells are also sensitive to cross-linking agents, this strengthens the notion that the capacity to repair cross-links determines hypoxic radiosensitivity.     

Response of subpopulations of the FSall C fibrosarcoma to low dose x-rays and various potential enhancing agents.

The effectiveness of various oxygen carrying treatments in sensitizing subpopulations of the FSallC fibrosarcoma to low doses of radiation was assessed, and compared with survivals obtained with the same cells after in vivo irradiation under normally oxygenated or hypoxic conditions. FSallC tumors were treated with 2-10 Gray then the Hoechst 33342 dye diffusion method was used to separate the tumor into bright (enriched in normally oxygenated cells) and dim (enriched in hypoxic cells) subpopulations. There was good agreement between the survival of normally oxygenated cells in culture and bright cells from tumors and between hypoxic cells in culture and dim cells from tumors over a radiation dosage range of 2-5 Gray. At 10 Gray bright cells from tumors were minimally less sensitive to the radiation dose than normally oxygenated cells in vivo. When maximally effective doses of perfluorochemical emulsions (F44E at 4 g PFC/kg or Fluosol-DA at 2.4 g PFC/kg) or a purified bovine hemoglobin solution (PBHS at 1.32 g protein/kg) were administered 1 hr. prior to radiation therapy with carbogen (95% O2, 5% CO2) breathing prior to and during radiation delivery, low single doses of x-ray (2-5 Gray) were measurably more cytotoxic toward both FSallC tumor cell subpopulations. These results indicate that perfluorochemical emulsions or purified bovine hemoglobin preparations along with carbogen breathing may be able to increase tumor radiosensitivity to the relatively low radiation doses per fraction used in the clinic.