Radiosensitizing and toxic properties of quinoline and nitroquinoline complexes of platinum [PtCl2(NH3)quinoline]

The assessment of drugs designed to be useful in the eradication of hypoxic (resistant) cells involves comparison of hypoxic and aerobic radiosensitization, cytotoxicities, as well as DNA binding and reduction potentials. Three pairs of isomers of quinoline complexes [amino dichloro quinoline platinum (II)] were studied in this context. For the cis 5- and 6-nitroquinoline complexes, the DNA binding and toxicity were higher with the 5-substituted ligand. Reduction potentials were similar (-260 and -280 mV). No selectivity for hypoxic toxicity was observed, but radiosensitizing ability by both complexes was greater in hypoxic (than oxic) Chinese hamster ovary (CHO) cells. The trans 5-nitroquinoline complex produced better sensitization in hypoxia than its cis isomer [enhancement ratio (ER) 1.7 at 10 microM versus 40 microM for cis]. However, this was accompanied by some aerobic sensitization. The trans isomer of the (unsubstituted) quinoline complex was considerably more toxic than its cis isomer. Neither showed selectivity for hypoxia, either as radiosensitizers or as cytotoxins, which may be attributable to the lack of a reducible (nitro) function. Four quinoline complexes showed high activity in cisplatin-resistant L1210 cells, with the lowest resistance factor being for the trans quinoline complex. Results suggest that trans complexes with one aromatic ring may have activity different from the cis geometry, which should be exploited with respect to cisplatin resistance and cross-resistance with radiation.     

RSR13, a synthetic allosteric modifier of hemoglobin, as an adjunct to radiotherapy: preliminary studies with EMT6 cells and tumors and normal tissues in mice

RSR13, 2[4-[[(3,5dimethylanilino)carbonyl]methyl]phenoxy]-2-methylpropion ic acid, a synthetic allosteric modifier of hemoglobin, reduces the affinity of hemoglobin for oxygen. The experiments reported here examined the effect of treatment with RSR13, combined with oxygen breathing, on the radiation response of EMT6 mammary tumors in BALB/c mice and of two normal tissues. RSR13 plus oxygen breathing increased the response of EMT6 tumors to irradiation. RSR13 had no discernible effects on tumors rendered maximally hypoxic by nitrogen asphyxiation, no discernible cytotoxic effects in EMT6 tumors, and no effect on the viability or radiation response of EMT6 cells in vitro under either aerobic or hypoxic conditions. The effects of RSR13 therefore reflect changes in tumor oxygenation, rather than a direct cytotoxic or radiosensitizing effect of the drug. RSR13 plus oxygen reduced the hypoxic fraction to 9% from the value of 24% found in both air-breathing and oxygen-breathing mice. Treatment with RSR13 plus oxygen did not alter the radiation response of the bone marrow progenitor cells (CFU-S) or acute radiation reactions in the skin. The improvement in tumor radiation response produced by treatment with RSR13 plus oxygen, combined with the absence of enhanced radiation reactions in the normal tissues, support further testing of RSR13 as an adjunct to radiotherapy.     

Redox generation of nitric oxide to radiosensitize hypoxic cells

PURPOSE: Previous studies have shown that nitric oxide (NO) delivered from NO donor agents sensitizes hypoxic cells to ionizing radiation. In the present study, nitroxyl (NO-), a potential precursor to endogenous NO production, was evaluated for hypoxic cell radiosensitization, either alone or in combination with electron acceptor agents. METHODS AND MATERIALS: Radiation survival curves of Chinese hamster V79 lung fibroblasts under aerobic and hypoxic conditions were assessed by clonogenic assay. Hypoxia induction was achieved by metabolism-mediated oxygen depletion in dense cell suspensions. Cells were treated with NO- produced from the nitroxyl donor Angeli's salt (AS, Na2N2O3, sodium trioxodinitrate), in the absence or presence of electron acceptor agents, ferricyanide, or tempol. NO concentrations resulting from the combination of AS and ferricyanide or tempol were measured under hypoxic conditions using an NO-sensitive electrode. RESULTS: Treatment of V79 cells under hypoxic conditions with AS alone did not result in radiosensitization; however, the combination of AS with ferricyanide or tempol resulted in significant hypoxic radiosensitization with SERs of 2.5 and 2.1, respectively. Neither AS alone nor AS in combination with ferricyanide or tempol influenced aerobic radiosensitivity. The presence of NO generated under hypoxic conditions from the combination of AS with ferricyanide or tempol was confirmed using an NO-sensitive electrode. CONCLUSION: Combining NO- generated from AS with electron acceptors results in NO generation and substantial hypoxic cell radiosensitization. NO- derived from donor agents or endogenously produced in tumors, combined with electron acceptors, may provide an important strategy for radiosensitizing hypoxic cells and warrants in vivo evaluation.     

Evaluation of the radiosensitizing effects of RK28 intravenous, intraarterial, and intratumoral injections on the rabbit VX2 tumor system

PURPOSE: To evaluate the differences in the radiosensitizing effects of intravenous (i.v.) injection, intraarterial (i.a.) injection, and intratumoral (i.t.) injections of the hypoxic cell radiosensitizer RK28 ([1-(4'-hydroxy-2'-butenoxy)methyl-2-nitroimidazole], a 2-nitroimidazole with an acyclic sugar analogue substituted at the N-1 position of the imidazole ring) using an animal experimental system. METHODS AND MATERIALS: Rabbit VX2 tumors, which were implanted in the muscle of left hind legs and grown to 3 cm in diameter, were treated with RK28 (80 mg/kg x b.wt.) before 15 Gy of local x-ray irradiation. The auricular vein and the left saphenous artery were used for systemic injection and regional injection, respectively. For i.t. injection, a 21-gauge needle with three lateral holes was positioned in the central area of the tumor. Tumor regression was precisely evaluated by computed tomograpy (CT), and survival time was also studied. Using high-performance liquid chromatography (HPLC), pharmacokinetic studies for RK28 and its seven major metabolites were performed in tumor and serum at 0, 10, 20, 30, and 60 min after drug injection was completed. RESULTS: Radiosensitizing effects of RK28 were considered present after i.a. injection (p < 0.05) and i.t. injection (p < 0.05) after analyzing tumor volumes on day 21 after treatment. Increased survival was not observed in any group with RK28 injection compared with survival in the group treated by x-ray irradiation alone. Pharmacokinetic studies showed the average concentration of RK28 in the tumor during x-ray irradiation was 1.3 times higher after i.a. injection and 3.5 times higher after i.t. injection than that after i.v. injection. The time modifying factor50 (TMF50: ratio of time for tumor to decrease by 50%, radiation alone vs. radiation plus drug) was calculated to be 1.5 after i.v. injection, 1.7 after i.a. injection, and 2.3 after i.t. injection. The values of TMF50 correlated to the average concentrations of RK28 in the tumor. As to metabolites of RK28, beta-glucuronated compound and cysteine conjugate were highly detected. The concentrations of cysteine conjugate were higher in the tumor than in serum via i.v. injection. CONCLUSIONS: Radiosensitizing effects of RK28 were observed on the rabbit VX-2 tumor system after i.a. or i.t. injection. Pharmacokinetic studies proved that radiosensitizing effects depended on the concentration in the tumor, though the administration routes were different. Combined forms with nonprotein thiols were detected. However, survival benefits were not obtained by RK28. For clinical applications of RK28, i.a. or i.t. injection could facilitate better local control of cancer.     

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.     

The clinical rationale for S-phase radiosensitization in human tumors

Nonhypoxic cell radiosensitizers, principally the halogenated pyrimidines and hydroxyurea, have been studied in the laboratory and clinical setting for more than 30 years. Early clinical experience in the 1960s and 1970s with the thymidine analogs 5-bromodeoxyuridine (BUdR) and 5-iododeoxyuridine (IUdR) was disappointing because normal tissue toxicity eliminated any potential for therapeutic gain. Inadequate delivery systems for intravenous and intraarterial infusions also contributed to the decline of this strategy. More recently, laboratory investigations have revealed further information regarding the mechanism of IUdR/BUdR radiosensitization. This knowledge provided a rationale for the sequence and timing of drug and radiation exposure, which could be both effective and tolerable. Advancing technology also provided safer infusion devices, and a resurgence in clinical trials combining IUdR or BUdR and radiation resulted. Current laboratory studies are now providing data on tumor cell kinetics, which is being applied to ongoing clinical trials. Fluoropyrimidines, principally 5-fluorouracil (5-FU), were also used in early clinical trials and unlike IUdR/BUdR were found to have significant activity as single agents against a variety of tumor types. The clinical integration of 5-FU and radiation occurred more slowly, but recent trials have demonstrated a therapeutic gain. Improved rates of local control and survival with combined 5-FU and radiation versus radiation alone have now been demonstrated in patients with rectal, esophageal, and anal carcinomas. However, the mechanism of interaction between the fluoropyrimidines and radiation remains uncertain and continues to be investigated with the hope of improved clinical outcome. As the cellular pathways influenced by the halogenated pyrimidines have been defined, the potential for biochemical modulation of these agents has been recognized. Leucovorin, the most commonly applied modulator, has been shown to enhance the activity of 5-FU in patients with metastatic colorectal carcinoma. These studies serve as an example for current trials that use biochemical modulators of IUdR, BUdR, and 5-FU as radiosensitizers. Hydroxyurea, currently used in the treatment of chronic leukemia, has also been considered a radiosensitizer. As with IUdR/BUdR, the clinical trials have often been inconclusive and interest in this radiosensitizer has waned. A poor understanding of the mechanism of action and tumor cell/normal tissue kinetics may be responsible for the lack of overall success with this strategy. Current investigations of cell kinetics in humans and potential mechanisms of hydroxyurea action could provide information critical to future trials of hydroxyurea radiosensitization.     

Leucovorin modulation of 5-iododeoxyuridine radiosensitization: a phase I study

Evidence for clinically significant radiosensitization by the halogenated pyrimidine 5-iododeoxyuridine (IdUrd) continues to accumulate. In vitro radiosensitization has been demonstrated in human colon tumor cell lines following exposure to 1-10 micrometer. Coadministration of leucovorin (LV) increases radiosensitization, which correlates directly with increased IdUrd DNA incorporation. Clinical data regarding proliferation rates and thymidine kinase levels in tumors versus normal tissues suggest selective incorporation of IdUrd into gastrointestinal tumors may occur. The objectives of this Phase I study were: (a) to assess the feasibility of LV modulation of IdUrd radiosensitization by determining the maximum tolerated dose (MTD) of IdUrd plus LV; and (b) to perform correlative laboratory studies to investigate the potential of IdUrd plus LV to increase radiosensitization in vivo. Seventeen patients with unresectable or recurrent gastrointestinal adenocarcinomas received a 14-day course of continuous i.v. infusion of IdUrd prior to initiation of radiotherapy. Two additional 14-day infusions of IdUrd with LV were given during the course of radiotherapy (60 Gy in 6 weeks). The initial dose of IdUrd was 250 mg/m2/day and was escalated in subsequent patients to 400 and 600 mg/m2/day. The LV dose remained fixed at 250 mg/m2/day. Leukopenia was the dose-limiting toxicity, and 400 mg/m2/day was the MTD for this trial. At the MTD, the mean +/- SD steady-state plasma concentration of IdUrd during the infusion, measured by high-performance liquid chromatography, was 0.66 +/- 0.23 micrometer. There was no significant influence of LV on IdUrd DNA incorporation in peripheral blood granulocytes as measured by high-performance liquid chromatography. Based on toxicity data and correlative laboratory studies, a meaningful increase in radiosensitization would not be achieved with the IdUrd infusion schedule and dose of LV investigated compared with IdUrd alone.     

Preclinical assessment of hypoxic marker specificity and sensitivity

PURPOSE: In the search for a sensitive, accurate, and noninvasive technique for quantifying human tumor hypoxia, our laboratory has synthesized several potential radiodiagnostic agents. The purpose of this study was to assess and compare the hypoxic marking properties of both radioiodinated and Tc-99m labeled markers in appropriate test systems which can predict for in vivo activity. MATERIALS AND METHODS: Preclinical assessment of hypoxic marker specificity and sensitivity employed three laboratory assays with tumor cells in vitro and in vivo. Radiolabeled marker uptake and/or binding to whole EMT-6 tumor cells under extremely hypoxic and aerobic conditions was measured and their ratio defined hypoxia-specific factor (HSF). Marker specificity to hypoxic tumor tissue was estimated from its selective avidity to two rodent tumors in vivo, whose radiobiologic hypoxic fractions (HF) had been measured. The ratios of % injected dose/gram (%ID/g) of marker at various times in EMT-6 tumor tissue relative to that in the blood and muscle of scid mice were used to quantify hypoxia-specific activity. This tumor in this host exhibited an average radiobiologic HF of approximately 35%. As well, nuclear medicine images were acquired from R3327-AT (HF approximately =15%) and R3327-H (no measurable HF) prostate carcinomas growing in rats to distinguish between marker avidity due to hypoxia versus perfusion. RESULTS: The HSF for FC-103 and other iodinated markers were higher (5-40) than those for FC-306 and other Tc-99m labeled markers. The latter did not show hypoxia-specific uptake into cells in vitro. Qualitative differences were observed in the biodistribution and clearance kinetics of the iodinated azomycin nucleosides relative to the technetium chelates. The largest tumor/blood (T/B) and tumor/muscle (T/M) ratios were observed for compounds of the azomycin nucleoside class in EMT-6 tumor-bearing scid mice. These markers also showed a 3-4 x higher uptake into R3327-AT tumors relative to the well-perfused R3327-H tumors. While both FC-306 and CERETEC rapidly distributed at unique concentrations to different tissues, their avidity to EMT-6 and R3327-AT tumors did not correlate with tumor HF. CONCLUSIONS: The halogenated azomycin nucleosides with the lowest lipid/water partition coefficient values were found to yield the optimal hypoxia-specific signal in these animal tumors. Our Tc-99m-labeled azomycin chelates showed little or no hypoxia-specific uptake and had in vivo biodistribution and clearance kinetics similar to those of CERETEC, a perfusion agent with no known hypoxic binding activity.     

Exploiting tumor hypoxia through bioreductive release of diffusible cytotoxins: the cobalt(III)-nitrogen mustard complex SN 24771

PURPOSE: To assess the oxygen dependence of a novel cobalt-nitrogen mustard complex, SN 24771, designed to release a diffusible cytotoxic metabolite in hypoxic tumor microenvironments. METHODS AND MATERIALS: Oxygen dependence of cell killing was assessing in well-stirred single cell suspensions obtained by enzymatic dissociation of EMT6 spheroids, using a sensitive oxygen electrode to measure oxygen concentrations in solution. Cell killing in intact EMT6 spheroids was also compared with that in single cell suspensions. RESULTS: Cytotoxicity of SN 24771 in single cell suspensions was inhibited by very low concentrations of oxygen. The C50 value (O2 required for 50% inhibition of log cell kill) was ca. 0.02% O2 at 1 h, and the K value (O2 required to give a cytotoxic potency equal to the average of that at zero and infinite O2) was of a similar order. However, intact spheroids were much more sensitive to SN 24771 than could be accounted for by the K curve for single cell suspensions, this estimate being based on published data for the oxygen concentration profile in these spheroids. CONCLUSION: The cytotoxicity of SN 24771 is inhibited appreciably at oxygen concentrations which are too low to provide radiosensitization. In this respect, SN 24771 resembles organic bioreductive drugs such as quinones and nitroaromatic compounds. However, the extensive killing observed in multicellular spheroids is consistent with release of a diffusible nitrogen mustard on reduction. Bioreductive drugs with a low K value for activation, but which release a diffusible cytotoxin, may have desirable properties as tumor radiosensitizers.     

Cells at intermediate oxygen levels can be more important than the "hypoxic fraction" in determining tumor response to fractionated radiotherapy

The presence of hypoxic cells in human tumors is thought to be one of the principal reasons for the failure of radiation therapy. Intensive laboratory and clinical efforts to overcome tumor hypoxia have focused on oxygenating, radiosensitizing or killing the maximally radioresistant fraction of tumor cells. This "hypoxic fraction" dominates the single-dose radiation response, irrespective of the oxygenation status of the remainder of the tumor cell population. However, at doses that are typical of those delivered in a daily radiotherapy protocol, we show that the tumor response is highly dependent upon the cells at oxygen levels intermediate between fully oxygenated and hypoxic (0.5-20 mm Hg). For most tumors, these cells are more important than the radiobiologically hypoxic cells in determining treatment outcome after 30 fractions of 2 Gy. We also show that under conditions of diffusion-limited hypoxia, the impact of full reoxygenation between fractions is much smaller than previously realized. Together, the results imply that tumor hypoxia plays a more significant role in determining the outcome of fractionated radiotherapy than previous measurements and assumptions of hypoxic fractions have indicated. Therefore, the concept of a hypoxic fraction in human tumors is less meaningful when pertaining to a fractionated radiotherapy regimen, and should not be expected to be useful for predicting tumor responses in the clinic. This implies the need to characterize tumor oxygenation in a manner that reflects the true oxygenation status of all the tumor cells, not just the ones most refractory to the effects of ionizing radiation. Furthermore, effective therapeutic agents must have the ability to specifically sensitize or kill those cells at intermediate levels of oxygen in addition to the radiobiologically hypoxic cells.     

A 2-week pretreatment with 13-cis-retinoic acid + interferon-alpha-2a prior to definitive radiation improves tumor tissue oxygenation in cervical cancers

BACKGROUND: We have evaluated the tumor tissue pO2 in cervical cancers in patients treated with 13-cis-retinoic acid and interferon-alpha-2a prior to and during radiotherapy. PATIENTS AND METHODS: From June 1995 through April 1997, 22 patients with squamous cell carcinoma FIGO IIB/III of the cervix who were scheduled for definitive radiotherapy with curative intent received additional treatment with 13-cis-retinoic acid (cRA, isotretinoin) plus interferon-alpha-2a (IFN-alpha-2a) as part of a phase-II protocol. cRA/IFN-alpha-2a started 14 days prior to radiotherapy (1 mg per kilogramme body weight cRA orally daily plus 6 x 10(6) IU IFN-alpha-2a subcutaneously daily). After this induction period, standard radiotherapy was administered (external irradiation with 50.4 Gy in 28 fractions of 1.8 Gy plus HDR-brachytherapy). During radiotherapy, cRA/IFN-alpha-2a treatment was continued with 50% of the daily doses. Tumor tissue pO2-measurements were performed prior to and after the cRA/IFN-induction period as well as at 20 Gy and at the end of radiotherapy with an Eppendorf-pO2-histograph. RESULTS: In 11 out of the 22 patients, pO2-measurements were performed prior to the cRA/IFN-induction therapy. The median pO2 of these untreated tumors was 17.7 +/- 16.3 mm Hg. The relative frequency of hypoxic readings with pO2-values below 5 mm Hg ranged from 0% to 60.6% (mean 24.3 +/- 21.0%). After the 2-week induction period with cRA/IFN, the median pO2 had increased from 17.7 +/- 16.3 mm Hg to 27.6 +/- 19.1 mm Hg (not significant). In all 5 patients with hypoxic tumors prior to cRA/IFN (median pO2 of 10 mm Hg or less), the median pO2 was above 20 mm Hg after the 2-week cRA/IFN-induction. In this subgroup of hypoxic tumors, the median pO2 increased from 6.3 +/- 2.7 mm Hg to 27.0 +/- 5.6 mm Hg (p = 0.004, t-test for paired samples). The frequency of hypoxic readings (pO2-values < 5 mm Hg) decreased from 44.7 +/- 17.1% to 2.0 +/- 2.5% (p = 0.012, t-test for paired samples). There was, however, no obvious volume reduction after 14 weeks of cRA/IFN on clinical examination. A complete clinical remission of the local tumor was observed in 19/22 patients after radiotherapy and additional cRA/IFN-alpha-2a-treatment. In primarily hypoxic tumors (with a median pO2 below 10 mm Hg prior to treatment), 4/5 achieved complete remission. CONCLUSIONS: Pretreatment with cRA/IFN improves oxygenation of primarily hypoxic cervical cancers. The mechanisms of action remain unclear and further investigation of the combination regimen is recommended.     

Radiation rendered more cytotoxic by fludarabine monophosphate in a human oropharynx carcinoma cell-line than in fetal lung fibroblasts

PURPOSE: Fludarabine monophosphate (fludarabine-P) is a relatively new drug in the treatment of different haematological diseases. The mechanism of action also implies a possible role of this drug as a radiosensitizer. Up to now no in vitro investigations dealing with radiosensitizing effects of fludarabine-P in carcinoma cell lines and fibroblasts have been published. The aim of our studies was to analyse the cytotoxic and radiosensitizing effects of different dosages and application schedules of fludarabine-P in a human squamous carcinoma cell line of the oropharynx (ZMK-1) and of fetal lung fibroblasts (MRC-5) in vitro. Possible mechanisms of interaction of fludarabine-P and radiation were investigated. METHODS: ZMK-1 and MRC-5 cells were cultured under standard conditions with different concentrations of fludarabine-P in combination with escalating doses of radiation. Cytotoxic effects were measured by colony-forming assays. Induction and rejoining of radiation-induced DNA double-strand breaks after incubation with fludarabine-P were measured using constant-field gel electrophoresis. Incubation times for rejoining varied from 0 h to 24 h. RESULTS: Fludarabine-P showed a radiosensitizing activity in ZMK-1 tumour cells and MRC-5 fibroblasts. The observed effects depended on the concentration and the incubation time. The largest effect was demonstrable for an incubation of 5 days, which started shortly before irradiation, whereas an incubation solely before irradiation did not have a clear effect on the cellular survival. The sensitizer enhancement ratio, at the 10% survival level, in the ZMK-1 cells was 2.2 in comparison to 1.6 in MRC-5 cells. The analysis of the interaction of fludarabine-P and ionising radiation by means of the isobologram approach, revealed an overadditive effect in the tumour cell line and an additive effect in the lung fibroblasts. Fludarabine-P did not modify the rejoining of radiation-induced DNA double-strand breaks in either cell line. CONCLUSIONS: We conclude that fludarabine-P in clinically attainable doses is a strong radiosensitizer in ZMK-1 cells and has a lower activity in the MRC-5 fibroblasts in vitro. The radiosensitization of fludarabine-P seems to be over additive in the malignant cells and additive in normal fetal fibroblasts. This would indicate that fludarabine-P might enhance the therapeutic ratio of radiation. Further investigations are warranted to identify the potential of this drug as a radiosensitizer in vivo and to elucidate the mechanism of interaction of the drug and radiation.     

Fetal islet xenotransplantation in rodents and primates

(E)-2'-deoxy-2'-(fluoromethylene) cytidine (FMdC), a novel inhibitor of ribonucleotide-diphosphate reductase, has been shown to have anti-tumor activity against solid tumors and sensitize tumor cells to ionizing radiation. Pentoxifylline (PTX) can potentiate the cell killing induced by DNA-damaging agents through abrogation of DNA-damage-dependent G2 checkpoint. We investigated the cytotoxic, radiosensitizing and cell-cycle effects of FMdC and PTX in a human colon-cancer cell line WiDr. PTX at 0.25-1.0 mM enhanced the cytotoxicity of FMdC and lowered the IC50 of FMdC from 79 +/- 0.1 to 31.2 +/- 2.1 nM, as determined by MTT assay. Using clonogenic assay, pre-irradiation exposure of exponentially growing WiDr cells to 30 nM FMdC for 48 hr or post-irradiation to 0.5 to 1.0 mM PTX alone resulted in an increase in radiation-induced cytotoxicity. Moreover, there was a significant change of the radiosensitization if both drugs were combined as compared with the effect of either drug alone. Cell-cycle analysis showed that treatment with nanomolar FMdC resulted in S-phase accumulation and that such an S-phase arrest can be abrogated by PTX. Treatment with FMdC prior to radiation increased post-irradiation-induced G2 arrest, and such G2 accumulation was also abrogated by PTX. These results suggest that pharmacological abrogation of S and G2 checkpoints by PTX may provide an effective strategy for enhancing the cytotoxic and radiosensitizing effects of FMdC.     

Kinetics of mouse jejunum radiosensitization by 2',2'-difluorodeoxycytidine (gemcitabine) and its relationship with pharmacodynamics of DNA synthesis inhibition and cell cycle redistribution in crypt cells

Gemcitabine (dFdC), a deoxycitidine nucleoside analogue, inhibits DNA synthesis and repair of radiation-induced chromosome breaks in vitro, radiosensitizes various human and mouse cells in vitro and shows clinical activity in several tumours. Limited data are however available on the effect of dFdC on normal tissue radiotolerance and on factors associated with dFdC's radiosensitization in vivo. The purpose of this study was to determine the effect of dFdC on mouse jejunum radiosensitization and to investigate the kinetics of DNA synthesis inhibition and cell cycle redistribution in the jejunal crypts as surrogates of radiosensitization in vivo. For assessment of jejunum tolerance, the mice were irradiated on the whole body with 60Co gamma rays (3.5-18 Gy single dose) with or without prior administration of dFdC (150 mg kg-1). Jejunum tolerance was evaluated by the number of regenerated crypts per circumference at 86 h after irradiation. For pharmacodynamic studies, dFdC (150 or 600 mg kg-1) was given i.p. and jejunum was harvested at various times (0-48 h), preceded by a pulse BrdUrd labelling. Labelled cells were detected by immunohistochemistry on paraffin-embedded sections. DNA synthesis was inhibited within 3 h after dFdC administration. After an early wave of apoptosis (3-6 h), DNA synthesis recovered by 6 h, and crypt cells became synchronized. At 48 h, the labelling index returned almost to background level. At a level of 40 regenerated crypts, radiosensitization was observed for a 3 h time interval (dose modification factor of 1.3) and was associated with DNA synthesis inhibition, whereas a slight radioprotection was observed for a 48-h time interval (dose modification factor of 0.9) when DNA synthesis has reinitiated. In conclusion, dFdC altered the radioresponse of the mouse jejunum in a schedule-dependent fashion. Our data tend to support the hypothesis that DNA synthesis inhibition and cell cycle redistribution are surrogates for radiosensitization. More data points are however required before a definite conclusion can be drawn.     

The influence of hypoxia on the cytotoxicity of concomitant KW-2149 and ionizing irradiation in Chinese hamster fibroblasts

7-N-((2-((2-(gamma-L-glutamylamino)ethyl)dithio)ethyl))-mitomycin C (KW-2149) is a newly synthesized water-soluble mitomycin C (MMC) analog. Preclinical testing showed an interesting activity profile and a superior hematological tolerance in murine models. The aim of this study was to investigate the interaction of this compound with ionizing radiation, both under normoxic and hypoxic conditions, in Chinese hamster fibroblasts (V79). V79 cells were irradiated both under normoxic conditions and after a 1 h period of hypoxia. Paired irradiation dose-response curves confirmed the significance of radioresistance under hypoxia with an oxygen enhancement ratio of approximately 3. In contrast to MMC, KW-2149 showed no increased cytotoxic effect on hypoxic V79 cells. The cytotoxic effect of KW-2149 increased with increasing concentration, irrespective of the ambient oxygen pressure. When KW-2149 was combined with irradiation under hypoxic conditions, cytotoxicity was significantly enhanced under these conditions. The difference in survival between normoxic and hypoxic conditions was statistically significant (p < 0.004). These data suggest a radiosensitizing effect of KW-2149, more pronounced under hypoxic conditions. This effect increases with radiation dose. It also corroborates earlier suggestions of a different mode of action of KW-2149 as compared to MMC.     

Interstitial fluid pressure in cervical carcinoma: within tumor heterogeneity, and relation to oxygen tension

BACKGROUND: Interstitial fluid pressure (IFP) is elevated in many animal and human tumors. The authors assessed tumor IFP and its relation to tumor oxygenation in a prospective clinical study of patients with cervical carcinoma. METHODS: Measurements were made in 77 patients with cervical carcinoma prior to treatment. IFP was measured in normal paravaginal submucosal tissue and at one to five positions in the visible tumor with the patients anesthetized and in the lithotomy position. Tumor oxygen tension was measured immediately prior to IFP using a polarographic needle electrode. Patients were treated with radiotherapy only. Response was evaluated 3 months after the completion of radiotherapy. RESULTS: There was substantial variation in IFP from region to region in some tumors. The mean IFP in individual tumors ranged from 3 to 48 millimeters of mercury (mmHg). The overall mean and median values for the entire patient group were 19 mmHg and 17 mmHg, respectively. IFP was significantly higher in tumor tissue than in normal tissue (P < 0.0001). Tumors with high IFP were more likely to be hypoxic (P < 0.007) and less likely to regress completely with radiotherapy (P < 0.04). CONCLUSIONS: IFP in cervical carcinoma is elevated above normal tissue values. Multiple measurements are needed to evaluate IFP in these tumors. High IFP is associated with hypoxia and may provide information about the mechanism of hypoxia on which treatment can be based.     

Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression

Tumor hypoxia and high levels of expression of the urokinase-type plasminogen activator (uPA) receptor (uPAR) represent a poor clinical outcome for patients with various cancers. Here, we examined the effect of hypoxia on in vitro invasion of extracellular matrix and uPAR expression by human carcinoma cells. Compared with culture under 20% O2, culture for up to 24 hr under 1% or 4% O2 resulted in increased cell surface uPAR. However, the highest uPAR levels were observed in cells cultured under 1% O2. Culture of MDA-MB-231 breast carcinoma cells under hypoxia also resulted in increased uPAR mRNA levels. Furthermore, incubation with cobalt chloride or with an iron chelator also resulted in elevated uPAR expression, while presence of 30% carbon monoxide in the hypoxic atmosphere reduced the hypoxia-mediated uPAR mRNA upregulation. Increased uPAR expression was paralleled by higher cell-associated uPA levels and lower levels of secreted uPA as determined by gel zymography performed on cell extracts and culture-conditioned media. In addition, the in vitro invasiveness of MDA-MB-231 breast carcinoma cells was significantly higher when the invasion assay was performed under hypoxic conditions. This effect of hypoxia on invasion was abrogated by including in the assay a monoclonal, function-blocking anti-u PAR antibody or by the presence of 30% carbon monoxide in the hypoxic atmosphere. Our findings indicate that hypoxia stimulates carcinoma cell invasiveness by upregulating uPAR expression on the cell surface through a mechanism that requires a putative heme protein. Through a similar mechanism, hypoxia may stimulate tumor invasion and metastasis in vivo.     

Evaluation of 64Cu-ATSM in vitro and in vivo in a hypoxic tumor model

We have evaluated Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM), an effective marker for the delineation of hypoxic but viable tissue, in vitro in the EMT6 carcinoma cell line under varying degrees of hypoxia and compared it with the flow tracer 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (Cu-PTSM) and the hypoxic tracer 18F-fluoromisonidazole (MISO). We have also compared the uptake of Cu-ATSM and Cu-PTSM in vivo and ex vivo in a murine animal model bearing the EMT6 tumor. METHODS: Uptake of 64Cu-ATSM, 64Cu-PTSM and 18F-MISO in vitro into EMT6 cells was investigated at the dissolved oxygen concentrations of 0, 1 x 10(3), 5 x 10(3), 5 x 10(4) and 2 x 10(5) ppm. Biodistribution performed at 1, 5, 10, 20 and 40 min compared 64Cu-ATSM with 64Cu-PTSM in BALB/c mice bearing EMT6 tumors. To determine long-term retention of 64Cu-ATSM, biodistribution was also performed at 1, 2 and 4 h. Ex vivo autoradiography of tumor slices after co-injection of 60Cu-PTSM (60Cu, T1/2 = 23.7 min) and 64Cu-ATSM (64Cu, t1/2 = 12.7 h) into the same animal was performed. RESULTS: After 1 h, 64Cu-ATSM was taken up by EMT6 cells: 90% at 0 ppm, 77% at 1 x 10(3) ppm, 38% at 5 x 10(3) ppm, 35% at 5 x 10(4) ppm and 31% at 2 x 10(5) ppm. 18F-MISO also showed oxygen concentration dependent uptake, but with lower percentages than 64Cu-ATSM. 64Cu-PTSM showed 83%-85% uptake into the cells after 1 h, independent of oxygen concentration. Biodistribution data of 64Cu-ATSM and 64Cu-PTSM showed optimal tumor uptake after 5 and 10 min, respectively (0.76% injected dose (ID)/organ for 64Cu-ATSM and 1.11%ID/organ for 64Cu-PTSM). Ex vivo imaging experiments showed 60Cu-PTSM uniform throughout the EMT6 tumor, but heterogeneous uptake of 64Cu-ATSM, indicative of selective trapping of 64Cu-ATSM into the hypoxic tumor cells. CONCLUSION: Cu-ATSM exhibits selectivity for hypoxic tumor tissue both in vivo and in vitro and may provide a successful diagnostic modality for the detection of tumor ischemia.     

Augmentation in chemosensitivity of intratumor quiescent cells by combined treatment with nicotinamide and mild hyperthermia

C3H/He and Balb/c mice bearing SCC VII and EMT6/KU tumors, respectively, received continuous administration of 5-bromo-2'-deoxyuridine (BrdU) for 5 days using implanted mini-osmotic pumps to label all proliferating (P) cells. Nicotinamide was administered intraperitoneally before cisplatin injection and/or tumors were locally heated at 40 degrees C for 60 min immediately after cisplatin injection. The tumors were then excised, minced and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (a cytokinesis-blocker), and the micronucleus (MN) frequency in cells without BrdU labeling (quiescent (Q) cells) was determined using immunofluorescence staining for BrdU. The MN frequency in total (P+Q) tumor cells was determined from tumors that had not been pretreated with BrdU labeling. The sensitivity to cisplatin was evaluated in terms of the frequency of induced micronuclei in binuclear tumor cells (MN frequency). In both tumor systems, the MN frequency in Q cells was lower than that in the total cell population. Nicotinamide treatment elevated the MN frequency in total SCC VII cells. Mild heating raised the MN frequency more markedly in Q cells than in total cells. The combination of nicotinamide and mild heat treatment increased the MN frequency more markedly than either treatment alone. In total SCC VII cells, nicotinamide increased 195mPt-cisplatin uptake. Mild heating elevated 195mPt-cisplatin uptake in total EMT6/KU cells. Cisplatin-sensitivity of Q cells was lower than that of total cells in both tumor systems. Nicotinamide sensitized tumor cells including a large acutely hypoxic fraction, such as those of SCC VII tumors, through inhibition of the fluctuations in tumor blood flow. Tumor cells including a large chronically hypoxic fraction such as Q cells were thought to be sensitized by mild heating through an increase in tumor blood flow.     

Choice-sensitive health costs

Gemcitabine, a cytidine nucleoside analogue, has schedule-dependent antitumor activity in vitro and in vivo. Gemcitabine also has dose- and time-dependent radiosensitization properties in vitro. Thus it may have therapeutic application in combination with radiation. The aims of this study were to investigate whether gemcitabine could enhance radiation-induced tumor regrowth delay in a human squamous carcinoma (FaDu) xenograft in nude mice and to examine the effect of gemcitabine on radiation-induced apoptosis in in vivo tumors. Radiation was given locally to the tumors twice daily in 2 Gy fractions over 2 weeks for 5 days/week. Significant regrowth delay enhancement was observed which was dependent on gemcitabine schedule. Effective schedules using maximum tolerated gemcitabine doses were twice weekly and once weekly, but not daily. Significant toxicity occurred with radiation plus twice weekly gemcitabine, but enhancement was seen using gemcitabine doses well below the maximum tolerated dose. Both gemcitabine and radiation led to apoptotic cell death, but this was not increased when both treatments were combined. These results indicate that gemcitabine may be of therapeutic value as a radiation enhancer in the treatment of human cancers. Preliminary studies suggest that increased apoptotic cell death is not a mechanism leading to this enhancement.