Detection of sequence-specific antitumor alkylating agent DNA damage from cells treated in culture and from a patient.

Detection of sequence-specific DNA damage induced by antitumor alkylating agents might provide a mechanism for detecting and discriminating damage specific to one or more of these drugs. Using repetitive primer-extension and human alphoid DNA as a substrate, lesions specific for an activated form of cyclophosphamide, 4-hydroperoxycyclophosphamide, were detected at 32 of 33 guanines within a 200-base pair region in DNA from cells treated in culture. There was a marked variation in lesion site intensity among affected guanines. For instance, guanines flanked by cytosine were weak sites of 4-hydroperoxycyclophosphamide-induced damage. Damage at bases other than guanine induced by cisplatin, UV irradiation, and adozelesin were compared to drug-DNA lesions induced by 4-hydroperoxycyclophosphamide. Using this method it was possible to detect, and at some sites distinguish, between cyclophosphamide- and cisplatin-induced DNA damage within WBC DNA from a patient treated with both agents. There was a different damage pattern for DNA derived from cells treated in culture compared to DNA derived from the patient sample.

Reduced oxygenation in a rat mammary carcinoma after chemo- or radiation therapy and reoxygenation with perflubron emulsion/carbogen breathing.

Rat 13672 mammary carcinoma tumors were grown subcutaneously in the hind legs of female Fischer 344 rats to a volume of about 1 cm3. Tumor oxygenation was measured using an Eppendorf PO2 histograph. Tumor oxygen measurements were made under four conditions: (a) normal air breathing, (b) carbogen breathing, (c) after intravenous administration of a perflubron emulsion (8 ml/kg) with air breathing and (d) after intravenous administration of a perflubron emulsion (8 ml/kg) with carbogen breathing. Tumor oxygenation was examined without treatment or 24 h and 48 h after treatment with cyclophosphamide (300 mg/kg, i.p.) or cisplatin (8 mg/kg, i.p.] or after the fifth dose of a daily regimen of 3-Gy irradiation (5 x 3 Gy). Under normal air-breathing conditions 49% of the tumor had a PO2 < or = 670 Pa (5 mm Hg). The degree of hypoxia in the tumors increased after each treatment such that 24 h after treatment 65%-85% of the oxygen readings were < or = 670 Pa and 48 h after treatment 60%-74% of the oxygen readings were < or = 670 Pa. Administration of the perflubron emulsion/carbogen atmosphere increased the oxygen content of the tumors both without treatment and after each of the treatments. A knowledge of tumor oxygen content over the course of treatment and the ability to increase tumor oxygen should allow for the development of more rational treatment combinations and better treatment outcomes.

Oxygenation of tumors by a hemoglobin solution.

Tumor oxygen tensions were measured using a computer-controlled PO2 microelectrode in two preclinical solid tumor models, the rat 9L gliosarcoma and the rat 13672 mammary carcinoma. Tumor oxygenation profiles were determined under four conditions: (a) during normal air breathing, (b) during carbogen breathing, (c) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with normal air breathing and (d) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with carbogen breathing. Both tumors had severely hypoxic regions under normal air-breathing conditions. Although carbogen breathing increased the oxygenation of the better-oxygenated portions of the tumor, it made no impact on the severely hypoxic tumor regions. Administration of the hemoglobin solution was effective in increasing the oxygenation throughout both tumors under normal air-breathing conditions. The addition of carbogen breathing to administration of the hemoglobin solution eliminated severe hypoxia in the 9L gliosarcoma and markedly reduced the severely hypoxic regions of the 13672 mammary carcinoma. At 24 h after administration of the hemoglobin solution the 13672 mammary carcinoma showed greater hypoxia than before treatment, which was partially corrected with carbogen breathing.

Cytotoxicity of antitumor platinum complexes with L-buthionine-(R,S)-sulfoximine and/or etanidazole in human carcinoma cell lines sensitive and resistant to cisplatin.

Human 2008 ovarian carcinoma cells and the C13 CDDP-resistant subline and human MCF-7 breast carcinoma cells and the MCF-7/CDDP CDDP-resistant subline were exposed to L-buthionine-(S,R)-sulfoximine (50 microM) for 48 h prior to and during exposure for 1 h to the antitumor platinum complexes, cis-diamminedichloroplatinum(II), carboplatin or D,L-tetraplatin and/or to etanidazole (1 mM) for 2 h prior to and during exposure for 1 to the antitumor platinum complexes. These modulators alone did not significantly alter the cytotoxicity of CDDP toward either parental line. A twofold enhancement in cytotoxicity was observed with carboplatin in the 2008 cells and with D,L-tetraplatin in both parental lines with the single modulators. The modulator combination (buthionine sulfoximine/etanidazole) was very effective along with D,L-tetraplatin in both the MCF-7 parent and MCF-7/CDDP cell lines where at the higher platinum complex concentrations there was 1.5 to 3 logs increased killing of cells by the drug plus the modulators compared with the drug alone. Similarly, when C13 cells were exposed to CDDP (100 microM) or D,L-tetraplatin (100 microM) along with buthionine sulfoximine and etanidazole there was a 2-log increase in cell killing compared with exposure to the platinum complex alone. Treatment of each of the four cell lines with buthionine sulfoximine decreased both the non-protein and total sulfhydryl content of the cells. Treatment with the combination of modulators did not produce a further decrease in cellular sulfhydryl content compared with buthionine sulfoximine alone. The total sulfhydryl content in MCF-7 cells and 2008 cells exposed to buthionine sulfoximine and etanidazole was 58% and 31% of normal and the total sulfhydryl content of MCF-7/CDDP cells and C13 cells treated the same way was 54% and 23% of normal, respectively. DNA alkaline elution was used to assess the impact of exposure to the modulators, buthionine sulfoximine and etanidazole, alone and in combination on the cross linking of DNA by the antitumor platinum complexes in the MCF-7 and MCF-7/CDDP cell lines. Overall, the increases in DNA cross linking factors were greater in the MCF-7 cells than in the MCF-7/CDDP cells. These results indicate a possible clinical potential for this modulator combination.

Antiangiogenic treatment (TNP-470/minocycline) increases tissue levels of anticancer drugs in mice bearing Lewis lung carcinoma.

Although the antiangiogenic agent TNP-470 does not, in general, increase the cytotoxicity of anti-cancer therapies in cell culture, the antiangiogenic agents TNP-470 and minocycline individually and especially in combination have been shown to increase the tumor growth delay produced by several standard cytotoxic therapies in the Lewis lung carcinoma. In an effort to understand the mechanism by which the antiangiogenic agent combination TNP-470/minocycline potentiates the antitumor activity of cytotoxic therapeutic agents in vivo, the biodistribution of [14C]-cyclophosphamide and cis-diamminedichloroplatinum(II) was determined 6 h after cytotoxic drug administration in animals bearing Lewis lung carcinoma pretreated with TNP-470/minocycline and in animals without pretreatment. Higher levels of 14C and platinum were found in 9 tissues (including tumor) except blood in animals pretreated with TNP-470/minocycline. The increased drug levels in the tumors may be sufficient to account for the increased tumor growth delays observed previously. DNA alkaline elution of tumors from animals pretreated with TNP-470/minocycline showed increased DNA cross-linking by both cyclophosphamide and cis-diamminedichloroplatinum(II). The possible implications of these results are discussed.

Increased efficacy of chemo- and radio-therapy by a hemoglobin solution in the 9L gliosarcoma.

Tissue oxygen tensions were measured in the rat 9L gliosarcoma under conditions of normal air breathing or carbogen breathing and after intravenous administration of a hemoglobin solution with air breathing or carbogen breathing. Administration of the hemoglobin decreased the level of hypoxia in the tumors. Treatment of the animals with the antiangiogenic combination of TNP-470 and minocycline also increased tumor oxygenation compared with untreated controls. Treatment with the antiangiogenic agents along with administration of the hemoglobin solution/carbogen breathing decreased the hypoxic fraction (% pO2 readings < or = 5 mmHg) from 71 % to 30%. Treatment of the tumor-bearing animals with BCNU or adriamycin modestly reduced hypoxia in the tumors, while treatment with fractionated radiation markedly increased hypoxia in the tumors. Tumor growth delay was used to assess the response of the subcutaneous tumor to the various treatment combinations. There was a strong correlation between increased therapeutic response and decreased tumor hypoxia. Tumor growth delay from BCNU increased from 5.3 days to 16.4 days with TNP-470/-minocycline/hemoglobin solution/carbogen. Similarly, the tumor growth delay from adriamycin increased from 3.9 days to 17.0 days with TNP-470/minocycline/hemoglobin solution/carbogen. Finally, the tumor growth delay from fractionated radiation increased from 4.8 days to 13.3 days with TNP-470/minocycline/hemoglobin solution/carbogen. When etanidazole was added to the complete radiation regimen, the tumor growth delay increased further to 20.5 days. These data show that the addition of non-toxic agents that increase tumor oxygenation to cytotoxic therapies can markedly increase therapeutic response.

Reduced oxygenation in a rat mammary carcinoma post-radiation and reoxygenation with a perflubron emulsion/carbogen breathing.

Oxygen profiles of the rat mammary 13672 carcinoma were determined using a pO2 histograph prior to treatment and 24 hrs. and 48 hrs. after 2 or 5 fractions of 3 gray given once daily. The tumors were much more hypoxic at 24 hrs. post radiation therapy than prior to therapy. There was little increase in the oxygenation of the tumors at 48 hrs. post therapy compared with 24 hrs. post therapy indicating that reoxygenation was occurring very slowly in this tumor. Carbogen breathing improved the oxygenation of the tumors under each of the conditions studied. Administration of a perflubron emulsion (8 ml/kg) produced little or no change in the oxygenation of the tumors under normal air breathing conditions. However, with the addition of carbogen breathing to administration of the perflubron emulsion there was an increase in the mean/median pO2's of the tumors to levels equal to or greater than carbogen breathing alone. Perhaps most significantly administration of the perflubron emulsion with carbogen breathing increased the oxygenation of the most hypoxic regions of the tumors but carbogen breathing alone did not.

Restoration of tumor oxygenation after cytotoxic therapy by a perflubron emulsion/carbogen breathing.

PURPOSE: Hypoxic cells are presumed to be an obstacle to successful cancer treatment because these cells are protected from the cytotoxic effects of radiotherapy and certain anti-cancer drugs. The current study was conducted to determine the effect of cytotoxic therapy on tumor oxygenation and the effect of administration of a perfluorochemical emulsion/carbogen breathing treatment on tumor oxygenation after cytotoxic therapy. MATERIALS AND METHODS: Female Fisher 344 rats bearing 13762 mammary carcinoma cells implanted subcutaneously in a hindlimb were treated with standard therapeutic single doses of anti-tumor treatments of several types, including alkylating agents (cisplatin, melphalan, cyclophosphamide); natural products (doxorubicin, paclitaxel, etoposide); antimetabolites (fluorouracil); hypoxic cell-selective agents (mitomycin, SR-4233); and fractionated irradiation (3 Gy/day for 5 days). The oxygen levels in the tumors were measured with an Eppendorf PO2 histograph before the treatment and 24 hours after treatment under air breathing and carbogen breathing conditions, and after administration of a perflubron emulsion under air breathing and carrbogen breathing conditions. Fifty to 60 points were measured per tumor, and 8 to 10 tumors made up each group. RESULTS: The tumors were more hypoxic after treatment with every anticancer treatment. The percentage of PO2 readings < or = 5 mmHg in the untreated tumors was 49% and ranged from 85% (radiotherapy) to 59% (etoposide) in the treated tumors. Administration of the perflubron emulsion (8 mL/kg) and carbogen breathing (95% O2/5% CO2) increased the oxygenation of the tumors such that the percentage of PO2 readings < or = 5 mmHg was 32% in the untreated control tumors and ranged from 27% (radiotherapy) to 56% (doxorubicin) in the treated tumors. There was a direct correlation between the level of tumor cell killing and the increased oxygenation observed in the tumor. CONCLUSION: Tumors may be more hypoxic after an effective dose of a cytotoxic therapy, and administration of a perflubron emulsion/carbogen mixture can increase the tumor oxygen content when hypoxia is the result of cytotoxic therapy. Hypoxia produced by therapy may be regarded as a mechanism of resistance that leads to diminished tumor cell killing with subsequent doses of drugs or radiation. The restoration of tumor oxygenation by the perflubron emulsion/carbogen breathing may provide a clinically relevant means of overcoming at least in part hypoxia-related resistance.

Increased tumor oxygenation and radiation sensitivity in two rat tumors by a hemoglobin-based, oxygen-carrying preparation.

The rat 13762 mammary carcinoma and the rat 9L gliosarcoma were grown subcutaneously in a hind limb of female, Fisher 344 rats. The oxygen content of the tumors was determined using an Eppendorf pO2 histograph. Fifty-to-sixty oxygen measurements were made per tumor and there were 8-to-10 animals per group. The percent of pO2 readings < or = 5 mmHg in the mammary carcinoma was 49%, this was decreased to 34% by administration of the hemoglobin preparation (8 ml/kg) and further decreased to 29% when carbogen (95% O2/5% CO2) breathing was added to administration of the hemoglobin preparation. The percent of pO2 readings < or = 5 mmHg in the gliosarcoma was 49%, this was decreased to 24% by administration of the hemoglobin preparation and further decreased to 0% when carbogen breathing was added to administration of the hemoglobin preparation. Therapeutic response was assessed over a single-dose range of radiation therapy (10, 20 and 30 Gray). The dose modifying factor produced by the hemoglobin preparation/air was 1.6 and by the hemoglobin preparation/carbogen was 2.7 in the rat 13762 mammary carcinoma. The dose modifying factor produced by the hemoglobin preparation/air was 1.9 and by the hemoglobin preparation/carbogen was 2.9 in the rat 9L gliosarcoma. Administration of a hemoglobin-based oxygen carrier reduced tumor hypoxia and increased tumor response to radiation therapy.

Restoration of tumor oxygenation after cytotoxic therapy by a perflubron emulsion/carbogen breathing.

Female, Fisher 344 rats bearing 13762 mammary carcinoma implanted subcutaneously in a hind limb were treated with standard therapeutic single doses of antitumor treatments of several types including: 1) antitumor alkylating agents (cisplatin, cyclophosphamide); 2) natural products (adriamycin, taxol and etoposide); 3) antimetabolites (5-flourouracil); 4) hypoxic cell selective agents (mitomycin C, SR-4233) as well as 5) fractionated radiation therapy (3 Gray daily for 5 days). The oxygen levels in the tumors were measured in the absence of treatment and 24 hrs. after treatment using an Eppendorf p02 histograph. Fifty- to sixty-points were measured per tumor and 8-10 tumors comprised each group. The tumors were more hypoxic post treatment with every anticancer drug or radiation. The percent of p02 readings < or = 5 mmHg in the untreated tumors ranged from 85% (x-rays) to 59% (etoposide). Administration of the perflubron emulsion (8 ml/kg) and carbogen breathing (95% O2/5% CO2) increased the oxygenation of the tumors such that the percent of pO2 readings < or = 5 mmHg was 32% in the untreated controls and ranged from 27% (x-rays) to 56% (adria) in the treated tumors. These results indicate that administration of a perflubron emulsion/carbogen can increase the oxygen content of tumors when hypoxia is the result of cytotoxic therapy.

Oxygenation of the rat 9L gliosarcoma and the rat 13672 mammary carcinoma with various doses of a hemoglobin solution.

Tumor oxygen tensions were measured using a computer controlled pO2 microelectrode in two preclinical solid tumor models, the rat 9L gliosarcoma and the rat 13672 mammary carcinoma. Tumor oxygenation profiles were determined under four conditions: 1) normal air breathing, 2) carbogen (95% O2/5% CO2) breathing, 3) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with normal air breathing and 4) after intravenous administration of a solution of ultrapurified polymerized bovine hemoglobin with carbogen breathing. Both tumors had severely hypoxic regions under normal air breathing conditions. Although carbogen breathing increased the oxygenation of the better oxygenated portions of the tumor, it did not impact on the severely hypoxic tumor regions. Administration of the hemoglobin solution was effective in increasing the oxygenation throughout both tumors under normal air breathing conditions. The addition of carbogen breathing to administration of the hemoglobin solution eliminated severe hypoxia in the 9L gliosarcoma and markedly reduced the severely hypoxic regions of the 13672 mammary carcinoma.

Potentiation of cytotoxic therapies by TNP-470 and minocycline in mice bearing EMT-6 mammary carcinoma.

The ability of the antiangiogenic agents TNP-470 and minocycline, singly or in combination, to potentiate the antitumor effects of several cytotoxic therapies was assessed in the murine EMT-6 mammary carcinoma as well as in two drug resistant sublines of that tumor designated EMT-6/CTX and EMT-6/CDDP. The antiangiogenic agents alone or in combination did not alter the growth of the tumors. However, their administration along with cyclophosphamide, CDDP, or thiotepa substantially increased the tumor growth delay produced by these cytotoxic therapies in tumors responsive to the drugs--the increase was about 2-fold for TNP-470 and minocycline together. In drug resistant tumors, treatment with the antiangiogenic agents did not reverse drug resistance but did increase the effect of the cytotoxic drugs. Treatment with TNP-470/minocycline also increased the oxygenation of each of the three tumors. Thus, TNP-470/minocycline administration increased the efficacy of fractionated radiation therapy, especially when used along with a perflubron emulsion oxygen delivery agent/carbogen. These results indicate that treatment regimens including therapies directed toward the proliferating normal cells within a tumor mass as well as therapies directed toward the malignant cells can produce improved outcomes.

Oxygenation of human tumor xenografts in nude mice by a perfluorochemical emulsion and carbogen breathing.

Human solid tumors (prostate carcinomas PC-3 and DU-145, breast carcinoma MX-1, cervical carcinoma ME-180, small cell lung carcinoma SW2, and glioblastoma T98G) were grown as xenografts in nude mice. Using the Eppendorf pO2 histograph microelectrode system, the oxygen profiles of the tumors were determined while the animals breathed air or carbogen (95% O2/5% CO2), and after administration of the perfluorochemical emulsion Oxygent-CA (8 ml/kg) under air breathing and carbogen breathing conditions. Under normal air breathing with or without Oxygent-CA administration the mean oxygen tensions were between 4.9 and 9.3 mmHg and each tumor had severely hypoxic regions where the pO2 was less than 5 mmHg. The severely hypoxic regions comprised 41-71% of the oxygen tension measurements under normal air breathing conditions. Carbogen breathing alone increased the mean oxygen tensions to 10.9-23.9 mmHg. Administration of Oxygent-CA and carbogen breathing increased the mean oxygen tensions over the levels of carbogen breathing alone to varying degrees. The highest mean oxygen tensions were 40.8 mmHg in the T98G glioblastoma and 24.5 mmHg in the ME-180 cervical carcinoma. Investigation of the use of Oxygent-CA/carbogen to increase the oxygenation of clinical tumors is warranted.

Influence of an anti-angiogenic treatment on 9L gliosarcoma: oxygenation and response to cytotoxic therapy.

Tissue oxygen tensions were measured in subcutaneously growing rat 9L gliosarcoma under normal air and carbogen breathing conditions prior to and after i.v. administration of a perflubron emulsion. When these animals were treated with the anti-angiogenic agents TNP-470 and minocycline for 5 days prior to oxygen measurement, tumor hypoxia was decreased compared with untreated tumors. Hypoxia, defined as the percent of pO2 readings < or = 5 mm Hg, was decreased from 71% in untreated air-breathing controls to 34% in animals treated with the anti-angiogenic agents, the perflubron emulsion and carbogen breathing. These effects were manifest in the increased response of the tumor to single-dose (10, 20 and 30 Gy) radiation therapy. Twenty-four hours after treatment with BCNU oxygenation of the tumors was not altered; however, 24 hr after administration of adriamycin oxygenation of the tumors was increased such that hypoxia in adriamycin-treated tumors in animals receiving the perflubron emulsion and carbogen was reduced to 21%. Tumor growth delay in the s.c. tumors was increased by the addition of treatment with the anti-angiogenic agents from day 4 through day 18 post-tumor cell implantation along with BCNU or adriamycin on days 7-11. Administration of the perflubron emulsion and carbogen breathing resulted in increased tumor growth delay with the chemotherapeutic agents alone and in combination with the anti-angiogenic agents. Life span in animals bearing intracranially implanted 9L gliosarcoma progressively increased with administration of the anti-angiogenic agents and then the anti-angiogenic agents and perflubron emulsion/carbogen compared to treatment with BCNU or adriamycin.