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This article details the journey of the Colorado State University (CSU) Libraries to sever their ties with the Swets subscription agency. Although the CSU Libraries had not participated in Swets early pre-payment discount program, the CSU account with Swets involved over $1M in subscriptions. As a result of the bankruptcy, the Libraries methodically performed an elaborate number of steps to divest themselves of their former agreement with Swets and to ensure continual delivery of the relevant subscription content from publishers. Recounted are the early stirrings of trouble; the policy legacy of the previous bankruptcy of a major subscription agent more than a decade before (the Rowe.com/Faxon agency collapse); the steps undertaken by the university legal counsel, the library administration and the Acquisitions and Metadata Services Department to disentangle from the Swets agreement and account; the workflow for set-up of new accounts to achieve fairly unbroken subscription service for affected journal ti...

We have collected lab notebooks from Rod Wither's many years of experimentation, from laboratories in Houston and Los Angeles, as well as from several of his collaborators in the USA and overseas. The contents have been digitized, and in this note we explain the mechanism that has been set up to make the 'Withers Archive' available online.

We describe a case of a 24-year-old male presenting urgently with a juvenile nasopharyngeal angiofibroma (JNA) with difficulty breathing, inability to swallow, and respiratory distress following throat swelling. The swelling was reduced with administration of dexamethasone and the JNA was surgically resected within 48 hours. This presentation was atypical given the acuity of presentation and the patient’s older age.

Purpose To test whether a cyclooxygenase-2 inhibitor (celecoxib) could reduce mortality resulting from radiation-induced pneumonitis. Methods and Materials Celecoxib was given to mice twice daily for 40 consecutive days starting on the day of local thoracic irradiation (LTI) or 40 or 80 days later. C3Hf/KamLaw mice were observed for morbidity, and time to death was determined. Results were analyzed using the Cox proportional hazards model. Results Timing of celecoxib relative to LTI determined efficacy. A significant reduction in time to death was achieved only when celecoxib was started 80 days after LTI, corresponding to the time when pneumonitis is expressed. For these mice the reduction in mortality was quantified as a hazard ratio for mortality of treated vs untreated of 0.36 (95% confidence interval [CI] 0.24-0.53), thus significantly less than 1.0. Correspondingly, the median lethal dose for treated mice (12.9 Gy; 95% CI 12.55-13.25 Gy) was significantly ( P =.026) higher than for untreated mice (12.4 Gy; 95% CI 12.2-12.65 Gy). Conclusions Celecoxib significantly reduced lung toxicity when administered months after LTI when the deleterious effects of radiation were expressed. The schedule-dependent reduction in fatal pneumonitis suggests that celecoxib could be clinically useful by reintroduction of treatment months after completion of radiation therapy. These findings may be important for designing clinical trials using cyclooxygenase-2 inhibitors to treat radiation-induced lung toxicity as a complement to concurrent radiation therapy of lung cancers.

The Metadata Best Practices Task Force (MBPTF) at the Colorado State University (CSU) Libraries developed a core set of metadata elements and an accompanying data dictionary to facilitate a coordinated metadata management approach for a central digital repository of diverse digital objects. This article describes the rationale for the Task Force and the process used for its work following a look at the background of digitization and past metadata practices at CSU. The article includes a literature review on institutional metadata projects and examples, and it ends with a description of the Task Force's ongoing work and plans for future assessment.

Poly(ADP-ribose) synthetase inhibitor, INO-1001, is known to sensitize cells to radiation in vitro by inhibiting the repair of DNA damage. Recent evidence has suggested that PARP inhibition may also be a way of selectively targeting p53 deficient cancer cells. The present study tested INO-1001 for its in vivo effect on the chemoresponse of two p53 deficient tumors, human breast cancer MDA-MB-231 and murine mammary carcinoma MCa-K. Doxorubicin was used as the DNA damaging agent and tumor growth delay assay was used as the endpoint. Results showed that INO-1001 was highly effective in enhancing the anti-tumor effects of Doxorubicin for both MDA-MB-231 (EF = 1.88) and MCa-K (EF = 1.64). We conclude that PARP inhibitor INO-1001 has high potential for enhancing the anti-tumor effects of chemotherapy agents such as Doxorubicin against p53 deficient breast cancer.

Purpose: 130-nm albumin–bound paclitaxel (nab-paclitaxel) is a novel solvent-free albumin-bound paclitaxel, designed to avoid solvent-related toxicity. Nab-paclitaxel has been successfully introduced into the clinic but its radiation-enhancing potential has not yet been evaluated. We conducted a preclinical evaluation of the radiation-modulating effects of nab-paclitaxel in tumor and normal tissues. Experimental Design: Mice bearing syngeneic ovarian or mammary carcinomas were treated with nab-paclitaxel, radiation, or combination of both. Nab-paclitaxel was administered at 90 mg/kg, 1.5 times the maximum tolerated dose for solvent-based paclitaxel. End points were antitumor efficacy (growth delay, radiocurability, and cellular effects) and normal tissue toxicity (gut and skin). Results: Nab-paclitaxel showed single-agent antitumor efficacy against both tumor types and acted as a radiosensitizer. Combined with radiation, nab-paclitaxel produced supra-additive effects when given before radiation. Nab-paclitaxel significantly increased radiocurability by reducing the dose yielding 50% tumor cure (TCD50) from 54.3 to 35.2 Gy. Tumor histology following nab-paclitaxel treatment was characterized by pronounced necrotic and apoptotic cell death and mitotic arrest. Nab-paclitaxel did not increase normal tissue radioresponse. Conclusions: Nab-paclitaxel exhibited strong antitumor efficacy against both tumors as a single agent and it improved radiotherapy in a supra-additive manner. These improved effects were achieved without increased normal tissue toxicity to either rapidly or slowly proliferating normal tissues although the drug dose was 1.5 times higher than the maximum tolerated dose of solvent-based paclitaxel. These preclinical findings show that combining nab-paclitaxel with radiotherapy would improve the outcome of taxane-based chemoradiotherapy. This novel taxane is thus a good candidate for testing in clinical chemoradiotherapy trials.

Purpose: Clinical trials of antiangiogenic agents used alone for advanced malignancy have been disappointing but preclinical studies suggest that the addition of radiation therapy could improve antitumor efficacy. To test the hypothesis that antiangiogenic therapy combined with radiation therapy can overcome the limitations of antiangiogenic monotherapy, we studied the effects of endostatin combined with radiation on the growth and vascularization of A431 human epidermoid carcinomas growing intramuscularly in the legs of mice. Methods and Materials: Mice with established A431 human epidermoid leg tumors were treated with radiation, endostatin, both radiation and endostatin, or vehicle control. The experiment was repeated and mice from each group were killed at 2, 7, and 10 days after irradiation so that tumor tissue could be obtained to further analyze the kinetics of the antitumor, antivascular, and antiangiogenic response to therapy. Results: Endostatin enhanced the antitumor effects of radiation, and prolonged disease-free survival was observed in the combined treatment group. Endothelial cell proliferation was increased in tumors after irradiation but was blocked by the concurrent administration of endostatin, and the combination of endostatin with radiation enhanced endothelial cell apoptosis within 48 h after irradiation. Expression of vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 were increased in tumors after irradiation, and this increase was blocked by concurrent administration of endostatin. Conclusion: These data indicate that endostatin can block tumor revascularization after radiation therapy and thereby augment radioresponse.

Purpose Recently we reported that inhibition of cyclin-dependent kinases (cdks) by flavopiridol enhanced the radiation response of murine ovarian carcinoma cells in culture. The purpose of this investigation was to extend these studies to in vivo tumor models and test whether flavopiridol increases the therapeutic ratio of radiotherapy. Methods and materials Three transplantable syngeneic mouse tumors were used: mammary carcinoma (MCa-29), ovarian carcinoma (OCa-I), and a lymphoma (Ly-TH). Tumor treatment endpoints included growth delay, cure, and spontaneous lung metastases (OCa-I tumor). The normal tissue endpoint was survival of jejunal crypt cells quantified microscopically. A range of flavopiridol doses from 0.625 to 5.0 mg/kg were given systemically once or twice daily over 5, 10, or 20 days. Combined therapy flavopiridol treatments were initiated either several days before or shortly after the start of single dose or daily fractionated radiotherapy. Results The major findings of this study are that all three tumors treated with flavopiridol alone responded by tumor growth delay. Two of the tumors (MCa-29 and Ly-TH) responded in a schedule-dependent manner with larger radiation enhancement factors when flavopiridol treatment was started a few hours after irradiation (radioenhancement factors [EF] Ly-TH = 2.04, EF MCa-29 = 1.50 for single dose irradiation). When combined with fractionated irradiation (2.6 Gy daily for 10 or 20 days), flavopiridol enhanced the response of the MCa-29 tumor by a factor of 1.25–1.46. A fractional radiation dose of 6 Gy in combination with flavopiridol produced a 62.5% cure rate compared with 25% tumor cure for radiation alone. A novel finding of this study was the demonstration of antimetastatic activity of flavopiridol in addition to its effect on the local primary tumor. Both the incidence and absolute number of lung metastasis were reduced when flavopiridol followed surgical removal of the large (10 mm) primary leg tumor. The normal jejunum treated with flavopiridol and radiation responded in a schedule independent manner and the degree of radioenhancement (EF, 1.05–1.06) was much less than for any of the tumors studied. Conclusions Therapeutic gain was achieved when flavopiridol treatment was initiated either before or after the start of radiotherapy. Flavopiridol shows promising clinical potential administered alone or in combination with other cytotoxic agents, including both chemotherapy and radiotherapy.

Cyclooxygenase-2 (COX-2) is an enzyme expressed primarily in pathologic states, such as inflammatory disorders and cancer, where it mediates prostaglandin production. Its overexpression is associated with more aggressive biologic tumor behavior and adverse patient outcome. Increasing evidence shows that agents that selectively inhibit COX-2 enhance tumor response to radiation or chemotherapeutic agents. This article gives an overview of some of this evidence. In addition, we describe new results showing that celecoxib, a selective COX-2 inhibitor, enhanced response of A431 human tumor xenografts in nude mice to radiation by an enhancement factor (EF) of 1.43 and to the chemotherapeutic agent docetaxel by an EF of 2.07. Celecoxib also enhanced tumor response when added to the combined docetaxel plus radiation treatment (EF = 2.13). Further experiments showed that selective COX-2 inhibitors enhanced tumor cell sensitivity to ionizing radiation, involving inhibition of cellular repair from radiation damage and cell cycle redistribution as mechanisms for some cell types. The results show that selective COX-2 inhibitors have the potential to improve tumor radiotherapy or radiochemotherapy, and this therapeutic strategy is currently under clinical testing.

Purpose The epidermal growth factor (EGF) receptor is frequently overexpressed in malignant tumors, and its level is correlated with increased cellular resistance to ionizing radiation. However, no precedent studies have investigated whether expression of EGF receptor would by itself confer on cancer cells resistance to radiation. The current study is aimed to address this question. Methods and materials A full-length human EGF receptor expression vector was transfected into the OCA-I murine ovarian carcinoma cells for stable clones expressing various levels of EGF receptors. Apoptosis and cell clonogenic survival assays were used to evaluate the sensitivity of the resulting cell clones to ionizing radiation. Results OCA-I cell clones expressing various levels of EGF receptor (OCA-I EGFR) were obtained. These clones showed an EGF receptor level–dependent increase in resistance to ionizing radiation, measured by apoptosis and cell clonogenic survival assays. Compared with the results for parental OCA-I and control vector-transfected OCA-I cells at the 10% cell survival level, the radioresistance was increased by a factor of 1.60 for EGFR-C5 (high level of EGF receptor expression), 1.37 for EGFR-C3 (intermediate level of EGF receptor expression), and 1.28 for EGFR-C1 (low level of EGF receptor expression). Treatment of the OCA-I EGF receptor transfectants with the anti-EGF receptor monoclonal antibody C225 downregulated the levels of EGF receptor, reduced the phosphorylation levels of EGF receptor downstream substrates (such as Akt and MAPK), and reversed the cellular radioresistance. Conclusion Our results demonstrate that overexpression of the EGF receptor conferred cellular resistance to ionizing radiation. The EGF receptor is thus a valid target for potential radiosensitization.

Purpose: Conjugating drugs with polymeric carriers is one way to improve selective delivery to tumors. Poly (L-glutamic acid)-paclitaxel (PG-TXL) is one such conjugate. Compared with paclitaxel, its uptake, tumor retention, and antitumor efficacy are increased. Initial studies showed that PG-TXL given 24 h before or after radiotherapy enhanced tumor growth delay significantly more than paclitaxel. To determine if PG-TXL-induced enhancement is obtained in a more clinically relevant setting, we investigated PG-TXL effects on tumor cure. Methods and Materials: Mice bearing 7-mm-diameter ovarian carcinomas were treated with PG-TXL at an equivalent paclitaxel dose of 80 mg/kg, single dose or 5 daily fractions of radiation or both PG-TXL and radiation. Treatment endpoint was TCD 50 (radiation dose yielding tumor control in 50% of mice). Acute radioresponse of jejunum, skin, and hair was determined for all treatments. Results: PG-TXL dramatically improved tumor radioresponse, reducing TCD 50 of single-dose irradiation from 53.9 (52.2–55.5) Gy to 7.5 (4.5–10.7) Gy, an enhancement factor (EF) of 7.2. The drug improved the efficacy of fractionated irradiation even more, reducing the TCD 50 of 66.6 (62.8–90.4) Gy total fractionated dose to only 7.9 (4.3–11.5) Gy, for an EF of 8.4. PG-TXL did not affect normal tissue radioresponse resulting from either single or fractionated irradiation. Conclusion: PG-TXL dramatically potentiated tumor radiocurability after single-dose or fractionated irradiation without affecting acute normal tissue injury. To our knowledge, PG-TXL increased the therapeutic ratio of radiotherapy more than that previously reported for other taxanes, thus, PG-TXL has a high potential to improve clinical radiotherapy.

Purpose To investigate differences in tumor histotype, incidence, latency, and strain susceptibility in mice exposed to single-dose or clinically relevant, fractioned-dose γ-ray radiation. Methods and Materials C3Hf/Kam and C57BL/6J mice were locally irradiated to the right hindlimb with either single large doses between 10 and 70 Gy or fractionated doses totaling 40 to 80 Gy delivered at 2-Gy/d fractions, 5 d/wk, for 4 to 8 weeks. The mice were closely evaluated for tumor development in the irradiated field for 800 days after irradiation, and all tumors were characterized histologically. Results A total of 210 tumors were induced within the radiation field in 788 mice. An overall decrease in tumor incidence was observed after fractionated irradiation (16.4%) in comparison with single-dose irradiation (36.1%). Sarcomas were the predominant postirradiation tumor observed (n=201), with carcinomas occurring less frequently (n=9). The proportion of mice developing tumors increased significantly with total dose for both single-dose and fractionated schedules, and latencies were significantly decreased in mice exposed to larger total doses. C3Hf/Kam mice were more susceptible to tumor induction than C57BL/6J mice after single-dose irradiation; however, significant differences in tumor susceptibilities after fractionated radiation were not observed. For both strains of mice, osteosarcomas and hemangiosarcomas were significantly more common after fractionated irradiation, whereas fibrosarcomas and malignant fibrous histiocytomas were significantly more common after single-dose irradiation. Conclusions This study investigated the tumorigenic effect of acute large doses in comparison with fractionated radiation in which both the dose and delivery schedule were similar to those used in clinical radiation therapy. Differences in tumor histotype after single-dose or fractionated radiation exposures provide novel in vivo evidence for differences in tumor susceptibility among stromal cell populations.

Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH. Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8–8.5 mm in diameter. Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases. Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules. Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs. 12 days for tumors to reach an average diameter of 8 mm. Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 ± 5.4 (SEM) to 2.9 ± 1.1 (SEM). The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis. Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in tumor-bearing and nontumor-bearing animals. The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography. Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-[(aminopropyl)amino]ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr. WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9. At a concentration and time of exposure likely to be achieved in vivo, that is, 40 μM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values. Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes. After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 μM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively. The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo. © 2002 Wiley-Liss, Inc.

Purpose: It has been shown that paclitaxel (TXL) can strongly enhance tumor cells’ sensitivity to radiation. We examined whether the radiosensitizing effect of paclitaxel can be further enhanced when it is delivered systemically as a polymer-drug conjugate that provides enhanced tumor uptake and prolonged release of TXL in the tumor. Methods and Materials: C3Hf/Kam mice bearing 8-mm murine ovarian OCa-1 tumors were treated with i.v.-injected Poly(L-glutamic acid)-paclitaxel (PG-TXL) at an equivalent TXL dose of 80 mg/kg, followed 24 h later by single doses of local radiation ranging from 5 to 15 Gy. To determine how long the radiopotentiation persisted at extended times after PG-TXL administration, mice with OCa-1 tumors were given i.v. PG-TXL and 4, 24, 48, 72, 120, or 168 h later their tumors were irradiated at a dose of 10 Gy. Antitumor activity was determined by delay in tumor growth. Cell cycle distribution was assayed using flow cytometry. Tumor vascular volume was estimated using Tc-99 m-labeled red blood cells. Results: PG-TXL strongly potentiated the radioresponse of the OCa-1 tumor. The enhancement factors ranged from 2.79 to 4.28, depending on radiation dose, when PG-TXL preceded radiation by 24 h. The enhancement factor derived from radiation dose–response curves was as high as 5.13. The radiosensitizing effect of PG-TXL was also dependent on the interval between PG-TXL administration and radiation delivery, with greater enhancement been observed when the interval was decreased. The percentage of G2/M cells was significantly increased to 21.4% 48 h after PG-TXL but declined to a preinjection level of 14.8% 72 h after PG-TXL. PG-TXL only moderately increased the tumor vascular volume by 37% 24 h after PG-TXL administration. Conclusion: PG-TXL markedly potentiated response of OCa-1 tumor to radiation. When compared to literature data obtained from the same tumor model used here, PG-TXL exhibited stronger radiosensitization effect than TXL. Although its action is possibly mediated by arrest of cells in G2/M phases of cell cycle and by increased tumor blood supply, PG-TXL may exert its radiopotentiation activity through increased tumor uptake of PG-TXL and sustained release of TXL in the tumor. Our results show that conjugation of TXL to a polymer has the potential to further enhance its radiosensitizing activity and that clinical trials of PG-TXL in combination with radiation is warranted.

Purpose: Previously, we have reported that the bcl-2-expressing murine lymphoma cell line LY-ar is resistant to chemotherapy-induced apoptosis when compared to the non-bcl-2-expressing LY-as cell line. The intent of the present study was to determine whether this relationship extends to lymphomas produced from these cell lines in syngeneic mice, after treatment with the same chemotherapy agents. Methods: LY-ar and LY-as tumors were grown in the hind legs of syngeneic mice. They were subsequently exposed to graded doses of cisplatin (CP), etoposide (VP-16), Adriamycin (ADR), cytarabine (ara-C), cyclophosphamide (CY), or camptothecin (CAM). Apoptotic bodies were scored in histological sections of tumors that had been stained with hematoxylin and eosin. Tumor growth delay was determined on tumors that were treated when they were 8 mm in diameter. Thereafter, tumor diameter was measured daily with a vernier caliper until they had grown to a maximum of 16 mm in diameter. Results: When transplanted into host animals, tumors derived from these two cell lines and treated in vivo with CP, VP-16, ADR, ara-C, CY, and CAM displayed apoptotic propensities similar to those seen in the same cell lines when treated in vitro. Generally, for all the drugs tested, apoptotic indices in LY-as tumors were significantly higher than in LY-ar tumors. However, tumor growth delay measurements could not be predicted with any accuracy from the apoptotic indices. For some drugs LY-ar tumors were more sensitive than LY-as tumors (CP, Vp-16, ADR, ara-C), yet LY-ar tumors were more resistant to CY. Conclusions: Despite considerable interest in using apoptotic indices as predictors of treatment outcome, the data presented here suggest that these relationships are very complex. This may be especially true for chemotherapy agents for which effects in vivo are complicated by pharmacokinetics, host effects, and tumor cell heterogeneity.

Prostaglandins are arachidonate metabolites produced in virtually all mammalian tissues and possess diverse biologic capabilities, including vasoconstriction, vasodilatation, stimulation or inhibition of platelet aggregation, and immunomodulation, primarily immunosupression (1–4). They are implicated in the promotion of development and growth of malignant tumors (4–7). They are also involved in the response of tumor and normal tissues to cytotoxic agents such as ionizing radiation (8). Prostaglandin production is mediated by two cyclooxygenase enzymes: cyclooxygenase-1 and cyclooxygenase-2. Cyclooxygenase-1 is constitutively expressed and is ubiqui tous , and cyclooxygenase-2 is induced by diverse inflammatory stimuli (7,9). Nonsteroidal anti-inflammatory drugs (NSAIDs) or agents inhibit cyclooxygenase enzymes and consequently can prevent, inhibit, or abolish the effects of prostaglandins. Increasing evidence shows that NSAIDs can inhibit the development of cancer in both experimental animals and in humans (7), can reduce the size of established tumors (6–8), and can increase the efficacy of cytotoxic anticancer agents (8). Our own investigations have demonstrated that the NSAID indomethacin prolongs tumor growth delay and increases the tumor cure rate in mice after radiotherapy (8,10,11). Commonly used NSAIDs, including indomethacin, inhibit both cyclooxygenase-1 and cyclooxygenase-2. However, treatment with these agents may be limited by toxicity to normal tissue, particularly by ulcerations and bleeding in the gastrointestinal tract ascribed to the inhibition of cyclooxygenase-1. Recently developed selective cyclooxygenase-2 inhibitors exert potent anti-inflammatory activity but cause fewer unwanted side effects (7,9,12,13). These compounds may thus be safer than those NSAIDs that are in common use. A recent report (7) shows that cyclooxygenase-2-specific inhibitors can prevent carcinogenesis in experimental animals, but their efficacy in enhancing in vivo tumor response to radiation has not been established. By use of the mouse sarcoma NFSA, we investigated the potential of 4-[5-(4chlorophenyl)-3-(trifluoromethyl)-1Hpyrazol-l-yl]benzenesulfonamide (SC8236), a selective cyclooxygenase-2 inhibitor (14,15) (supplied by Searle, G. D. & Co., Skokie, IL), to enhance response of tumor to local g-irradiation. All studies reported had institutional approval and all guidelines for appropriate animal treatment were followed. We have reported earlier (6) that the NFSA sarcoma is a nonimmunogenic and prostaglandin-producing tumor that spontaneously developed in C3Hf/Kam mice. This tumor exhibits an increased radioresponse if indomethacin is given prior to tumor irradiation (10,11). In experiments described in this communication, solitary tumors were generated in the right hind legs of mice by the injection of 3 × 10 viable NFSA tumor cells. When tumors were 8 mm in diameter, they were locally irradiated with 25–80 Gy single-dose g-radiation. Treatment with SC-8236 (6 mg/kg body weight, given in the drinking water) was started when tumors were approximately 6 mm in diameter, and the treatment was continued for 10 consecutive days. In some experiments, tumor irradiation was performed 3–8 days after initiation of the treatment with SC-8236. The end points of the treatment were tumor growth delay (days) and TCD50 (tumor control dose 50, defined as the radiation dose yielding local tumor cure in 50% of irradiated mice 120 days after irradiation). Treatment of mice with SC-8236 alone significantly inhibited tumor growth (inset in Fig. 1, A). Tumor diameter doubling time, based on tumor growth from 6 to 12 mm in diameter, was increased from 7.3 days (95% confidence interval [CI] 4 6.4–8.1 days) to 14.8 days (95% CI 4 11.5–18.1 days) (P

Purpose/Objective: The nucleoside analogue gemcitabine inhibits cellular repair and repopulation, induces apoptosis, causes tumor growth delay, and enhances radiation-induced growth delay. After single doses of drug and radiation, maximum enhancement of tumor response was obtained when gemcitabine preceded radiation by at least 24 h. Conversely, the cellular radioresponse of the normal gastrointestinal epithelium was slightly protected when gemcitabine and radiation were separated by 24 h. This differential response created a time frame within which therapeutic gain could be maximized. In our present investigation, we sought to define the most therapeutically beneficial scheme of gemcitabine administration when combined with fractionated radiotherapy. Methods and Materials: C3Hf/Kam mice were given identical drug and radiation schedules of administration, and both normal tissue (jejunal mucosa) and tumor (Sa-NH) responses were measured. Irradiation was given once per day for 5 days in normal tissue and tumor growth delay studies and twice per day for the tumor cure endpoint. A total dose of 25 mg/kg gemcitabine was given i.p. in 1 of 3 schedules: a single dose of 25 mg/kg 24 h before the start of fractionated irradiation, 12.5 mg/kg 24 h before the first and third radiation doses, or 24 h before each of 5 radiation doses. Groups of mice bearing 7- or 8-mm diameter tumors were treated with gemcitabine alone or in combination with fractionated irradiation under ambient or hypoxic conditions. The survival response of the jejunal mucosa was quantified by the microcolony assay and histologically by quantifying apoptosis, mitosis, S-phase fraction, and crypt cellularity. Results: For tumor growth delay, dose-modifying factors (DMFs) were similar (1.34–1.46) for all 3 schedules of drug administration. In contrast, the response of the jejunum was strongly dependent on the schedule of gemcitabine administration. A single dose of gemcitabine before the start of fractionated radiotherapy resulted in slight radioprotection (DMF 0.96). Two doses and 5 daily doses of gemcitabine enhanced radiation response by factors of 1.09 and 1.23, respectively. Major factors affecting the response of the jejunal mucosa were apoptotic death of S-phase cells exposed to gemcitabine and cell cycle synchrony of surviving cells. Tumor reoxygenation was found to be a major mechanism for tumor radioenhancement, in addition to those reported earlier. Conclusion: All 3 schedules of drug administration produced therapeutic gain; however, when gemcitabine was given more than once in a 5-fraction radiation treatment schedule, normal tissue toxicity increased. The highest therapeutic gain (1.4) was achieved by giving a single dose of gemcitabine (25 mg/kg) 24 h before the start of fractionated radiotherapy.

Purpose: The study investigated whether basal, constitutive levels of p21 WAF1/CIP1 protein in murine carcinomas are related to in vivo tumor radioresponse. The study is based on recent observations demonstrating that in vitro cancer cell lines are resistant to cytotoxic drugs when they express high basal levels of p21 WAF1/CIP1 protein, and that the loss of the p21 gene in the HCT116 human colorectal cancer cell line results in increased radioresponse of xenografts derived from that cell line. Methods and Materials: Protein levels of p21 WAF1/CIP1 , p53, bax, and bcl-2 were determined in 8 carcinomas (3 mammary carcinomas designated MCa-4, MCa-29, and MCa-35, 2 squamous cell carcinomas designated SCC-IV and SCC-VII, ovarian adenocarcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. The tumors, growing in the right hind legs of mice, were 8 mm in diameter at the time of analysis. These tumors greatly differ in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. Results: Protein levels of these oncogenes varied among tumors, with p21 WAF1/CIP1 showing the greatest variation: its mean densitometric value ranged from 1 to 19. Bcl-2 levels also showed broad variation in densitometric values, from 1 to 10. In comparison, bax and p53 (7 of 8 tumors contained wild-type p53) varied much less among different tumor types; their variation was within a 5-fold range, and the level of p53 was similar in 6 of 8 tumors. Tumor radioresponse correlated significantly (R = 0.77, p = 0.02) only with the magnitude of p21 WAF1/CIP1 expression: tumors with high levels of p21 WAF1/CIP1 were less radiocurable than those with lower levels. Tumor radiocurability showed a significant positive correlation ( p = 0.02) with the extent of radiation-induced apoptosis, indicating that tumors that responded to radiation with higher percentages of apoptosis were more curable by radiation. Despite a strong trend to correlation, ( p = 0.15), p21 WAF1/CIP1 expression did not correlate significantly with radiation-induced apoptosis, which suggested that p21 WAF1/CIP1 influenced tumor radioresponse by mechanisms beyond that of apoptosis induction. Conclusion: Our findings showed that murine tumors exhibit wide variation in constitutive levels of p21 WAF1/CIP1 which had a significant relationship with tumor radioresponse: tumors with high levels of p21 WAF1/CIP1 were less radiocurable than those with lower levels. These findings support the concept that p21 WAF1/CIP1 is a major determinant of tumor radioresponse in vivo , and may have important clinical implications. The pretreatment assessment of p21 WAF1/CIP1 protein could serve as a useful predictor of radiotherapy outcome and may assist in selecting an effective treatment modality.

Tumor necrosis and oxygen status were investigated as a function of tumor size in three syngeneic murine carcinomas, MCa-4, OCa-I, and SCC-VII, in C3Hf/Kam mice. Tumor necrosis was estimated histologically, and tumor oxygenation determined by direct polarographic histography. As tumor volume increased necrosis increased significantly in all three tumor types (p < 0.001). Similarly, as tumor volume increased from 200 to 1400 mm3, hypoxia, defined as the percentage of measured pO2 values < or = 5.0 mm Hg, increased from 55.1% to 95.9%, 70.3% to 81.4%, and 56.8% to 98.5% in MCa-4, OCa-I, and SCC-VII tumors respectively (p < 0.001). Correcting pO2 for necrosis reduced the tumor size dependence of measured tumor hypoxia in all three tumor types but in no case was the reduction significant. The main effect of correction was to shift the fitted curves of percent pO2 values < or = 5.0 mm Hg down toward lower percentages for all tumors. This change was significant for MCa-4 and OCa-1 tumors (p < 0.001), but not for SCC-VII (p = 0.054). Defining the influence of variables such as necrosis that affect polarographic assessment of tumor oxygenation is important to enhance the technique's reliability and prospect as an investigative and predictive tool.

We studied the combination of tumor necrosis factor (TNF) and paclitaxel. Our aim was to determine whether TNF increases the antitumor efficacy of paclitaxel and if so whether the increase is mediated through the enhancement of apoptosis induction by paclitaxel. Mice bearing 6 mm MCa-K or MCa-4 mammary carcinomas, OCa-I ovarian carcinomas, or HCa-I hepatocarcinomas in their legs were treated with TNF, paclitaxel of their combination. TNF was administered i.p. daily at a dose of 10 micrograms per mouse for 7 days; paclitaxel at a dose of 40 mg/kg per mouse was given as a single i.v. injection 1 h before the second dose of TNF. Tumor growth delay was used as the endpoint of tumor response to the treatments. The results showed that the combination was either additive or supraadditive; supraadditive action occurred in three of the four tumors tested. The enhancement factors (EFs) were 1.24 for MCa-K, 1.53 for MCa-4, 1.0 for OCa-I and 2.17 for HCa-I. Histological analysis of treated MCa-K tumors revealed that TNF alone did not induce apoptosis of tumor cells, but in the combination it enhanced the apoptotic response to paclitaxel. Thus, TNF increased the antitumor efficacy of paclitaxel by enhancing cellular sensitivity to paclitaxel's induction of apoptosis. The results imply that the combination of TNF and paclitaxel has potential as a treatment for cancer.

The kinetics of taxol-induced mitotic arrest and apoptosis in murine mammary carcinoma MCA-4 and ovarian carcinoma OCA-I tumors were determined to establish a possible causative relationship between mitotic arrest and apoptosis and to see whether these cellular effects of taxol would correlate with the extent of its antitumor efficacy. Mice bearing 8-mm tumors in a hind leg were given taxol i.v. at a dose of 10-80 mg/kg. Both tumors responded to taxol by significant growth delay or transient regression; in general, the response was greater as the dose of taxol was increased. For kinetics studies the mice were treated with 60 mg/kg taxol given once when tumors were 8 mm in size or twice, with the second dose being given 3 days after the first. At various times ranging from 1 to 96 h after treatment with taxol, tumors were histologically analyzed to quantify mitotic and apoptotic activity. After a single dose of taxol, mitotic arrest was visible at 1 h, and the mitotic index increased with time to reach peak values of 36% in MCA-4 tumors and 22% in OCA-I tumors at 9 h. The index then declined to a baseline of 1%-3% at 3 days for MCA-4 tumors and 1 day for OCA-I tumors. Apoptosis followed mitotic arrest, beginning at the time of peak mitotic arrest, increasing to the highest level of about 20% at 18-24 h after treatment and gradually declining to the normal level of 3%-6% after 3-4 days. Nuclear material progressively condensed in mitotically arrested cells, culminating in the frank appearance of multiple apoptotic bodies. The change in cell morphology plus the dynamics of apoptosis development imply that a large percentage of tumor cells arrested in mitosis by taxol die by apoptosis. Kinetic analysis undertaken after the second dose of taxol showed a considerably lower percentage of cells arrested in mitosis as compared with that seen after a single dose, and the induction of apoptosis by the second dose was minimal. However, the antitumor efficacy of the second dose of taxol was similar to or better than that of the first dose, implying that in addition to mitotic arrest and apoptosis, there exist other mechanisms by which taxol exerts its antitumor action.

There is increasing attention directed to the hypothesis that apoptosis plays a role in the response to cancer treatment including chemotherapy. However, the evidence to support this hypothesis has come almost entirely from experiments conducted in cultured cell systems. To extend this hypothesis to the therapeutic setting it is necessary to address this critical question in tumors treated in vivo. We have therefore evaluated the extent of apoptosis induced in murine tumors treated in vivo with cancer chemotherapy agents. Seven different murine tumors, comprising a mammary adenocarcinoma (MCa-4), an ovarian adenocarcinoma (OCa-1), a lymphoma (LY-TH), three sarcomas (FSA, NFSA and SA-NH) and a squamous cell carcinoma (SSC-7), were examined 8 and 24 h after treatment with cisplatin or cyclophosphamide (CY). Apoptosis was scored by morphometric analysis of histological sections of the tumors. The results showed that MCa-4, OCa-1 and LY-TH had a significant apoptotic response to both cisplatin and CY, and the other tumors had essentially no apoptotic response. In addition, two of these tumors, MCa-4 and OCa-1, underwent apoptosis in response to adriamycin, 5-fluorouracil, Ara-C, etoposide, camptothecin and fludarabine. These observations demonstrate that apoptosis may be a feature of tumor response to chemotherapy in vivo, and illustrate the heterogeneity of apoptotic response amongst different tumor types and to different cytotoxic agents.

Programmed cell death or apoptosis has recently been recognized as an important mode of cell death in the response of tumor cells to cytotoxic treatments associated with chemotherapy or radiation therapy (RT). We previously showed that both cisplatin and radiation induced substantial apoptosis in the murine mammary tumor MCA-4 when administered as single agents in vivo. Moreover, the levels of apoptosis correlated with tumor response measured as tumor growth delay. In the current study a role for apoptosis in a combined treatment with cisplatin and radiation was evaluated. Mice bearing MCA-4 tumors were irradiated at different times following an injection with cisplatin, and tumor growth delay was assessed. Apoptosis was scored from histological sections of the tumors. The results showed that whereas the cisplatin sensitized the MCA-4 tumor to a subsequent irradiation in terms of the growth delay, the apoptotic index never rose above the maximum level characteristic of either agent when used singly. Thus, there was no indication that cisplatin sensitized the tumor cells to radiation-induced apoptosis. We conclude that apoptosis is not the only important mechanism for cell killing in tumors following cytotoxic treatment and that tumor response is a complex composite of many different factors. © 1995 Wiley-Liss, Inc.

Studies performed three decades ago in our laboratory supported the hypothesis that radiation efficacy may be augmented by bacterial extracts that stimulate non-specific systemic antitumor immune responses. Application to the clinic was halted by unacceptable side effects and toxicities resulting from exposure to whole bacterial pathogens. Later scientific advances demonstrated that DNA isolated from bacteria was immunostimulatory and could be reproduced with synthetic oligodeoxynucleotides (ODNs), thus fueling the transition from bugs to drugs. Unmethylated CpG motifs within bacterial DNA induce activation of Toll-like receptor 9 and subsequently activate antigen-specific cellular immune responses. CpG ODNs have demonstrated favorable toxicity profiles in phase I clinical trials. We showed that this potent immunoadjuvant can be used in combination with radiation therapy to enhance local and systemic responses of several murine tumors. Studies demonstrated that enhanced tumor response is mediated in part by the host immune system. Antitumor efficacy was diminished in immunocompromised mice. Animals cured by combination of radiation and CpG ODN were resistant to subsequent tumor rechallenge. This body of work contributes to our understanding of the dynamic interplay between tumor irradiation and the host immune system and may facilitate translation to clinical trials.

Experiments were performed to establish the extent and kinetics of tumor cell repopulation in a murine sarcoma, designated SA-NH, treated with cyclophosphamide (CY). Mice bearing 8-mm leg tumors were treated with 200 mg/kg CY which caused a transient tumor regression. Changes in the absolute clonogen content of tumors was determined by the change in TCD50 values (50% tumor control) obtained under hypoxic conditions of local tumor irradiation at different times after CY treatment until tumors regrew to the pretreatment size. For comparison, hypoxic TCD50 values were determined during the growth of tumors not treated with CY. CY greatly depleted tumors of clonogenic cells as manifested by the reduction in the control TCD50 value of 64.5 Gy to 32.8 Gy 1 day after CY treatment. The reduced TCD50 value remained unchanged for 2 weeks after treatment with CY, at which time the TCD50 began to rapidly increase, continuing until the end of the observation period of 21 days when tumors reached the pretreatment size. In contrast, there was a constant but slower increase in TCD50 values during the growth of tumors not treated with CY. The daily increase in TCD50 was more than twice as high in CY-treated than in CY-untreated tumors: 4.5 Gy/day versus 2.1 Gy/day. This implies that the rate of clonogen production in CY-treated tumors was twice as high as that of unperturbed tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Fifteen different murine tumours were evaluated with respect to the degree of apoptosis development that occurs in the tumour tissue in the first few hours following irradiation in vivo. Animals were killed at 3 or 6 h following irradiation with 0, 2.5, 10 or 25 Gy. Apoptosis was scored as percent aberrant nuclei by microscopic examination of histological sections made from the tumour specimens. Results showed that three of four mammary adenocarcinomas, one ovarian adenocarcinoma, and one lymphoma displayed at least 10% apoptotic cells after 25 Gy, whereas five sarcomas, three squamous cell carcinomas, and a hepatocarcinoma did not. The time courses and dose responses were similar in those tumours that responded. These data were compared with the known response of these same tumours when analysed using conventional assays. The tumours that did respond by significant apoptosis had longer specific growth delays and lower TCD50 (dose to cure 50% of animals) doses, thus suggesting that an acute apoptotic response following irradiation may be a feature of certain tumours that respond well to irradiation. Additionally, this analysis revealed heterogeneity in the apoptotic response both within an individual tumour specimen and among different tumour types. These observations of intra and intertumour heterogeneity are consistent with the idea that the propensity for apoptosis in tumours is genetically regulated.

Protective effects of indomethacin, a prototype prostaglandin-inhibiting agent, against early and late sequelae of radiation injury (after X-rays or gamma rays) in mice were investigated. The following tissues or organs were examined: hematopoietic tissue, esophagus, jejunum, colon, lung, hair follicles, and tissues involved in the development of radiation-induced leg contractures. In addition, the effect of indomethacin was tested against radiation-induced carcinogenesis. In all experiments, the radiation was delivered as a single dose. Indomethacin led to significant protection of hematopoietic tissue, by a factor of 1.3. There was also some protection against radiation-induced pneumonitis and against radiation-induced carcinogenesis (protection factor of 1.2). The other tissues tested showed no change in their radioresponse after being treated with indomethacin. Thus, indomethacin can act as a radioprotective agent against both early and late sequelae of radiation, but its effect is dependent on the tissue tested. This protection is smaller than that observed with WR-2721. However, indomethacin combined with WR-2721 produced a radioprotective effect greater than the radioprotection achieved by individual treatments.

Several methods are described for analysing the results of in vivo colony assays using the statistical procedure called maximum-likelihood analysis. The methods differ in the way in which they take into account possible sources of variability in the data. The methods described here for analysing microcolony data are direct methods, in that they use the observed colony counts rather than transformed (e.g. Poisson-corrected) data. Each method can be used to estimate the average number of surviving cells per tissue structure (e.g. per jejunal crypt) in a single dose group, together with 95% confidence intervals, or to fit cell-survival models to data from a range of dose groups (e.g. to obtain estimates of D0 or of the linear-quadratic parameters alpha and beta). Experimental microcolony data from murine jejunum, colon, and hair follicles irradiated in anagen (proliferative) or telogen (resting) phase have been analysed. Estimates of D0 have been derived from single-dose data and estimates of alpha, beta, and the initial number of clonogenic cells per structure have been derived from fractionation data. For hair follicles, the half-time of repair of sublethal radiation injury has also been derived from fractionation data.

Purpose: C225 strongly enhances tumor radioresponse when given concurrently with radiotherapy. We investigated whether additional therapeutic benefit could be achieved by continuing maintenance treatment with C225 after the completion of fractionated radiotherapy. Methods and Materials: A431 xenografts were treated with local irradiation or combined with C225 by two different schedules: (1) 6 h before the first dose of irradiation and at 3-day intervals for a total of 3 doses during the 7-day fractionated radiotherapy, or (2) 6 doses of C225 given both during radiotherapy and continuing for 3 additional doses after radiotherapy. Tumor cure was assessed by the radiation dose yielding local tumor control in 50% of animals (TCD{sub 50}), and time to recurrence was also determined. Results: Both treatment schedules increased radiocurability as evidenced by reductions in TCD{sub 50}, but the effect was greater when C225 was given both during and after radiotherapy. C225 reduced the TCD{sub 50} of 83.1 (73.2-124.8) Gy by radiation only to 46.2 (39.1-57.5) Gy when given during radiotherapy and to 30.8 (22.2-38.0) Gy when given during and after radiotherapy. Dose modification factors were 1.8 when C225 was given during radiotherapy and 2.7 when given both during and after radiotherapy. C225 was also effective inmore » delaying the onset of tumor recurrences, and was more effective when given as both concurrent and maintenance therapy. Conclusions: Data showed that C225 strongly enhanced the curative effect of fractionated radiation, and its effect was greater if administration was extended beyond the end of radiotherapy. This important finding may influence future designs of clinical trials combining anti-EGFR (anti-epidermal growth factor receptor) agents with radiotherapy.« less

Background and purpose Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine-guanine (CpG) motifs bind to Toll-like receptor 9 (TLR9) and stimulate both innate and adaptive immune reactions and possess anti-tumor activity. We recently reported that CpG ODN 1826 strongly enhances radioresponse of both immunogenic [Milas L, Mason K, Ariga H, et al. CpG oligodeoxynucleotide enhances tumor response to radiation. Cancer Res 2004;64:5074–7] and non-immunogenic [Mason KA, Ariga H, Neal R, et al. Targeting toll-like receptor-9 with CpG oligodeoxynucleotides enhances tumor response to fractionated radiotherapy. Clin Cancer Res 2005;11:361–9] murine tumors. Using two immunogenic murine tumors, a fibrosarcoma (FSa) and a mammary carcinoma (MCa-K), the present study explored whether CpG ODN 1826 also improves the response of murine tumors to the chemotherapeutic agent docetaxel (DOC). Materials and methods CpG ODN 1826 (100 μg) was given sc three times: when leg tumors were 6 mm, when they grew to 8 mm and again 1 week later. DOC (33 mg/kg iv) and local tumor radiation (10 Gy) were given when tumors were 8 mm. Effects of the treatments were assayed by tumor growth delay, defined as days for tumors to grow from 8 to 12 mm in diameter. Results Treatment with CpG ODN 1826 resulted in strongly enhanced response of FSa tumors to radiation and MCa-K tumors to the chemotherapeutic agent DOC. Enhancement of tumor treatment response was demonstrated by a strong prolongation in the primary tumor treatment endpoint, tumor growth delay. Coincidentally, this treatment also resulted in a higher rate of tumor cure than that observed after tumor radiotherapy or chemotherapy alone. When all three agents were combined the effect was comparable to that of the combination of CpG ODN 1826 with radiation in the case of FSa or of the combination of CpG ODN 1826 with DOC in the case of MCa-K. Conclusion Overall results show that CpG ODN 1826 can markedly improve tumor response to radiation and chemotherapy (DOC), suggesting that CpG ODNs have potential to be beneficial when used singly or in combination with other standard treatment modalities such as taxane chemotherapy, radiotherapy or both.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs detected by Toll-like receptor 9 of dendritic cells and B cells have potent immunomodulatory effects. CpG oligodeoxynucleotides induce cytokines, activate natural killer cells, and elicit T-cell responses leading to antitumor effects, including improved efficacy of chemotherapeutic agents and, as we reported recently, synergy between CpG oligodeoxynucleotide 1826 and single-dose radiotherapy of an immunogenic mouse fibrosarcoma. The present study extends this finding to the fractionated radiotherapy of the fibrosarcoma tumor and assesses the ability of CpG oligodeoxynucleotide 1826 to increase the radioresponse of a tumor (nonimmunogenic fibrosarcoma). The experiments used a murine immunogenic fibrosarcoma tumor, fibrosarcoma growing in the leg of mice, and response to radiotherapy was assessed by tumor growth delay and tumor cure rate (TCD50, radiation dose yielding 50% tumor cure). Multiple s.c. peritumoral or i.t. administrations of CpG oligodeoxynucleotide 1826 at a dose of 100 μg per mouse were given when established tumors were 6 mm in diameter. Local tumor irradiation was initiated when tumors grew to 8 mm in diameter; radiation was delivered in 1 to 9 Gy fractions given twice daily separated by 6 to 7 hours for 5 consecutive days to achieve a total dose of 10 to 90 Gy. CpG oligodeoxynucleotide 1826, given as a single agent, had only a small antitumor effect, but it dramatically enhanced fibrosarcoma response to radiotherapy. Although 83.1 (79.2-90.0) Gy total dose were needed to achieve tumor cures in 50% of mice treated with radiotherapy alone, only 23.0 (11.5-32.7) Gy total dose were needed in mice treated with both CpG oligodeoxynucleotide 1826 and radiotherapy. The magnitude of potentiation of tumor radioresponse at the TCD50 level was by a factor of 3.61, a much higher value than that (a factor of 1.93) that we reported for single-dose radiotherapy. Mice cured of their tumors by combined CpG oligodeoxynucleotide 1826 plus radiotherapy were highly resistant to s.c. tumor take or development of tumor nodules in the lung from i.v. injected tumor cells when rechallenged with fibrosarcoma cells 100 to 120 days after the treatment, suggesting the development of a memory response. CpG oligodeoxynucleotide 1826 also increased radioresponse of the nonimmunogenic fibrosarcoma tumor by a factor of 1.41 and 1.73 when CpG oligodeoxynucleotide 1826 was given s.c. and i.t., respectively. These findings show that CpG oligodeoxynucleotides are highly potent enhancers of tumor response to both single-dose and fractionated radiation and as such have potential to improve clinical radiotherapy.

Purpose C225 anti-EGFR (epidermal growth factor receptor) antibody has been shown to enhance tumor response to radiation and a number of chemotherapeutic agents. Because of increased use of concurrent chemoradiotherapy in cancer treatment, it is important to determine whether C225 enhances also the antitumor efficacy of radiation when combined with chemotherapy. This study assessed the effect of C225 on tumor response when combined with docetaxel plus single or fractionated radiation. Methods and materials MDA468 human adenocarcinoma and A431 human epidermoid carcinoma cells growing as xenografts in the right hind leg of nude mice were used. Mice bearing 8-mm tumors were treated with C225 antibody at a dose of 1 mg given i.p. once, twice, or three times 3 days apart, 10 or 30 mg/kg docetaxel given i.v., and/or local tumor irradiation of 8 or 10 Gy single dose or fractionated irradiation consisting of 2 Gy daily for 5 days. When all three agents were combined, C225 was given 6 h before or 18 h after docetaxel, and radiation was given 24 h after docetaxel. The treatment end point was tumor growth delay. Results C225 enhanced the antitumor efficacy of docetaxel, local tumor irradiation, and docetaxel combined with radiation. The response of both MDA468 and A431 carcinomas was enhanced. The enhancement factors ranged from 1.19 to 8.52, the degree of the enhancement depending on experimental conditions such as administration of multiple vs. single dose C225 or single or fractionated irradiation. C225 given twice or 3 times was more effective than when administered as a single dose. The effect of C225 was more pronounced when combined with single than fractionated irradiation with or without docetaxel. The triple-agent therapy was more effective than a single agent or double combination therapies, expressed by both increased tumor growth delay and the rate of tumor cure. Conclusions Our results show that C225 anti-EGFR antibody is a potent enhancer of tumor response to docetaxel or radiation as single agents, and to docetaxel when combined with radiation. Thus, these findings provide strong preclinical evidence in support of combination of anti-EGFR blockade with chemoradiotherapy.

The effects of dose per fraction on the ability of amifostine exposure to elevate angiostatin levels in the serum of mice and to inhibit spontaneous metastases formation using the well-characterized murine Sa-NH sarcoma were investigated. Amifostine was administered intraperitoneally at doses of 50, 100, or 200 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8 mm in diameter. Amifostine was again administered immediately following surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Nontumor-bearing control animals were treated using the same dosing and surgery schedules. The average number of pulmonary metastases per animal was determined for each experimental group. A significant reduction (P

Purpose: We recently reported that overexpression of epidermal growth factor receptor (EGFR) positively correlated with radioresistance of murine carcinomas. Because cyclin D1 is a downstream sensor of EGFR activation, the present study investigated whether a relationship exists between the extent of cyclin D1 expression and in vivo radiocurability of murine tumors. We further investigated the influence of radiation on cyclin D1 expression and the expression of p27, an inhibitor of the cyclin D1 downstream pathway, as well as the relationship of these molecular determinants to cell proliferation and induced apoptosis in tumors exposed to radiation. Methods and Materials: Cyclin D1 expression was assayed in nine carcinomas syngeneic to C3Hf/Kam mice using Western blot analysis. These tumors greatly differed in their radioresponse as assessed by TCD 50 . The expression of cyclin D1 and p27 proteins was determined by Western blotting. Cell proliferative activity in tumors was determined by proliferating cell nuclear antigen (PCNA) immunochemistry. The effect of irradiation on the expression of cyclin D1 or p27 proteins and on PCNA positivity was determined in the radiosensitive OCa-I and in the radioresistant SCC-VII tumors. Results: Cyclin D1 expression varied among tumors by 40-fold, and its magnitude positively correlated with poorer tumor radioresponse (higher TCD 50 values). The level of cyclin D1 expression paralleled that of EGFR. A 15-Gy dose reduced constitutive expression of cyclin D1 in the radiosensitive OCa-I tumors, but had no influence on expression of cyclin D1 in the radioresistant SCC-VII tumors. In contrast, 15 Gy increased the expression of p27 in radiosensitive tumors and reduced it in radioresistant tumors. Radiation induced no significant apoptosis or change in the percentage of PCNA-positive (proliferating) cells in SCC-VII tumors with high cyclin D1 levels, but it induced significant apoptosis and a decrease in the percentage of proliferating cells in OCa-I tumors with low cyclin D1 expression. Conclusion: Our findings show a positive correlation between cyclin D1 expression and tumor radioresistance. The expression of cyclin D1 and p27 was modified by radiation and was associated with cellular response to radiation, but this depended on the pretreatment level of cyclin D1 expression. These findings may have important clinical implications: The pretreatment assessment of cyclin D1 expression could serve as a useful predictor of radiotherapy outcome and assist in selecting an effective treatment modality.

Docetaxel, a potent chemotherapeutic drug and a strong enhancer of tumor radioresponse, possesses immunomodulating properties. We previously reported that 40% of murine tumors responding to docetaxel by growth delay showed heavy infiltration with macrophages or lymphocytes. The present study explored the effect of whole body irradiation on antitumor action of docetaxel alone or docetaxel plus tumor irradiation. Mice bearing 8-mm MCa-K mammary carcinoma in the leg received 33 mg/kg docetaxel i.v., 5 to 65 Gy tumor irradiation, or both (radiation given 24 h after docetaxel). Docetaxel delayed tumor growth and enhanced the efficacy of radiation: it dramatically reduced TCD50 (radiation dose yielding 50% tumor cure) from the control value of 38.6 Gy to 11.8 Gy, for an enhancement factor of 3.27. In addition to enhancing tumor radioresponse, docetaxel decreased the lung metastatic rate in mice with local tumor control from 26% in mice receiving radiation alone to 11% in mice receiving docetaxel plus radiation. Docetaxel induced heavy infiltration of tumors with lymphocytes, determined 2-4 days after treatment: the percentage of lymphocytes increased from the control value of

This study investigated P-glycoprotein (Pgp) expression by murine tumors with and without resistance to paclitaxel and the role of (99m)Tc-2-methoxyisobutylisonitrile (MIBI)/(201)Tl imaging in predicting the effect of paclitaxel. Antitumor effect of paclitaxel and biodistribution of the radiopharmaceuticals were evaluated in mice bearing four tumor types. Pgp expression did not correlate with the antitumor efficacy of paclitaxel. Although the absolute uptake of (99m)Tc-MIBI did not correlate with Pgp expression, (99m)Tc-MIBI could predict paclitaxel sensitivity by its higher uptake.

One objective of the NGLI project was to establish a sedimentological and physical property database for Northern Gulf of Mexico that can be immediately utilized by the existing Navy R&D activities within the area Results from the core and grab sample analyses will be used to populate this database.

Our goal was to determine whether scintigraphy with111In-DTPA-paclitaxel could predict the response to chemotherapy with paclitaxel.Methods: Ovarian carcinoma (OCA 1), mammary carcinoma (MCA-4), fibrosarcoma (FSA) and squamous cell carcinoma (SCC VII) were inoculated into the thighs of female C3Hf/Kam mice. Mice bearing 8 mm tumors were treated with paclitaxel (40 mg/kg). The growth delay, which was defined as the time in days for tumors in the treated groups to grow from 8 to 12 mm in diameter minus the time in days for tumors in the untreated control group to reach the same size, was measured to determine the effect of paclitaxel on the tumors. Sequential scintigraphy in mice bearing 10 to 14 mm tumors was conducted at 5, 30, 60, 120, 240 min and 24 hrs postinjection of111In-DTPA-paclitaxel (3.7MBq) or111In-DTPA as a control tracer. The tumor uptakes (% injection dose/pixel) were determined.Results: The growth delay of OCA 1, MCA-4, FSA and SCC VII tumors was 13.6, 4.0, −0.02 and −0.28 days, respectively. In other words, OCa 1 and MCA-4 were paclitaxel-sensitive tumors, whereas FSA and SCC VII were paclitaxel-resistant tumors. The tumor uptakes at 24 hrs postinjection of In-111 DTPA paclitaxel of OCA 1, MCA-4, FSA and SCC VII were 1.0 × 10−3, 1.6 × 10−3,2.2 × 10−3 and 9.0 × 10−3 % injection dose/ pixel, respectively. There was no correlation between the response to chemotherapy with paclitaxel and the tumor uptakes of111In-DTPA-paclitaxel.Conclusions: Scintigraphy with111In-DTPA-paclitaxel could not predict the response to paclitaxel chemotherapy. Although there was significant accumulation of the paclitaxel in the tumor cells, additional mechanisms must be operative for the agent to be effective against the neoplasm.111In-DTPA-paclitaxel activity is apparently different from that of paclitaxel with Cremophor.

Purpose: To determine, as we did for paclitaxel, whether mitotic arrest and apoptosis induced in murine tumors in vivo by docetaxel correlate with the drug's antitumor effect and whether the antitumor efficacy of docetaxel depends on p53 mutational status of tumors. Methods: C3Hf/Kam mice were implanted with one of the following 15 syngeneic tumors: seven adenocarcinomas (MCa-4, MCa-29, MCa-35, MCa-K, OCa-I, ACa-SG, and HCa-I), two squamous cell carcinomas (SCC-IV and SCC-VII), five sarcomas (FSa, FSa-II, Sa-NH, NFSa, and Sa-4020) and one lymphoma (Ly-TH). When the tumors had grown to 8 mm in diameter, the mice were treated with 31.3 mg/kg docetaxel i.v. Tumor growth delay was the endpoint of docetaxel's antitumor effect. In separate groups of mice, mitotic arrest and apoptosis were determined micromorphometrically 1 to 72 h after docetaxel treatment. Tumors were assayed for their p53 status by sequence analysis of RNA prepared from freshly excised tumors. Results: Docetaxel caused statistically significant growth delay in six of seven adenocarcinomas, three of five sarcomas, and the lymphoma, but not in either of the squamous cell carcinomas. The drug induced mitotic arrest in all tumor types, but to various degrees ranging from 6.4 +/− 0.4% to 25.1 +/− 0.1%. In contrast, docetaxel induced appreciable apoptosis in only 5 of 15 tumors, with 10.3 +/− 1.6% being the highest apoptotic value. Neither mitotic arrest nor apoptosis were significantly correlated with tumor growth delay. However, tumors that responded to docetaxel by significant tumor growth delay histologically displayed massive cell destruction by cell lysis, and four of these tumors also showed marked infiltration with mononuclear lymphoid cells. Of the 15 tumors only 3 had mutant p53. Conclusions: Docetaxel exhibited a strong antitumor effect in two-thirds of murine tumors, and on a milligram per kilogram basis was more effective than paclitaxel against the same tumors. The drug was a potent inducer of mitotic arrest but a weak inducer of apoptosis, neither of which correlated with its antitumor effect. Tumor cell lysis appeared to be a major mode of tumor cell destruction and can be regarded as the main mechanism underlying antitumor efficacy of docetaxel. In contrast, paclitaxel's antitumor efficacy is related to its ability to induce apoptosis. At the molecular level, there was no dependency of antitumor efficacy of docetaxel on p53 mutational status of tumors.

Paclitaxel is a potent chemotherapeutic drug and also has the potential to act as a radioenhancing agent. The latter is based on its ability to arrest cells in the radiosensitive G2M phases of the cell cycle; the weight of supporting evidence is derived mainly from in vitro studies. Our previous in vivo experiments identified enhanced tumour radioresponse predominantly attributable to tumour reoxygenation occurring as a result of paclitaxel-induced apoptosis. The current study investigated whether paclitaxel enhanced the radioresponse of tumours which are insensitive to apoptosis induction, but exhibited mitotic arrest, and compared the degree and kinetics of the response to that in tumours which develop apoptosis. The mouse mammary carcinoma MCa-29 (apoptosis sensitive) and the squamous cell carcinoma SCC-VII (apoptosis resistant) were used. In addition, the study investigated whether paclitaxel affected normal skin radioresponse to determine if a therapeutic gain could be achieved. Paclitaxel enhanced the radioresponse of both types of tumours. In the SCC-VII tumour, radiopotentiation occurred within 12 h of paclitaxel administration coincident with mitotic arrest, where enhancement factors (EFs) ranged from 1.15 to 1.37. In MCa-29 tumour, the effect was greater, EFs ranging from 1.59 to 1.91 and occurred between 24 and 72 h after paclitaxel when apoptosis was the predominant microscopic feature of treated tumours and when tumour oxygenation was found to be increased. The acute skin radioresponse and late leg contracture response were essentially unaffected by prior treatment with paclitaxel. Therefore, by two distinct mechanisms, paclitaxel was able to enhance the radioresponse of paclitaxel-sensitive and -resistant tumours, but not the normal tissue radioresponse, thus providing true therapeutic gain.

The aim of this study was to evaluate whether radiolabeled iodoerythronitroimidazole (IETNIM) could predict the radiosensitization effect on tumors. Tumor-bearing mice were irradiated at a dose of 25, 31 and 37 Gy after the injection of IETNIM. They were also exposed to 37 Gy radiation at 35, 70, 140 and 240 min after the i.p. injection of IETNIM. After the irradiation, tumor growth assays were conducted and the effect of IETNIM as a radiosensitizer was estimated as enhancement factor (EF). Tumor uptake was measured at 35, 70, 140 and 240 min after i.p. injection of [131I]IETNIM, which were the same intervals used in the radiosensitization study. EF of IETNIM in mice treated with 25, 30 and 37 Gy irradiation was 0.72, 0.98 and 1.28, respectively. EF of IETNIM in mice irradiated at 35, 70, 140 and 240 min after the injection was 1.50, 1.69, 1.46 and 1.08, which corresponded to the tumor uptake and blood clearance of [131I]IETNIM. [131I]IETNIM may be a suitable radiopharmaceutical to predict the radiosensitization effect of misonidazole analogs on tumors.

Background . Paclitaxel has been shown to radiosensitize tumor cells in culture by arresting them in the most radiosensitive G 2 and M cell cycle phases. In vivo preclinical studies are now necessary to obtain full insight into the radiopotentiating potential of this drug and its ability to increase the therapeutic gain of radiotherapy. We tested its ability to enhance the tumor radioresponse of an ovarian carcinoma and to influence the normal tissue radioresponse of recipient mice. Methods . Mice bearing 8-mm isotransplants of a syngeneic ovarian carcinoma, designated OCA-I, in their legs were treated with 40 mg/kg paclitaxel i.v., 14–60 Gy single-dose local tumor irradiation, or both; radiation was given under ambient conditions 1–96 h after paclitaxel. Tumor growth delay, tumor cure rate (TCD 50 assay), and delay in tumor recurrences were measured. Normal tissue radioresponse was determined using jejunal crypt cell survival at 3.5 days after exposure of mice to 9–14 Gy single dose of total body irradiation; the mice were untreated or treated with 40 mg/kg i.v. paclitaxel 4–96 h before irradiation. Results . Paclitaxel alone was effective against OCA-I, but its combination with irradiation produced supra-additive tumor growth delay. It also reduced TCD 50 values and delayed tumor recurrences. The enhancement of tumor radioresponse ranged from 1.33 to 1.96; the value increased as the time between paclitaxel administration and tumor irradiation increased up to 48 h, but then decreased again at 96 h. In contrast, paclitaxel protected jejunum against radiation damage by factors of 1.03 to 1.07 when given 24–96 h before irradiation. It showed some potentiation of damage (by a factor of 1.07), but only when given 4 h before irradiation. Conclusions . Paclitaxel potentiated tumor radioresponse if given within 4 days before irradiation, whereas it caused radioprotection of normal tissue (jejunum) at that time. Therefore, paclitaxel significantly increased therapeutic gain and so has potential for use in combination with radiotherapy for pelvic malignancies.

Water-soluble paclitaxel may cause less side effects and be less costly to administer in comparison to a taxol formulation using a cremophor EL/alcohol vehicle. In this study, polyethylene glycol (PEG; MW 5000) was conjugated to the 2' position of paclitaxel through a spacer succinyl group. PEG-paclitaxel as a non-ionic paclitaxel prodrug was highly water soluble (> 20 mg equiv. paclitaxel/ml). The release of paclitaxel from phosphate-buffered solution was pH dependent. The half-life of PEG-paclitaxel was 7.6, 54 and 311 min at pH 9.0, 7.4 and 6.0, respectively. PEG-paclitaxel inhibited the growth of B16 melanoma cells to an extent similar to that of paclitaxel. In MCA-4 mammary tumor-bearing mice, a single dose of PEG-paclitaxel (40 mg equiv. paclitaxel/kg body weight) significantly delayed tumor growth. The average number of days for the tumor to reach 12 from 8 mm in diameter increased from 6.5 days for control animals to 8.5 days for PEG-paclitaxel-treated animals and 9.4 days for paclitaxel-treated animals. These studies demonstrated that PEG may be used as an effective solubilizing carrier for paclitaxel.

Paclitaxel is a novel chemotherapeutic agent that arrests cells in the radiosensitive G2 and M phases of the cell cycle and as such may act as a specific cell cycle radiosensitizer. We recently reported that paclitexel induces mitotic arrest in the MCA-4 murine mammary carcinoma and enhances radio-response of this tumor. However, the greatest enhancement was observed not when radiation was given at the time of peak mitotic arrest, which was 9 h after paclitaxel administration, but when it was given 24 h after paclitaxel. This implied the involvement of other mechanisms in radiosensitization; we hypothesized that tumor reoxygenation was a likely mechanism based on the observed massive loss of mitotically arrested cells at 24 h. The present study shows that paclitaxel greatly enhanced MCA-4 tumor radioresponse when radiation was given under air-breathing conditions (DMF = 1.74), but not when it was performed under hypoxic conditions. This observation supports the hypothesis of tumor reoxygenation as a mechanism of enhancement of tumor radioresponse. That reoxygenation occurred in tumors treated with paclitaxel 24 h earlier was confirmed by direct measurements of pO2 values, using the Eppendorf pO2 histograph. Median pO2 values increased from 6.2 mmHg in untreated tumors to 10.0 mmHg in tumors treated with paclitaxel. These observations emphasize the importance of timing of paclitaxel administration in relation to radiation treatment.

Purpose: The purpose of this investigation was to follow up our previous studies on the development of apoptosis in irradiated murine tumors by testing whether an apoptotic subpopulation of cells reemerges between fractionated exposures. Methods and Materials: Mice bearing a murine ovarian carcinoma, OCa-I, were treated in vivo with two fractionation protocols: two doses of 12.5 Gy separated by various times out to 5 days and multiple daily fractions of 2.5 Gy. Animals were killed 4 h after the last dose in each protocol, and the percent apoptosis was scored from stained histological sections made from the irradiated tumors according to the specific features characteristic of this mode of cell death. Results: The 12.5+12.5 Gy protocol yielded a net total percent apoptosis of about 45% when the two doses were separated by 5 days (total dose=25 Gy), whereas the 2.5 Gy per day protocol yielded about 50% net apoptotic cells when given for 5 days (total dose=12.5 Gy). These values are to be compared to the value of 36% apoptotic cells that is yielded by large single doses (> 25 Gy). Thus, these results indicate that an apoptotic subpopulation of cells reemerged between the fractions in both protocols, but the kinetics appeared to be delayed in the 12.5+12.5 Gy vs. the multiple 2.5 Gy protocol. Conclusion: This reemergence of cells with the propensity for radiation -induced apoptosis between fractionated exposures is consistent with a role for this mode of cell death in the response of tumors to radiotherapy and may represent the priming of a new subpopulation of tumor cells for apoptosis as part of normal tumor homeostasis to counterbalance cell division.

A number of radiobiologic parameters of radiation-induced apoptosis were investigated in a syngeneic murine B-cell lymphoma, designated LY-TH. These included radiation dose effect, kinetics of apoptosis development, the effect of hypoxia and split-dose recovery. Tumors, 8 mm in size, were locally irradiated with graded doses ranging from 1 to 10-Gy gamma rays. Radiation-induced apoptosis was observed as early as 1 h after irradiation, peaked between 4 and 6 h and could no longer be detected 24 h later. The magnitude of the apoptotic response generally increased with radiation dose, but lower doses seemed to be relatively more effective than higher doses. Tumor hypoxia, produced by tumor clamping, inhibited induction of apoptosis by a factor of about 2.5. When two doses of radiation were separated by times of 1-10 days, the proportion of apoptotic cells induced by the second dose was greatly reduced compared to the initial dose. This reduction was the greatest when the second dose was given 2 days after the first dose. The proportion of apoptotic cells induced by the second dose slowly recovered after 2 days but it did not return to the initial levels after as long as 10 days.