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Purpose: To discuss the role of postoperative radiation therapy (RT) for patients with squamous cell carcinoma of the head and neck., Results: Patients with adverse pathologic features have a high likelihood of local-regional recurrence and a decreased probability of survival after surgery alone. Postoperative RT reduces the risk of local-regional failure and probably improves survival. Patients who are at high risk for recurrence after surgery benefit from more aggressive dose-fractionation schedules that may include altered fractionation to decrease the overall time from surgery to the completion of RT. Adjuvant chemotherapy also appears to improve the probability of cure in high risk patients., Conclusion: Patients who have a high likelihood of local-regional recurrence after surgery have improved disease control and survival after postoperative RT., (Copyright 2003, Elsevier Science (USA). All rights reserved.))

Background and Purpose: To evaluate whether sustained expression of mouse endostatin by adeno-associated virus (AAV)-mediated gene transfer can enhance the treatment efficacy of ionizing radiation., Materials and Methods: Mouse endostatin was cloned into recombinant AAV (rAAV) under the control of CMV beta-actin promoter. Recombinant mouse endostatin expressed via AAV gene transfer was tested for biological activity in endothelial cells. The impact of elevated serum levels of endostatin on tumor-induced angiogenesis was evaluated using an in vivo angiogenesis assay. The anti-tumor efficacy of combining rAAV-mediated endostatin delivery with radiation was evaluated in a human colorectal tumor model (HT29)., Results: Recombinant mouse endostatin expressed through an AAV vector (rAAV-mEndo) inhibited endothelial cell proliferation (by 40-45%) and migration (by 22-33%). Intramuscular injection of rAAV-mEndo (1x10(9) i.u.) led to a sustained serum endostatin level of approximately 500 ng/ml. Compared to control animals this endostatin level was sufficient to inhibit tumor cell-induced vessel formation (37 vs. 28.5, P<0.05) and delay the growth of HT29 xenografts (time from 200 to 1,000 mm(3), 21 vs. 34.5 days, P<0.05). When combined with ionizing radiation, elevated serum endostatin levels significantly enhanced the time for tumors to grow from 200 to 1,000 mm(3) (radiation, 34 days; endostatin plus radiation, 50 days, P<0.05)., Conclusion: The delivery of endostatin via rAAV vectors may provide an effective means of enhancing the anti-tumor efficacy of radiation therapy.

Purpose: The present report reviews the preclinical data on combined chemotherapy/vascular targeting agent treatments. Basic principles are illustrated in studies evaluating the antitumor efficacy of the vascular targeting agent ZD6126 (N-acetylcochinol-O-phosphate) when combined with the anticancer drug cisplatin in experimental rodent (KHT sarcoma) and human renal (Caki-1) tumor models., Methods and Materials: C3H/HeJ and NCR/nu-nu mice bearing i.m. tumors were injected i.p. with ZD6126 (0-150 mg/kg) or cisplatin (0-20 mg/kg) either alone or in combination. Tumor response to treatment was assessed by clonogenic cell survival., Results: Treatment with ZD6126 was found to damage existing neovasculature, leading to a rapid vascular shutdown. Histologic evaluation showed dose-dependent morphologic damage of tumor cells within a few hours after drug exposure, followed by extensive central tumor necrosis and neoplastic cell death as a result of prolonged ischemia. ZD6126 doses that led to pathophysiologic effects also enhanced the tumor cell killing of cisplatin when administered either 24 h before or 1-24 h after chemotherapy. In both tumor models, the administration of a 150 mg/kg dose of ZD6126 1 h after a range of doses of cisplatin resulted in an increase in tumor cell kill 10-500-fold greater than that seen with chemotherapy alone. In contrast, the inclusion of the antivascular agent did not increase bone marrow stem cell toxicity associated with this anticancer drug., Conclusion: The results obtained in the KHT and Caki-1 tumor models indicate that ZD6126 effectively enhanced the antitumor effects of cisplatin therapy. These findings are representative of the marked enhancements generally observed when vascular targeting agents are combined with chemotherapy in solid tumor therapy.

Background: Silencing of the viral CMV immediate early enhancer promoter can be a problem in certain cell types when engineering stable cell lines., Materials and Methods: We compared the efficacy of the CMV promoter to the promoter of the elongation factor-1 alpha (EF-1 alpha) for the generation of stable colon carcinoma cell lines (HT-29). Green fluorescent protein (GFP) expression cassettes were delivered by recombinant adeno-associated virus (AAV) which is known for its ability to stably transduce cells. Stable cell lines were characterized in vitro by FACS and in vivo after HT-29 clones were grown as xenografts in nude mice., Results: Stable HT-29 clones with > 97% of all cells homogeneously expressing GFP were generated with the EF-1 alpha promoter. In contrast in clones carrying the CMV promoter, only up to 60% of the cells were GFP-positive with expression levels varying widely between cells. Superinfection with wild-type adenovirus induced GFP expression in more than 90% of the cells indicating that the CMV promoter was silenced. In vivo the tumors carrying the EF-1 alpha promoter were homogeneously GFP-positive, whereas the CMV promoter gave rise to a scattered pattern of GFP expression., Conclusion: This study underlines the importance of the promoter for the generation of stable cell lines. In addition it demonstrates that recombinant AAV can effectively be used as a gene delivery system for this purpose.

The tumor vessel support network offers a tantalizing target for cancer therapy, given the complete dependence of a solid neoplasia on the vasculature for both the delivery of oxygen and other nutrients, as well as the effective removal of wasteproducts. Attacking a tumor's supportive blood vessel network offers ameans of improving cancer cure rates on the basis of two principles. The first reflects evidence indicating that physiological conditions in tumors, arising primarily as a consequence of inadequate and non-uniform vascular networks, are significant contributors to resistance to non-surgical anticancer treatments. The second involves the recognition that the inherent differences between blood vessels in tumors and those associated with normal tissues provide a variety of unique targets for the design of novel therapeutics, highly selective for neoplastic growth. Therapeutic approaches that aim to destroy the tumor endothelium are being actively pursued. The application of such vascular targeting strategies as adjuvants to conventional therapeutics such as radiotherapy, offers unique opportunities to develop even more effective cancer therapies.

Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of approximately 200 mm(3) significantly increased the time for tumours to grow to 1000 mm(3)., (Copyright 2002 Cancer Research UK)

A variety of approaches has demonstrated that interfering with tumor-induced angiogenesis may be an effective strategy in cancer therapy. However, it is likely that to be most effective such strategies will require extended suppression of the angiogenic process. Gene therapy offers a possible approach to achieve sustained release of a therapeutically potent transferred gene product. In the present study the angiogenesis inhibitor endostatin was expressed through a recombinant adeno-associated viral (rAAV) vector and shown to be biologically active in vitro and in vivo. Intramuscular injection of rAAV-HuEndo (1 x 10(9) i.u.) led to a sustained serum endostatin level of approximately 35-40 ng/mL. This endostatin level was sufficient to inhibit tumor cell-induced angiogenesis and to suppress both the initiation and subsequent growth of a human colorectal cancer model.

The American Association for Cancer Research held its 93rd Annual Meeting on 6 - 10 April 2002. This was an enormous meeting with approximately 14,000 registrants and some 5750 abstracts, the vast majority of which were presented as posters. Those abstracts judged to be of particular significance or interest were presented as 15-min platform presentations in minisymposia, of which there were 36 during the meeting. In addition to this, there was a full day of 15 educational sessions (each with three to four speakers), 11 award lectures, 47 early morning Meet-the-Expert Sunrise Sessions. There was a mind-boggling array of choices for those interested in cancer research. This meeting was the largest ever convened on a single disease. We will endeavour to provide some of the highlights of this exciting meeting.

The utility of combining the vascular targeting agents 5,6-dimethyl-xanthenone-4 acetic acid (DMXAA) and combretastatin A-4 disodium phosphate (CA4DP) with the anticancer drugs cisplatin and cyclophosphamide (CP) was evaluated in experimental rodent (KHT sarcoma), human breast (SKBR3) and ovarian (OW-1) tumor models. Doses of the vascular targeting agents that led to rapid vascular shutdown and subsequent extensive central tumor necrosis were identified. Histologic evaluation showed morphologic damage of tumor cells within a few hours after treatment, followed by extensive hemorrhagic necrosis and dose-dependent neoplastic cell death as a result of prolonged ischemia. Whereas these effects were induced by a range of CA4DP doses (10-150 mg/kg), the dose response to DMXAA was extremely steep; doses < or = 15 mg/kg were ineffective and doses > or = 20 mg/kg were toxic. DMXAA also enhanced the tumor cell killing of cisplatin, but doses > 15 mg/kg were required. In contrast, CA4DP increased cisplatin-induced tumor cell killing at all doses studied. This enhancement of cisplatin efficacy was dependent on the sequence and interval between the agents. The greatest effects were achieved when the vascular targeting agents were administered 1-3 hr after cisplatin. When CA4DP (100 mg/kg) or DMXAA (17.5 mg/kg) were administered 1 hr after a range of doses of cisplatin or CP, the tumor cell kill was 10-500-fold greater than that seen with chemotherapy alone. In addition, the inclusion of the antivascular agents did not increase bone marrow stem cell toxicity associated with these anticancer drugs, thus giving rise to a therapeutic gain., (Copyright 2002 Wiley-Liss, Inc.)

Purpose: The aim of this study was to evaluate the antitumor efficacy of the novel vascular targeting agent ZD6126 (N-acetylcochinol-O-phosphate) in the rodent KHT sarcoma model, either alone or in combination with single- or fractionated-dose radiation therapy., Methods: C3H/HeJ mice bearing i.m. KHT tumors were injected i.p. with ZD6126 doses ranging from 10 to 150 mg/kg. Tumors were irradiated locally in unanesthetized mice using a linear accelerator. Tumor response to ZD6126 administered alone or in combination with radiation was assessed by clonogenic cell survival assay or tumor growth delay., Results: Treatment with ZD6126 led to a rapid tumor vascular shutdown as determined by Hoechst 33342 diffusion. Histologic evaluation showed morphologic damage of tumor cells within a few hours after drug exposure, followed by extensive central tumor necrosis and neoplastic cell death as a result of prolonged ischemia. When combined with radiation, a 150 mg/kg dose of ZD6126 reduced tumor cell survival 10-500-fold compared with radiation alone. These enhancements in tumor cell killing could be achieved for ZD6126 given both before and after radiation exposure. Further, the shape of the cell survival curve observed after the combination therapy suggested that including ZD6126 in the treatment had a major effect on the radiation-resistant hypoxic cell subpopulation associated with this tumor. Finally, when given on a once-weekly basis in conjunction with fractionated radiotherapy, ZD6126 treatment was found to significantly increase the tumor response to daily 2.5 Gy fractions., Conclusion: The present results demonstrated that in the KHT sarcoma, ZD6126 caused rapid tumor vascular shutdown, induction of central tumor necrosis, tumor cell death secondary to ischemia, and enhancement of the antitumor effects of radiation therapy.

Combretastatin A4 disodium phosphate (CA4DP) was evaluated in a xenograft model of AIDS-KS. KS xenografts were highly vascular, showing brisk mitotic activity, focal areas of necrosis, and intervening fibrovascular septae. Neoplastic cells were large or spindle-shaped, with vesicular nuclei and modest pleomorphism. Multiple junctions, microvillous-like projections, abortive lumina and rare Weibel Palade bodies were revealed by electron microscopy. Treatment with CA4DP (100 mg/kg) resulted in rapid onset of vascular effects that within 4 h resulted in an almost complete vascular shutdown in these tumors. Histological evaluation showed morphological damage within a few hours after treatment, followed by extensive necrosis which increased to approximately 90% by 24 h. At this time, viable tumor cells were evident only at the periphery of the tumor. These findings demonstrate not only the marked susceptibility of the KS model to CA4DP but also its potential application in studies related to the pathogenesis and therapy of AIDS-KS.

The antitumor efficacy of the vascular targeting agent combretastatin A-4 disodium phosphate (CA4DP) was evaluated in a xenograft model of Kaposi's sarcoma (KS) grown in athymic mice. Response to CA4DP alone or in combination with localized radiation treatment or systemic chemotherapy (cisplatin or vinblastine) was assessed using a clonogenic cell survival or tumor growth delay assay. Administering increasing doses of CA4DP to tumor-bearing mice resulted in a dose-dependent increase in tumor cell kill. CA4DP also enhanced the antitumor effects of radiation and chemotherapy approximately 10-100-fold. Although single doses of CA4DP as large as 300 mg/kg failed to alter tumor growth, the same total dose, administered as 3 fractions in 5 or 9 days, resulted in significant growth delay. Such repeated CA4DP exposures also significantly increased the response of KS xenografts to cisplatin. These findings suggest that CA4DP ought to be considered as a candidate agent for therapeutic evaluation in AIDS-KS patients.

The interaction between 5,6-dimethylxanthenone-4-acetic acid (DMXAA) and radiation was investigated in two different mouse tumor models and a normal mouse tissue. C3H mouse mammary carcinomas transplanted in the feet of CDF1 mice and KHT mouse sarcomas growing in the leg muscles of C3H/HeJ mice were used. DMXAA was dissolved in saline and injected intraperitoneally. Tumors were irradiated locally in nonanesthetized mice, and response was assessed using tumor growth for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. DMXAA alone had an antitumor effect in both tumor types, but only at doses above 15 mg/kg. DMXAA also enhanced radiation damage, and again there was a threshold dose. No enhancement was seen in the C3H mammary carcinoma at 10 mg/kg and below, while in the KHT sarcoma, doses above 15 mg/kg were necessary. This enhancement of radiation damage was also dependent on the sequence of and interval between the treatments with DMXAA and radiation. Combining radiation with DMXAA at the maximum tolerated dose (i.e., the highest dose that could be injected without causing any lethality) of either 20 mg/kg (CDF1 mice) or 17.5 mg/kg (C3H/HeJ mice) gave an additive response when the two agents were administered simultaneously. Even greater antitumor effects were achieved when DMXAA was administered 1-3 h after irradiation. However, when administration of DMXAA preceded irradiation, the effect was similar to that seen for radiation alone, suggesting that appropriate timing is essential to maximize the utility of this agent. When such conditions were met, DMXAA was found to increase the tumor response significantly in the absence of an enhancement of radiation damage in normal skin, thus giving rise to therapeutic gain.

Background and Purpose: The ability of combretastatin A-4 disodium phosphate (CA4DP) to induce vascular damage and enhance the radiation response of murine tumors was investigated., Materials and Methods: A C3H mouse mammary carcinoma transplanted in the foot of CDF1 mice and the KHT mouse sarcoma growing in the leg muscle of C3H/HeJ mice were used. CA4DP was dissolved in saline and injected intraperitoneally. Tumor blood perfusion was estimated using 86RbCl extraction and Hoechst 33342 fluorescent labelling. Necrotic fraction was determined from histological sections. Tumors were locally irradiated in non-anaesthetised mice and response assessed by local tumor control for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma., Results: CA4DP decreased tumor blood perfusion and increased necrosis in a dose-dependent fashion in the C3H mammary carcinoma, which was maximal at 250 mg/kg. The decrease in perfusion and induction of necrosis by CA4DP was more extensive in the KHT sarcoma. CA4DP enhanced radiation damage in both tumor types. In the KHT sarcoma this enhancement was independent of whether the drug was given before or after irradiating, whereas for C3H mammary carcinoma the enhancement was only significant when administered at the same time or after the radiation, with no enhancement seen if CA4DP was given before. These effects were drug-dose dependent. CA4DP did not enhance radiation damage in normal skin., Conclusions: CA4DP enhanced radiation damage in the two tumor models without enhancing normal tissue damage. These radiation effects were clearly consistent with the anti-vascular action of CA4DP.

Background and Purpose: The neovascularization of tumor cells is a prerequisite if a clinically relevant tumor size is to be reached. A continuously expanding vessel network supplying nutritional requirements and removing waste products is essential for continued tumor development, growth and survival., Results: In many tumors, the growing endothelium is unable to fully support the demands of the neoplastic cell population. As a consequence of the inadequacies of the resulting aberrant vasculature, microenvironmental conditions develop in tumors which are not only detrimental to the response of tumors to conventional anticancer treatments, but may lead to or predispose cells to genetic modifications resulting in more aggressive phenotypes and higher metastatic potential. Yet the utter dependence of the tumor on its induced vessel formation for growth, survival and spread has also created a great deal of enthusiasm for developing therapeutic approaches to specifically targeting the tumor microcirculation., Conclusions: The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies.

The likelihood of local control after radiation therapy may be improved by increasing total dose or decreasing overall time. The probability of late complications increases with dose per fraction. Altered fractionation techniques usually employ two or more fractions per day using a dose per fraction that is similar or less than that employed in conventional fractionation. Altered fractionation may be broadly classified as hyperfractionation or accelerated fractionation. Data suggest that altered fractionation schedules may improve local control (and to a lesser extent, survival) compared with conventional irradiation.

Purpose: The aim of this study was to evaluate the antitumor efficacy of combretastatin A-4 disodium phosphate (combretastatin prodrug) in the rodent KHT sarcoma model either alone or in combination with radiation therapy., Methods: KHT tumors were grown in C3H/HeJ mice. Combretastatin A-4 prodrug was injected intraperitoneally at doses ranging from 10 to 100 mg/kg. Tumors were irradiated in unanesthetized mice using a 137Cs source. Tumor response to combretastatin A-4 prodrug was assessed by histological evaluations as well as an in vivo to in vitro cell survival assay., Results: Histological evaluation showed morphological damage of tumor cells within a few hours after drug treatment, followed by extensive central necrosis. Administering increasing doses of combretastatin A-4 prodrug to tumor-bearing mice resulted in a dose-dependent increase in cell killing irrespective of whether the tumors were irradiated or not. When combined with radiation, a 100 mg/kg dose of combretastatin A-4 prodrug reduced tumor cell survival 10-500-fold lower than that seen with radiation alone. Further, the shape of the cell survival curve observed following the combination therapy suggested that including combretastatin in the treatment had a major effect on the radiation-resistant hypoxic cell subpopulation associated with this tumor., Conclusion: The present results demonstrated that in the KHT sarcoma, combretastatin A-4 prodrug caused rapid vascular shutdown, a concentration-dependent direct cell killing, and effective enhancement of the antitumor effects of radiation therapy.

Purpose: One enzyme/prodrug combination that has the potential to be used in virally directed enzyme/prodrug therapy (VDEPT) is the obligate 2-electron reducing enzyme, DT-diaphorase (NQO1), with bioreductive agents such as EO9. The present studies were undertaken to determine if this enzyme, as well as the reporter molecule, green fluorescent protein (GFP), could be expressed from a single dicistronic unit under control of the CMV promoter in an adeno-associated virus (AAV) background., Methods: The human ovarian tumor cell line, SAU, and the mouse sarcoma cell line, KHT/iv, were studied due to their low level of NQO1 expression. These cells were transfected with pTRUF3-NQO1 using a liposome-mediated protocol., Results: The results indicate that this construct has the ability to increase the total protein level of NQO1 by 66-fold in SAU and 102-fold in KHT/iv after 24 h. Furthermore, the level of NQO1 activity in SAU increased from undetectable levels to approximately 200 nmol/min/mg, and the NQO1 activity in KHT/iv increased approximately 10-fold following transfection. Expression of the GFP reporter was readily detectable in both cell types using FACS analysis., Conclusions: Taken together, these results indicate that this proviral AAV vector plasmid will allow for the production of a recombinant AAV, which can coordinately express the enzyme NQO1 and the GFP reporter for use in vivo in VDEPT studies with various bioreductive agents which are substrates for NQO1.

Purpose: The chemosensitizing potential of the benzotriazine-N-oxide tirapazamine was determined in rodent mammary tumor cells grown as solid tumors., Materials and Methods: C3H/HeJ mice bearing i.m. transplanted 16C mammary carcinomas were treated with varying doses of either cisplatin alone or cisplatin in combination with a 0.27 mmol/kg dose of tirapazamine. Tumor response to single agent or combination therapy was assessed using an in situ tumor growth delay assay. Normal tissue toxicity resulting from the tirapazamine, cisplatin, or tirapazamine plus cisplatin was determined by measuring bone marrow stem cell (CFU-GM) toxicities and blood urea nitrogen (BUN) levels., Results: Tirapazamine itself had no measurable effect on the growth of this tumor. However, when administered from 3 h before to simultaneously with a single dose of cisplatin, the resultant tumor growth delay was significantly increased as compared to that seen with cisplatin alone. The administration of tirapazamine 3 h prior to a range of doses of cisplatin was found to result in a dose modifying factor (DMF) of approximately 1.7 in tumor response compared to cisplatin alone. Tirapazamine did demonstrate some hematologic toxicity on its own but it did not potentiate the toxicity of cisplatin when the two agents were administered in combination. BUN analysis showed that tirapazamine had little effect on BUN levels but did suppress the BUN values of mice treated with the combination of tirapazamine and 15 mg/kg cisplatin as compared to cisplatin alone., Conclusions: The present findings suggest that the addition of tirapazamine to cisplatin therapy may lead to a therapeutic benefit.

Purpose: To investigate whether electrode measurements of tumor oxygenation obtained under a range of different treatment conditions designed to alter the degree of tumor hypoxia could be correlated with estimates of radiobiological hypoxia measured under the same conditions., Methods and Materials: Experiments were performed in restrained, nonanesthetized, female C3H/He mice, which had approximately 0.5 g KHT sarcomas growing intramuscularly in the hind limbs. The treatments used to modify tumor oxygenation status included breathing gas mixtures of varying oxygen content, altering tumor blood flow, and shifting the hemoglobin oxygen dissociation curve. Radiobiological hypoxic fraction was estimated using the paired survival curve assay, while electrode measurements of tumor oxygenation were obtained with an Eppendorf histograph., Results: With the selected manipulations it was possible to vary the radiobiological hypoxic fraction in the tumors from approximately 1 to approximately 100% of the total viable cell population. Furthermore, these changes in radiation response were directly reflected in the changes in tumor oxygenation measurements made with the Eppendorf histograph., Conclusion: These findings suggest that in the KHT tumor model the Eppendorf electrode measurements could predict the response of the tumors to radiation as determined by the proportion of hypoxic cells.

Proliferating cells in tumors may be of considerable relevance in cancer therapy. Not only do such cells dictate the rate of tumor progression, but evidence exists that they may also play an important role in the diagnosis and prognosis of tumor regrowth. Consequently, the identification of this subset of cells in the overall neoplastic cell population is of considerable importance. The aim of the present investigations was to compare four flow cytometric methodologies commonly used to study cell proliferation. These included nuclear antigen Ki67 detection, acridine orange (AO) and bromodeoxyuridine (BrdUrd) staining, and percent S-phase determinations. Three human tumor cell lines (HEp3, A549, H226) were examined in various stages of growth. Further, a direct comparison was made of the proliferation activities of HEp3 cells grown in culture or as xenografts in nude mice. The results showed that of the techniques investigated, detection of the nuclear antigen Ki67 may be most useful for marking proliferating tumor cells and determining tumor growth fractions.

Background and Purpose: Nicotinamide is a radiation sensitizer currently undergoing clinical testing. This was an experimental study to determine the importance of drug dose and time interval between drug administration and irradiation for radiosensitization., Materials and Methods: Nicotinamide (50-500 mg/kg) was injected intraperitoneally into CDFI or C3H mice and drug plasma pharmacokinetics were determined by HPLC. Radiosensitization was measured in tumours and normal tissues after local irradiation. The tumours were a C3H mammary carcinoma, the KHT sarcoma and the SCCVII carcinoma. Tumour response was assessed using either growth delay (C3H) or clonogenic survival (KHT/SCCVII). Normal tissue toxicities evaluated included early responding skin (development of moist desquamation of the foot) and late responding bladder (reservoir function estimated by cystometry) and lung (ventilation rate measured by plethysmography)., Results: All nicotinamide peak plasma concentrations were seen within 30 min after injection. Irradiating tumours at peak times resulted in enhancement ratios (ERs) of 1.27 (C3H), 1.75 (KHT) and 1.45 (SCCVII) with high nicotinamide doses and 1.27 (C3H), 1.28 (KHT) and 1.36 (SCCVII) after giving clinically relevant doses (100-200 mg/kg). Lower ERs were observed when the time interval between drug injection and irradiation was increased beyond the peak time. Irradiating normal tissues at peak times after injecting 100-200 mg/kg nicotinamide gave ERs of 1.20 (skin), 0.90 (bladder) and 1.02 (lung)., Conclusions: Clinically achievable doses of nicotinamide will enhance tumour radiation damage while having minimal effects in normal tissues, but for the best tumour effect radiation should be given at the time of peak plasma drug concentrations.

Purpose: The aim of these studies was to determine whether chemotherapy-resistant tumor cell sublines derived from a single starting cell population with identical treatment protocols, have the same mechanism of resistance., Methods and Materials: Twelve cyclophosphamide-resistant sublines were derived from KHT-iv murine sarcoma cells by repeated exposures to 2, 4, or 8 microg/ml doses of 4-hydroperoxycyclophosphamide (4-OOHCP). To investigate possible mechanisms of resistance, glutathione (GSH) levels, glutathione S-transferase (GST) activity, and aldehyde dehydrogenase (ALDH) activity were determined. In addition, studies with the GSH depletor buthionine sulfoximine (BSO) and the ALDH inhibitor diethylamino-benzaldehyde (DEAB) were undertaken., Results: Resistant factors to 4-OOHCP, assessed at 10% clonogenic cell survival, ranged from 1.5-7.0 for the various cell lines. Crossresistance to melphalan and adriamycin also were commonly observed. Increased GSH levels, GST activity and ALDH activity were detected in the sublines but not all exhibited the same pattern of biochemical alterations. The response to GSH and ALDH inhibitors also varied among the sublines; the resistance being reversible in some cell lines but not others., Conclusion: The present results indicate that when resistant sublines are derived simultaneously from the same starting cell population, the observed mechanisms of resistance may not be the same in each of the variants. These findings support the hypothesis that preexisting cellular heterogeneity may affect mechanisms of acquired resistance.

A centrifugation assay was used to determine the effects of ionizing radiation on the adhesive interaction of A549 human lung adenocarcinoma tumor cells and human umbilical vein endothelial cells (HUVEC). The tumor cells were fluorescently labeled and divided into control (sham-irradiated) and irradiated groups. The irradiated groups were exposed to irradiation levels ranging from 5 to 20 Gy using a 137Cs source. A specified number of these A549 tumor cells were then delivered into each well of 96-well cell culture plates containing confluent monolayers of human umbilical cord vein endothelial cells (HUVEC), and were given time to adhere to the endothelial cells. The wells were then sealed and were exposed to an acceleration field varying from 1 to 42 g (0-500 rpm) for 10 min. Finally, the wells were drained, and the number of tumor cells adhering to the endothelial monolayer were counted using a fluorescent microscope system. Our results indicate that the irradiation of A549 tumor cells significantly increased their adhesive interaction with endothelial cells (number of adhering irradiated cells/number of adhering control cells = 1.0, 1.3, 1.9, 2.2 for 0, 5, 10, 20 Gy respectively). In contrast, when endothelial cells were irradiated, rather than tumor cells, adhesive interaction decreased with an increase in the radiation dose (irradiated/control = 1.0, 0.9, 0. 8, 0.5 for 0, 5, 10, 20 Gy respectively). Simultaneous irradiation of both the tumor cells and the endothelial cells did not alter their adhesive interaction significantly. These findings may have important implications for the metastatic ability of irradiated tumor cells.

While the potential importance of hypoxia in limiting the sensitivity of tumor cells to ionizing radiation has long been appreciated, methods for accurately quantifying the number of radiation-resistant hypoxic cells within tumors have been lacking. We have used the pentafluorinated derivative [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acet amide] of etanidazole (EF5), which binds selectively to hypoxic cells. The adducts formed between EF5 and cellular proteins in the hypoxic cells were detected using the specific monoclonal antibody (MAb), ELK3-51 conjugated to the flurochrome Cy3, and the number of hypoxic cells was quantified by flow cytometry. To verify the validity of this technique for the detection of hypoxic cells, mice bearing KHT sarcomas were treated with various agents to alter tumor oxygenation and hence vary the fraction of radiobiologically hypoxic tumor cells. The percentage of EF5 binding cells was then compared directly with the clonogenic survival of the tumor cells following radiation treatment under the various pretreatment conditions. The results showed that allowing the mice to breathe carbogen (5% CO2/95% O2) prior to irradiation reduced clonogenic cell survival approx. 6-fold and led to an absence of cells binding high levels of EF5. In contrast, pretreating the tumor-bearing animals with either hydralazine, which decreased tumor blood flow, or phenylhydrazine hydrochloride, which made the mice anemic, increased tumor cell survival following irradiation 2- to 4-fold, indicative of an increase in the fraction of hypoxic tumor cells. EF5 measurements made under identical conditions illustrated a shift in the cells in the tumor to high EF5 binding. Our results demonstrate that flow cytometric measurement by fluorescent MAb binding to EF5 adducts may relate directly to radiobiological hypoxia in KHT tumors measured by conventional methods.

The potential of tirapazamine to enhance the in situ efficacy of the anti-cancer drug cyclophosphamide (CP) was evaluated in two rodent tumours (KHT sarcoma, 16C mammary carcinoma) and one human ovarian tumour xenograft (MLS) using end points of in vivo to in vitro cell survival or in situ tumour growth delay. For comparison, bone marrow toxicity under similar treatment conditions was determined using a CFU-GM stem cell survival assay. The results showed that a 0.27 mmol kg-1 dose of tirapazamine alone had little anti-tumour effect. However, when given prior to a range of CP doses, tirapazamine increased the efficacy of this chemotherapeutic agent in all three tumour models investigated. CFU-GM stem cell toxicity, assessed under similar treatment conditions, demonstrated that this dose of tirapazamine (1) led to some bone marrow toxicity on its own; and (2) increased the toxicity of CP beyond that seen with CP alone. The present findings demonstrate that the bioreductive agent tirapazamine can potentiate the in situ anti-tumour efficacy of the alkylating agent CP. However, in the preclinical models investigated, the enhanced anti-tumour effect did not translate into a therapeutic benefit because a similar increase in bone marrow toxicity also resulted from this treatment combination.

The impact of neck stage (N stage) on local control after treatment for head and neck cancer is controversial. This article reviews the pertinent literature. Based on this review, the authors conclude that although N stage may be inversely related to local control, the relationship is relatively weak. Hence, current data are insufficient to justify altering treatment of the primary lesion based solely on N stage.

Purpose: Numerous previous studies have attempted to relate the radiobiological hypoxic fraction (HF) to direct measures of tumor oxygenation such as HbO2 saturations, tumor pO2 levels, or hypoxic cell labeling. Although correlations have been found within tumor lines, no overall relationships were seen across tumor lines. The current objective was to examine the effect on HF of changes in the fractions of the oxygenated and anoxic tumor cells that remain clonogenic., Methods and Materials: A mathematical model was developed that relates the HF to direct measures of tumor oxygenation. The primary assumptions were that: (a) the tumor is divided into distinct compartments of either fully oxygenated or fully anoxic cells, and (b) the survival of the oxygenated cells is negligible compared to that of the anoxic cells. Based on these assumptions, the HF is plotted as a function of the fractions of clonogenic or nonclonogenic, and oxygenated or anoxic cells., Results: If all cells are clonogenic, then the HF equals the fraction of anoxic cells. If a higher fraction of anoxic than oxygenated cells are nonclonogenic, then the HF will be overestimated by the fraction of the tumor measured to be anoxic using direct measuring techniques. If a higher fraction of the oxygenated than anoxic cells are nonclonogenic, the HF will be underestimated by the fraction of anoxic cells., Conclusion: Correlations between the HF and direct measures of tumor oxygenation have been described within tumor lines evaluated under different physiological condition. However, such relationships can be totally unpredictable between different tumors if the fraction of the anoxic cells that is clonogenic varies substantially. Clearly, if tumor anoxia cannot be detected using direct measures, this is an accurate indication that the tumor is well oxygenated. When tumor anoxia is present, however, the conclusions are ambiguous. Even when a small fraction of the tumor is measured as anoxic, direct measures of tumor oxygenation may not be representative of the HF if a substantial proportion of nonclonogenic cells is present.

The relationship between DNA damage removal and cellular sensitivity to the alkylating agent cyclophosphamide (CP), currently one of the most effective drugs in the treatment of human tumors, has not been well established. In the present study, we have analyzed drug-induced DNA cross-link persistence in the CP-resistant subline KHT-rep/iv and its sensitive counterpart KHT-iv. The alkaline elution technique was used to detect both DNA-DNA and DNA-protein cross-links formed following exposure to clinically achievable CP concentrations. In addition, the role of glutathione (GSH) in the cross-link persistence was analyzed. The KHT-rep/iv cell line was chosen for study because compared to the parent KHT-iv line, this cell line demonstrates 2-3-fold increased GSH levels, and resistance to CP is known to be mediated by thiols. Following exposure to 4-hydroperoxy-cyclophosphamide (4-OOHCP), an active form of CP, the KHT-rep/iv cells exhibited fewer total cross-links and a shift in the time of maximal cross-link formation (6.2 at 2 h versus 2.9 at 4 h for the parent and resistant lines, respectively). When the GSH content of the resistant cells was reduced to equal that of the parent cell line prior to the alkaline elution assay, both the total number and time course of development of cross-links were similar between the two lines. Also, when the cells were exposed to mechlorethamine, an alkylating agent which forms cross-links at a much faster rate than 4-OOHCP, the time course of cross-link formation after mechlorethamine exposure was similar between the two sublines, with maximal cross-link formation occurring after a 1-h incubation. The present findings suggest that (a) the increased GSH levels in KHT-rcp/iv cells may be responsible for the delay in maximal cross-link development after treatment with 4-OOHCP, possibly by binding with the monoadduct produced by the drug; and (b) the faster reaction rate of mechlorethamine cross-link formation may prevent the tripeptide from binding to the monoadduct. These results are consistent with the hypothesis that GSH may "quench" the formation of cross-links following 4-OOHCP exposure.

The present study investigates the correlation between tumor oxygen availability and radiosensitivity following oxygen manipulation. Previous work has shown that tumors may contain both diffusion- and perfusion-limited hypoxic cells. Recently, the combination of nicotinamide (NIC) administration plus carbogen breathing has been proposed as a means of targeting both hypoxic cell subpopulations. Intravascular HbO2 saturations were measured for KHT murine sarcomas following either NIC, carbogen breathing, or the combination, and compared with determinations of tumor cell survival under matched conditions. The percentage of vessels > or = 25% HbO2 increased significantly for both the carbogen and NIC-carbogen combination, while remaining unchanged from controls following NIC. These findings contrast with the survival data, where all treatments showed identical cell survival. A possible explanation is that different proportions of clonogenic versus nonclonogenic cells may be oxygenated by the alternative treatments. Thus direct determinations of alterations in tumor oxygenation may not reflect corresponding changes in radiosensitivity.

The cytotoxicity and chemosensitizing potential of the nitrohetercyclic agent RB 6145 and its R enantiomer PD 144872 were determined in rodent tumor cells grown in tissue culture or as solid tumors. Using a clonogenic cell survival assay the degree of selective cytotoxicity of these bioreductive drugs was first determined in KHT/iv cells. Cells treated under hypoxic conditions were observed to be approximately 50-80-fold more susceptible to the action of RB 6145 or PD 144872 than were cells exposed under air. To assess the in vitro chemosensitizing potential of RB 6145 and PD 144872, doses of these agents which led to survival values between 0.5 and 1.0 under hypoxic conditions were administered, and were then combined concomitantly with variable doses of the nitrosourea CCNU. Exposure to the nitrosourea was for 1 h. The results showed that inclusion of either sensitizer enhanced the cell killing of the chemotherapeutic agent 2.4-2.6-fold. Subsequent experiments evaluated the therapeutic benefit of combining these bioreductive agents with CCNU in KHT sarcoma-bearing C3H/HeJ mice. When given at times ranging from 90 min before to 60 min after CCNU exposure, these bioreductive drugs increased the tumoricidal activity of the chemotherapeutic agent. Complete dose response curves combining RB 6145 and PD 144872 and a range of CCNU doses also were evaluated. The sensitizers (0.75 mmol/kg) were administered 30 min prior to the chemotherapeutic agent and survival of clonogenic tumor cells 22-24 h after treatment was used to assay tumor response.(ABSTRACT TRUNCATED AT 250 WORDS)

Purpose: The definition and quantitation of radiation-induced morphologic alterations in murine lungs is presented., Methods and Materials: The extent of injury to the lung, which is the dose-limiting organ in the thorax, may be reduced by fractionating the total radiation exposure to permit partial repair of radiation-induced damage between fraction administration and also to permit a larger total exposure to be administered. We previously reported that, following fractionated radiation exposures, as the dose/fraction decreases, the total dose to reach an isoeffect increases, with an alpha/beta ratio of 3.2 and 3.0 for breathing rates and lethality, respectively. In the present report, we provide comparative morphologic evaluation of the effects of weekly fractionated (three doses at one dose/week), daily fractionated (15 doses at 1/diem), and hyperfractionated (30 doses at 2/diem) radiation exposures. The doses administered within each group were uniform. To determine morphologic alterations, LAF1 mice were irradiated with 3, 15, and 30 fractions delivered in 19 days overall treatment time. In the hyperfractionation schedule, the two fractions per day were separated by a 6-h time interval. Total doses were as follows: 15-21 Gy for weekly fractionation, 30-41.5 Gy for daily fractionation, and 30-49.5 Gy for hyperfractionated schedules. Lung tissue, recovered either 24 or 72 weeks following the final exposure, was evaluated by transmission and scanning electron microscopy and light microscopy., Results: Using a series of morphologic parameters, a total dose of 15 Gy in the weekly treatment schedule was found to be equivalent to a total dose of 30 Gy in the daily fractionation schedule and 37 Gy in the hyperfractionated treatment regimen at 24 weeks postirradiation. Measured at 72 weeks postirradiation, total exposures of 15 Gy on the weekly fractionation regimen corresponded to total exposures of approximately 30 Gy in both the daily fractionated and hyperfractionated regimens. Morphological damage was not uniform throughout the exposed lung and tended to be concentrated in lobes or portions of lobes., Conclusions: In the three fractionation regimens studied, there is progressive sparing of the lung with increased fractionation (i.e., weekly < daily < twice daily) during the pneumonitic stage (24 weeks postirradiation). Both daily and twice daily fractionations provide increased sparing over weekly fractionation during the fibrotic stages (72 weeks postirradiation), but were not markedly different from each other (i.e., weekly < daily = twice daily).

Purpose: To determine whether electrode measurements of tumor oxygenation, made in a variety of murine tumor models, correlate with estimates of radiobiological hypoxia in the same tumor systems., Methods and Materials: The tumor models used were a C3H mammary carcinoma grown in the feet of CDF1 mice; the SCCVII, KHT and RIF-1 tumors grown in the feet or flanks of C3H/Km mice; and the CaNT and SaF tumors grown on the backs of CBA mice. All treatments were performed when tumors were about 200 mm3 in size. Radiobiological hypoxic fractions were determined using either a paired survival curve assay, with survival measured 0-24 h after irradiation, or using a clamped tumor control assay, with percent local tumor control estimated 90 days after treatment. Measurements of tumor oxygen partial pressure (pO2) distributions were performed using Eppendorf oxygen electrodes., Results: The hypoxic fractions determined from the radiation response data were about 1% in RIF-1 and SCCVII, 12% in C3H and KHT, 28% in CaNT and up to 38% in SaF tumors. When this data was compared with the tumor oxygenation measurements it was found that as hypoxic fraction increased the mean, median, and the percentage of pO2 values < or = 5 mmHg showed a trend towards poorer oxygenation status. However, none of these pO2 changes were significantly correlated with hypoxia. Moreover, the pO2 values < or = 2.5 mmHg indicated an improvement in oxygen status with increasing hypoxic fraction., Conclusion: Electrode measurements of tumor oxygenation alone may, therefore, not be a good indicator of tumor hypoxia across different tumor cell lines.

Purpose: To determine the cytotoxicity and chemosensitizing potential of RG 6145 in mouse KHT/iv and human A549 tumor cells., Methods and Materials: RSU 1069, the lead compound in a series of nitroimidazoles containing an alkylating aziridine function, has been shown to possess a high degree of selective cytotoxicity for hypoxic cells in addition to being a potent sensitizer of radiation and chemotherapy. Unfortunately, preliminary clinical studies have revealed a dose-limiting gastrointestinal toxicity for RSU 1069. Recently RB 6145, the ring-opened analogue of RSU 1069, has been found to be less emetic than RSU 1069. In the present studies, we assessed both the differential hypoxic cell cytotoxicity of RB 6145 and its chemosensitizing potential when combined concomitantly with variable doses of the activated form of cyclophosphamide (4-hydroperoxy-cyclophosphamide, 4-OOH-CP) or the nitrosourea CCNU., Results: As we had observed previously for RSU 1069, RB 6145 was found to be less cytotoxic to human than rodent tumor cells. In addition, the degree of selective cytotoxicity toward hypoxic cells was (a) less in A549 than in KHT/iv cells (factor of 9 vs. 80) but (b) comparable to that seen with RSU 1069. For both cell lines, inclusion of the sensitizer enhanced the cell killing of the chemotherapeutic agent 4-OOH-CP by a factor of approximately 1.5-1.7-fold. When combined with CCNU, RB 6145 increased the killing of A549 cells approximately 1.8-fold. Similar hypoxic cell preferential cytotoxicity and enhancement in anti-tumor treatment efficacy were seen when A549 cells were exposed to the R enantiomer of RB 6145 (PD 144872) either alone or in combination with CCNU., Conclusion: These data support the notion that further consideration should be given to the clinical application of these bioreductive agents.

Purpose: The aim of these studies was to provide evidence supporting thiol-related alterations as a mechanism of resistance in the cyclophosphamide (CP)-resistant subline, KHT-rcp/iv, and to elucidate the mechanism by which the observed elevation in GSH is achieved., Methods and Materials: The response of the parental and resistant cells to three anticancer agents (adriamycin, cisplatin, and ionizing radiation), previously associated with thiol-related resistance, was determined using a clonogenic cell survival assay. Further, using spectrophotometric techniques, the activities of several glutathione (GSH)-related enzymes; glutathione S-transferase (GST), glutathione peroxidase (GSH-PX), glutathione reductase (GSR), gamma-glutamylcysteine synthetase (GCS) and gamma-glutamyl transpeptidase (GGT) were also measured., Results: The studies indicated that the CP-resistant cells were cross-resistant to adriamycin, cisplatin and ionizing radiation. In addition, the KHT-rcp/iv cells exhibited 2-3-fold increases in GST, GSR and GCS activity. Since GCS catalyzes the rate-limiting step of GSH synthesis, increased activity of this enzyme may be one mechanism by which intracellular GSH levels may be upregulated in treatment refractory tumor cells. No significant difference in GSH-PX activity was observed between the two cell lines, while GGT activity could not be detected in either cell line., Conclusion: Taken together, these results suggest that thiols could play a significant role in the acquired resistance and cross-resistance to chemotherapeutic agents. Modulation of these enzymes may be an effective means of enhancing anticancer therapies.

Preclinical investigations have demonstrated that both diffusion- and perfusion-limited hypoxic cells may exist in tumors. One approach to target such hypoxic cell subpopulations is through the combined application of nicotinamide (NIC) administration and carbogen (5% CO2:95% O2) breathing. Because carbogen pre-irradiation breathing time (PIBT) can markedly influence the radiosensitizing effectiveness of this gas mixture, in the present experiments the effect of localized radiation on the transplantable KHT sarcoma was investigated in mice receiving NIC while breathing carbogen for various periods of time. When mice were given carbogen prior to radiation therapy, there was a minimum in tumor cell survival for PIBTs of 2-30 min. Longer PIBTs led to a loss of the radiosensitizing effect. NIC, administered as a 1000-mg/kg dose, effectively enhanced radiation cell killing in this tumor if given 45 min to 2 h prior to radiotherapy. In experiments in which either agent was combined on its own under optimum conditions (carbogen, 10 min PIBT; or NIC, 1000 mg/kg 2 h prior to irradiation), with a range of single doses of radiation, the results showed an enhancement ratio of approximately 1.9 as determined from the ratio of the slopes of the cell survival curves obtained in the absence or presence of the radiation sensitizer. This sensitizing effect could not be increased further when NIC and carbogen breathing were combined under optimum conditions.

The ability of nicotinamide and carbogen breathing to improve the radiation response of a C3H mammary carcinoma by reducing both acute and chronic hypoxia was investigated. Using a tumour growth delay assay the response of 200 mm3 foot tumours to local irradiation was found to be increased by either injecting nicotinamide (100-1,000 mg/kg) 20 min prior to irradiation, or by allowing mice to breathe carbogen for 10 min before and during the radiation treatment. The greatest radiosensitization occurred when nicotinamide and carbogen were combined. With a histological fluorescent staining technique nicotinamide was shown to prevent transient stoppages in microregional blood flow, and also appeared to improve tumour oxygenation as measured with an Eppendorf oxygen electrode, both effects being consistent with its ability to decrease perfusion limited acute hypoxia. Carbogen had no effect on vessel closure, but it significantly improved tumour oxygenation, which was indicative of it reducing diffusion limited chronic hypoxia.

The effect of administering the thiol modulating agent buthionine sulfoximine (BSO) in conjunction with alkylating chemotherapy was investigated in vivo in the mouse KHT sarcomas and bone marrow stem cells. Tumour response to treatment was assessed by an in vivo to in vitro excision assay and bone marrow survival was determined in vitro by CFU-GM. Glutathione (GSH) depletion and recovery kinetics were determined at various times after treatment using high performance liquid chromatography (HPLC) techniques. Following a single 2.5 mmol kg-1 dose of BSO, tumour GSH reached a nadir of approximately 40% of control 12-16 h after treatment. Bone marrow GSH was depleted to approximately 45% of control 4-8 h after treatment but recovered to normal by 16 h. When a range of doses of CCNU, mitomycin C, cyclophosphamide or melphalan (MEL) were given 16 h after mice were exposed to a 2.5 mmol kg-1 dose of BSO, only the antitumour efficacy of MEL was effectively enhanced (by a factor of approximately 1.4). This BSO-MEL combination appeared to be selective for the tumour as the bone marrow toxicity was not increased beyond that seen for MEL alone. Since increasing the administered dose of BSO neither increased the extent of thiol depletion in the tumour nor enhanced the antitumour efficacy of MEL, three other protocols for delivering the thiol depletor were explored. BSO was given either as multiple 2.5 mmol kg-1 doses administered at 6 or 16 h intervals or continuously at a concentration of 30 mM supplied in the animals' drinking water. Both multi-dose BSO pretreatments were found to increase both the antitumour efficacy and normal tissue toxicity of MEL such that no advantage compared to the single dose combination was achieved. In contrast, maintaining the thiol depletor in the drinking water led to an approximately 1.7-fold increase in the antitumour efficacy of MEL without any corresponding increase in bone marrow stem cell toxicity. For the various pretreatment strategies it was possible, in all cases, to account for the presence or absence of a net therapeutic benefit on the basis of the tumour and bone marrow GSH depletion and recovery kinetics.

The combination of nicotinamide and carbogen breathing is awaiting clinical evaluation as a strategy to overcome tumour hypoxia and thus enhance radiation response. We have continued our evaluation of this approach in the murine SCCVII tumour with the aim of determining the importance of nicotinamide dose and the pre-irradiation breathing time (PIBT) for carbogen. For carbogen breathing alone maximal enhancement of radiation response was observed with PIBT's of between 5 and 30 min. When nicotinamide (1,000 mg kg-1 IP) was administered 60 min prior to irradiation little or no variation in radiation response was observed for all the PIBT's examined (5-90 min). Indeed at all PIBT's the cell survival obtained for the carbogen nicotinamide and radiation combination was indistinguishable from that expected for a fully aerobic response. For PIBT's of 15 and 60 min we examined the influence of nicotinamide doses between 50 and 1,000 mg kg-1. Significant radiosensitizing effects were observed for all nicotinamide doses tested above 50 mg kg-1. Moreover for doses of 250 mg kg-1 and above the cell survival data was consistent with that expected for a fully aerobic response. No additional benefit accrued from raising the nicotinamide dose above 250 mg kg-1. These results indicate that significant radiosensitization may be expected even with clinically achievable nicotinamide doses when it is combined with carbogen breathing. Furthermore, the use of nicotinamide may reduce the critical importance of PIBT on the radiosensitization observed with carbogen.

A disulfide, n-butyl 2-imidazolyl disulfide (III-2), recently reported to be more toxic to EMT6 tumor cells under hypoxia has also been shown to be preferentially toxic to KHT/iv cells under hypoxia with an IC90 value 3-fold lower versus that measured in air. The IC90 values for both cell lines were markedly affected when the pHe was decreased. Uptake studies revealed that the disulfide is metabolized at the cell membrane with only one of the metabolites, n-butanethiol, being taken up into the cell. There were no differences in the amount of uptake under oxia or hypoxia. Investigation of potential membrane damage revealed that III-2 was a mixed inhibitor of the enzyme Na+,K(+)-ATPase, from isolated plasma membrane. DNA damage, when examined using plasmid DNA in the absence or presence of glutathione, was absent. Minor single-strand breaks to EMT6 DNA could only be observed following exposure to III-2 at the highest concentrations tested, with no differences observed between treatments in air or under hypoxia.

The cytotoxicity and chemosensitizing potential of four dual function nitrofurans was determined in human HT-29 multi-cell spheroids and rodent KHT sarcoma solid tumors. Spheroids were treated with a range of doses of the bioreductive drugs for a period of up to 48 h and the extent of cell kill was assessed at various times after treatment. Cytotoxicity was determined using a clonogenic cell-survival assay. The results demonstrated that two of the nitrofurans were even more toxic to spheroid cells than was the potent bioreductive nitroimidazole aziridine RSU 1069. The dose of the nitrofuran which, after a 24-h exposure, led to a survival value between 0.5 and 1.0, then was chosen for subsequent studies aimed at assessing the ability of these agents to potentiate the efficacy of the nitrosourea CCNU. Exposure to this chemotherapeutic agent was for a period of 1 h. The results indicated that all four dual function nitrofurans enhanced the cell killing of the conventional chemotherapeutic agent by factors ranging from 1.1 to 1.7. Subsequent studies evaluated the therapeutic benefit of combining these bioreductive agents and CCNU in KHT sarcoma-bearing C3H/HeJ mice. The nitrofurans were administered i.p. 0.5 h prior to the chemotherapy and tumor response was assessed by measuring the survival of clonogenic KHT cells 22-24 h after treatment. Normal tissue toxicity was determined using a bone marrow stem cell (CFU-GM) assay. Combining these bioreductive agents with CCNU increased the tumor cell kill by factors of 1.2 to 1.7.(ABSTRACT TRUNCATED AT 250 WORDS)

Centrifugal elutriation was used to obtain synchronized cell populations in various cell cycle phases without prior growth-perturbing manipulation. Treatment of these subpopulations with novobiocin (NOVO), a putative inhibitor of the mammalian topoisomerase II enzyme, revealed a unique cell cycle phase-dependent cytotoxicity for this agent. At a concentration of 0.3 mM, NOVO was cytotoxic only to a specific cell subpopulation in the G1-S phase boundary. Cells in other cell cycle phases were completely unaffected. Additionally, S and G2M phase cells progressed through the cell cycle relatively unaffected by NOVO but were blocked at the G1-S boundary. NOVO treatment protected tumor cells from Adriamycin (ADR)-induced lethality but sensitized them to the toxic action of 4-hydroperoxycyclophosphamide, and alkylating agent. These opposing effects of NOVO were demonstrated in all of the four tumor cell lines investigated: A431 and HEp3 (derived from human squamous cell carcinomas); MLS, a human ovarian cancer cell line; and a Chinese hamster ovary cell line. The degree of protection against ADR was the greatest for S-phase cells, intermediate for cells in early G1 and M phases, and the least for late G1 cells. This cell cycle-dependent protection by NOVO, which is identical to the cell cycle-dependent cytotoxicity of ADR, was consistent with the idea that NOVO interfered directly with the cell-killing mechanism of ADR. In contrast, even though the cytotoxic activity of 4-hydroperoxycyclophosphamide exhibited significant cell cycle dependency, NOVO enhanced 4-hydroperoxycyclophosphamide lethality equally for all cell cycle phases.

The effects of etoposide (VP-16) and teniposide (VM-26) have been evaluated in human epidermoid carcinoma cells (A431, ME180 and HEp3) grown as exponential and plateau phase cultures. A significant increase in resistance to both these chemotherapeutic agents was observed in unfed plateau compared with exponential phase cells. The large differences in cell killing could not be explained by cell cycle specific toxicities resulting from variations in the cell cycle distributions. Rather the differences in the treatment efficacies probably reflect the 5- to 15-fold increase in the proportion of quiescent cells measured in the plateau phase cultures. These findings suggest that non-proliferating cells in tumors may be preferentially spared in treatments utilizing VP-16 and VM-26.

Acquired drug resistance is a major obstacle in the successful treatment of cancer by chemotherapy. To study mechanisms of resistance to alkylating agents, we have derived an in vitro subline of the murine KHT-iv sarcoma, KHT-rcp/iv, that has acquired resistance to cyclophosphamide. Compared to the parental line, KHT-rcp/iv exhibited an approximately 2.8-fold resistance, determined from the ratio of the slopes of the resistant to the parental survival curve. The subline also was found to be cross-resistant to other alkylating agents, as well as to the epipodophyllotoxin, etoposide, a structurally unrelated compound (resistance factor of approximately 2.2). In view of this broad spectrum of cross-resistance observed, we investigated the possibility that glutathione (GSH), an abundant tripeptide implicated in the detoxification of electrophilic species, may be involved. GSH levels were increased two-fold in the resistant line (16.0 +/- 1.0 fmol/cell) as compared to the parent line (8.1 +/- 0.7 fmol/cell). Also, when the GSH content was reduced to that of the parent with the addition of the thiol depletor buthionine sulfoxinine (BSO), the sensitivity of the subline to cyclophosphamide, as well as to the other agents, was enhanced so that it equaled that of the parent cell line. These results imply that enhanced GSH levels may be a major mechanism of resistance in the KHT-rcp/iv cells. Our findings further suggest that thiol manipulation may prove useful in overcoming this type of pleiotropic drug resistance.