Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors.

Patients with malignant gliomas have a very poor prognosis. To explore a novel and more effective approach for the treatment of malignant gliomas, a strategy that combined tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and radiation treatment (RT) was designed in this study. Plasmid pE4-GFP was constructed by including the radioinducible early growth response gene 1 (Egr-1) promoter, and it yielded the best response with fractionated RT. Plasmid pE4-TRAIL was constructed by including the Egr-1 promoter and evaluated using U251 and U87 glioma cells. In the assay of apoptosis and killing activities, pE4-TRAIL exhibited radioresponse. pE4-TRAIL combined with RT is capable of inducing cell death synergistically. The expression of TRAIL death receptors was evaluated; which may be influenced by RT. Glioma cells with wild-type p53 showed upregulated expression of death receptors, and more synergistic effects on killing activities are expected. pE4-TRAIL was transfected into the subcutaneous U251 glioma cells in nude mice by the in vivo electroporation method. In the mice treated with pE4-TRAIL and RT, apoptotic cells were detected in pathological sections, and a significant difference of tumor volumes was observed when compared with the other groups (P<0.001). Our results indicate that radioresponsive gene therapy may have great potential as a novel therapy because this therapeutic system can be spatially or temporally controlled by exogenous RT and provides specificity and safety.

IUdR polymers for combined continuous low-dose rate and high-dose rate sensitization of experimental human malignant gliomas.

Local polymeric delivery enhances IUdR radiosensitization of human malignant gliomas (MG). The combined low-dose rate (LDR) (0.03 Gy/h) and fractionated high-dose rate (HDR) treatments result in cures of experimental MGs. To enhance efficacy, we combined polymeric IUdR delivery, LDR, and HDR for treatments of both subcutaneous and intracranial MGs. In vitro: Cells (U251 MG) were trypsinized and replated in triplicate 1 day prior to LDR irradiation in media either without (control) or with 10 microM IUdR. After 72 hr, LDR irradiation cells were acutely irradiated (1.1 Gy/min) with increasing (0, 1.25, 2.5, 5.0, or 10 Gy) single doses. Implantable IUdR polymers [(poly(bis(p-carboxyphenoxy)-propane) (PCPP): sebaic acid (PCPP:SA), 20:80] (50% loading; 10 mg) were synthesized. In vivo: For flank vs. intracranial tumors, mice had 6 x 10(6) subcutaneous vs. 2 x 10(5) intracranial cells. For intracranial or subcutaneous MGs, mice had intratumoral blank (empty) vs. IUdR polymer treatments. One day after implantation, mice had immediate external LDR (3 cGy/h x 3 days total body irradiation) or HDR (2 Gy BID x 4 days to tumor site) or concurrent treatments. For the in vitro IUdR treatments, LDR resulted in a striking increase in cell-killing when combined with HDR. For the in vivo LDR treatments of flank tumors, the growth delay was greater for the IUdR vs. blank polymer treatments. For the combined LDR and HDR, the IUdR treatments resulted in a dramatic decrease in tumor volumes. On day 60 the log V/V0 were -1.7 +/- 0.22 for combined LDR + HDR + IUdR polymer (P < 0.05 vs. combined LDR + HDR + blank polymer). Survival for the intracranial controls was 22.9 +/- 1.2 days. For the blank polymer + LDR vs. blank polymer + LDR + HDR treatments, survival was 25.3 +/- 1.7 (P = NS) vs. 48.1 +/- 3.5 days (P < 0.05). For IUdR polymer + LDR treatment survival was 27.3 +/- 2.3 days (P = NS). The most striking improvement in survival followed the IUdR polymer + LDR + HDR treatment: 66.0 + 6.4 days (P < 0.05 vs. blank polymer + LDR + HDR). The polymeric IUdR delivery plus combined continuous LDR and HDR treatments results in growth delay and improved survival in animals bearing the MG xenografts. This treatment may hold promise for the treatment of human MGs.

Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha.

The switch to an angiogenic phenotype is a fundamental determinant of neoplastic growth and tumor progression. We demonstrate that homozygous deletion of the p53 tumor suppressor gene via homologous recombination in a human cancer cell line promotes the neovascularization and growth of tumor xenografts in nude mice. We find that p53 promotes Mdm2-mediated ubiquitination and proteasomal degradation of the HIF-1alpha subunit of hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that regulates cellular energy metabolism and angiogenesis in response to oxygen deprivation. Loss of p53 in tumor cells enhances HIF-1alpha levels and augments HIF-1-dependent transcriptional activation of the vascular endothelial growth factor (VEGF) gene in response to hypoxia. Forced expression of HIF-1alpha in p53-expressing tumor cells increases hypoxia-induced VEGF expression and augments neovascularization and growth of tumor xenografts. These results indicate that amplification of normal HIF-1-dependent responses to hypoxia via loss of p53 function contributes to the angiogenic switch during tumorigenesis.

Retinoic acid-mediated growth inhibition of small cell lung cancer cells is associated with reduced myc and increased p27Kip1 expression.

Human lung cancer cells, including small cell lung carcinoma (SCLC), frequently lose expression of retinoic acid receptor beta (RAR-beta) and are resistant to the growth inhibitory activity of all-trans retinoic acid (RA). To elucidate the role of RAR-beta in the growth regulation of SCLC by retinoids, we restored RAR-beta expression in RAR-beta-negative H209 SCLC cells by retroviral transduction (H209-RAR-beta). We found that H209-RAR-beta, but not parental H209 cells, underwent growth inhibition upon RA treatment. RA-treated H209-RAR-beta cells arrested in G1 and displayed reduced L-myc expression and cyclin-dependent kinase 2 (cdk2) activity compared with untreated cells. RA treatment of H209-RAR-beta cells was also accompanied by increased expression of the cdk inhibitor p27Kip1, whereas no differences in the expression of L-myc or p27Kip1 were detected upon RA treatment of parental H209 cells. The RA-induced growth arrest of H82 SCLC cells, which express endogenous RAR-beta, was also associated with reduced c-myc and increased p27Kip1 expression. We found that ectopic expression of p27Kip1 induced growth inhibition in both H209 and H82 cells, and that sustained myc expression in H209-RAR-beta cells promoted the induction of apoptosis upon RA addition. Our observations indicate that RAR-beta gene transfer can restore RA sensitivity in SCLC cells and suggest that myc and p27Kip1 may represent critical mediators of the RA-induced cell cycle arrest in SCLC cells expressing RAR-beta.

Raf-1-induced cell cycle arrest in LNCaP human prostate cancer cells.

Prostate cancer is the most commonly diagnosed neoplasm in men. LNCaP cells continue to possess many of the molecular characteristics of in situ prostate cancer. These cells lack ras mutations, and mitogen-activated protein kinase (MAPK) is not extensively phosphorylated in these cells. To determine the effects of ras/raf/MAPK pathway activation in these cells, we transfected LNCaP cells with an activatable form of c-raf-1(deltaRaf-1:ER). Activation of deltaRaf-1:ER, with resultant MAPK activation, reduced plating efficiency and soft agarose cloning efficiency 30-fold in LNCaP cells. Cell cycle distribution showed an accumulation of cells in G1 and was associated with the induction of CDK inhibitor p21WAF1/CIP1 at the protein and mRNA levels. p21WAF1/CIP1 mRNA stability was increased after deltaRaf-1:ER activation. In addition, activated deltaRaf-1:ER induced the senescence associated-beta-galactosidase in LNCaP cells. These data demonstrate that raf activation can activate growth inhibitory pathways leading to growth suppression in prostate carcinoma cells and also suggest that raf/MEK/MAPK pathway activation, rather than inhibition, may be a therapeutic target for some human prostate cancer cells.

Synthetic, implantable polymers for IUdR radiosensitization of experimental human malignant glioma.

BACKGROUND: Recently, polymeric controlled delivery of chemotherapy has been shown to improve survival of patients with malignant glioma. We tested the delivery of IUdR via polymers for radiosensitization of experimental intracranial human malignant glioma. To assess efficacy, we measured the in vitro release, the in vivo delivery of IUdR and the resultant radiosensitization of experimental human U251 glioblastoma xenografts. METHODS: In vitro: To measure release, increasing (10%, 30%, 50%) proportions of IUdR in synthetic [(poly(bis(p-carboxyphenoxy)-propane) (PCPP):sebacic acid (SA) polymer discs were serially incubated in buffered saline and the supernatant fractions were assayed. In vivo: To compare local vs. systemic delivery, mice bearing flank xenografts had intratumoral or contralateral flank IUdR polymer (50% loading) treatments. Mice bearing intracranial (i.c.) xenografts had i.c. vs. flank IUdR polymer treatments. Four or 8 days after implantation of polymers, mice were sacrificed and the percentage tumor cells that were labeled with IUdR was measured using quantitative microscopic immunohistochemistry. For comparisons of radiosensitization, mice bearing i.c. xenografts had i.c. vs. flank IUdR polymers and cranial fractionated external beam irradiation (2 Gy BID x 4 days). RESULTS: In vitro: Increasing percentage loadings of IUdR resulted in higher percentages of release: 43.7 +/- 0.1, 70.0 +/- 0.2, and 90.2 +/- 0.2 (p < 0.001 ANOVA) for the 10, 30, and 50% loadings, respectively. In vivo: For the flank tumors, both the ipsilateral and contralateral IUdR polymers resulted in similarly high percentages labeling of the tumors vs. time. For the ipsilateral IUdR polymers, the percentages of tumor cellular labeling after 4 vs. 8 days were 45.8 +/- 7.0 vs. 40.6 +/- 3.9 (p = NS. For the contralateral polymer implants, the percentages tumor cellular labeling were 43.9 +/- 10.1 vs. 35.9 +/- 5.2 (p = NS) measured 4 vs. 8 days after implantation. For the i.c. tumors treated with extracranial IUdR polymers, the percentages of tumor cellular labeling were low: 13.9 +/- 8.8 and 11.2 +/- 5.7 measured 4 and 8 days after implantation. For the i.c. tumors having the i.c. IUdR polymers, however, the percentages labeling were comparatively much higher: 34.3 +/- 4.9 and 35.3 +/- 4.0 on days 4 and 8, respectively. For the i.c. tumors, examination of the percentage cellular labeling vs. distance from the implanted IUdR polymer showed labeling was highest closest to the polymer disc. Radiosensitization: For mice bearing i.c. tumors and receiving flank vs. intracranial IUdR polymer treatments, the survival after external beam irradiation was significantly higher for the intracranial treatments: 49 + 8.9 vs. 80 + 4.1 (p = 0.03) days, respectively. CONCLUSIONS: Implantable biodegradable polymers provide the local, controlled release of IUdR and result in the high, local delivery of IUdR to experimental intracranial human malignant glioma. The local delivery and labeling result in improved survival following radiotherapy. This technique holds promise for the local delivery of IUdR for radiosensitization of human brain tumors.

Predicting cancer rates in astronauts from animal carcinogenesis studies and cellular markers.

The radiation space environment includes particles such as protons and multiple species of heavy ions, with much of the exposure to these radiations occurring at extremely low average dose-rates. Limitations in databases needed to predict cancer hazards in human beings from such radiations are significant and currently do not provide confidence that such predictions are acceptably precise or accurate. In this article, we outline the need for animal carcinogenesis data based on a more sophisticated understanding of the dose-response relationship for induction of cancer and correlative cellular endpoints by representative space radiations. We stress the need for a model that can interrelate human and animal carcinogenesis data with cellular mechanisms. Using a broad model for dose-response patterns which we term the "subalpha-alpha-omega (SAO) model", we explore examples in the literature for radiation-induced cancer and for radiation-induced cellular events to illustrate the need for data that define the dose-response patterns more precisely over specific dose ranges, with special attention to low dose, low dose-rate exposure. We present data for multiple endpoints in cells, which vary in their radiosensitivity, that also support the proposed model. We have measured induction of complex chromosome aberrations in multiple cell types by two space radiations, Fe-ions and protons, and compared these to photons delivered at high dose-rate or low dose-rate. Our data demonstrate that at least three factors modulate the relative efficacy of Fe-ions compared to photons: (i) intrinsic radiosensitivity of irradiated cells; (ii) dose-rate; and (iii) another unspecified effect perhaps related to reparability of DNA lesions. These factors can produce respectively up to at least 7-, 6- and 3-fold variability. These data demonstrate the need to understand better the role of intrinsic radiosensitivity and dose-rate effects in mammalian cell response to ionizing radiation. Such understanding is critical in extrapolating databases between cellular response, animal carcinogenesis and human carcinogenesis, and we suggest that the SAO model is a useful tool for such extrapolation.

Synthetic, implantable polymers for local delivery of IUdR to experimental human malignant glioma.

PURPOSE: Recently, polymeric controlled delivery of chemotherapy has been shown to improve survival of patients with malignant glioma. We evaluated whether we could similarly deliver halogenated pyrimidines to experimental intracranial human malignant glioma. To address this issue we studied the in vitro release from polymers and the in vivo drug delivery of IUdR to experimental human U251 glioblastoma xenografts. METHODS AND MATERIALS: In vitro: To measure release, increasing (10%, 30%, 50%) proportions of IUdR in synthetic [(poly(bis(p-carboxyphenoxy)-propane) (PCPP):sebacic acid (SA) polymer discs were serially incubated in buffered saline and the supernatant fractions were assayed. In vivo: To compare local versus systemic delivery, mice bearing flank xenografts had intratumoral or contralateral flank IUdR polymer (50% loading) treatments. Mice bearing intracranial (i.c.) xenografts had i.c. versus flank IUdR polymer treatments. Four or 8 days after implantation of polymers, mice were sacrificed and the percentage tumor cells that were labeled with IUdR was measured using quantitative microscopic immunohistochemistry. RESULTS: In vitro: Increasing percentage loadings of IUdR resulted in higher percentages of release: 43.7 + 0.1, 70.0 + 0.2, and 90.2 + 0.2 (p < 0.001 ANOVA) for the 10%, 30%, and 50% loadings, respectively. In vivo: For the flank tumors, both the ipsilateral and contralateral IUdR polymers resulted in similarly high percentages labeling of the tumors versus time. For the ipsilateral IUdR polymers, the percentage of tumor cellular labeling after 4 days versus 8 days was 45.8 +/- 7.0 versus 40.6 +/- 3.9 (p = NS). For the contralateral polymer implants, the percentage of tumor cellular labeling were 43.9 +/- 10.1 versus 35.9 +/- 5.2 (p = NS) measured 4 days versus 8 days after implantation. For the i.c. tumors treated with extracranial IUdR polymers, the percentage of tumor cellular labeling was low: 13.9 +/- 8.8 and 11.2 +/- 5.7 measured 4 and 8 days after implantation. For the i.c. tumors having the i.c. IUdR polymers, however, the percentage labeling was comparatively much higher: 34.3 +/- 4.9 and 35.3 +/- 4.0 on days 4 and 8, respectively. For the i.c. tumors, examination of the percentage cellular labeling versus distance from the implanted IUdR polymer showed that labeling was highest closest to the polymer disc. CONCLUSION: Synthetic, implantable biodegradable polymers provide the local, controlled release of IUdR and result in the high, local delivery of IUdR to experimental intracranial human malignant glioma. This technique holds promise for the local delivery of IUdR for radiosensitization of human brain tumors.

Sensitivity of human prostatic carcinoma cell lines to low dose rate radiation exposure.

PURPOSE: Low dose rate radioemitters, such as 125I, 103Pd, and 89Sr, have been used both for local and systemic treatment of prostate cancer. Most normal cells exposed to ionizing radiation characteristically activate cell cycle checkpoints, resulting in cell cycle arrest at the G1/S and G2/M transition points. Cancer cells are typically quite sensitive to radiation killing late in the G2 phase of the replicative cell cycle. Furthermore, most cancer cells accumulating at the G2/M transition point as a result of low dose rate radiation exposure appear to become sensitive to further low dose rate irradiation. For this reason, protracted exposure of cancer cells to low dose rate radiation has been proposed to result in increased cancer cell killing as compared with brief exposures of cancer cells to high dose rate radiation. Since many human prostatic carcinomas contain somatic genome alterations targeting genes which affect the cell cycle and radiation-associated cell cycle checkpoints, we evaluated the effects of low dose rate radiation exposure on the cell cycle and on clonogenic survival for various human prostatic carcinoma cell lines. MATERIALS AND METHODS: Human prostatic carcinoma cells from the LNCaP, DU 145, PC-3, PPC-1, and TSU-Pr1 cell lines were exposed to low dose rate (0.25 Gy/hour) or high dose rate (60 Gy/hour) radiation in vitro and then assessed for radiation cytotoxicity by clonogenic survival assay. Cell cycle perturbations following protracted exposure to low dose rate radiation were evaluated using flow cytometry. RESULTS: For LNCaP cells, low dose rate radiation exposure resulted in an accumulation of cells at both the G1/S and the G2/M cell cycle transition points. For DU 145, PC-3, PPC-1, and TSU-Pr1 cells, treatment with low dose rate radiation triggered G2/M cell cycle arrest, but not G1/S arrest. Unexpectedly, the cell cycle redistribution pattern phenotypes observed, G1/S and G2/M cell cycle arrest versus G2/M arrest alone, appeared to have little effect on low dose rate radiation survival. Furthermore, while PC-3, PPC-1, and TSU-Pr1 cells exhibited increased cytotoxic sensitivity to low dose rate versus fractionated high dose rate radiation treatment, DU 145 and LNCaP cells did not. CONCLUSIONS: Radiation-associated pertubations in replicative cell cycle progression were not dominant determinants of low dose rate radiation killing efficacy in human prostate cancer cell lines in vitro.

Effect of interstitial and/or systemic delivery of tirapazamine on the radiosensitivity of human glioblastoma multiforme in nude mice.

The purpose of this study was to investigate the feasibility and the efficacy of administering tirapazamine by a slow-releasing polymer disc that was implanted interstitially into a U251 (human glioblastoma multiforme) tumor grown in nude mice. Tumor-bearing animals, with a tumor nodule 0.8 cm3 in size, were distributed to groups receiving combinations of empty or drug-containing polymer implants in the tumor or contralateral leg, intraperitoneal (i.p.) drug, and/or irradiation. The drug (i.p.) alone (14 mg/kg x6) or in combination with tumor drug implant (2 mg) did not significantly increase the tumor volume doubling time compared to that of control animals. Given with 12 Gy of irradiation in twice a day 2-Gy fractions, combined i.p. drug and tumor drug implant significantly delayed tumor growth compared to irradiation alone, which was not achieved with either drug treatment alone added to irradiation. Toxicity, as manifested by transient weight loss, was primarily seen in animals receiving radiation and i.p. tirapazamine. These results indicated that a slow-releasing tirapazamine disc can be produced and the addition of an interstitially implanted tirapazamine disc further increased the effectiveness of i.p. tirapazamine.

Activated Raf-1 causes growth arrest in human small cell lung cancer cells.

Small cell lung cancer (SCLC) accounts for 25% of all lung cancers, and is almost uniformly fatal. Unlike other lung cancers, ras mutations have not been reported in SCLC, suggesting that activation of ras-associated signal transduction pathways such as the raf-MEK mitogen-activated protein kinases (MAPK) are associated with biological consequences that are unique from other cancers. The biological effects of raf activation in small cell lung cancer cells was determined by transfecting NCI-H209 or NCI-H510 SCLC cells with a gene encoding a fusion protein consisting of an oncogenic form of human Raf-1 and the hormone binding domain of the estrogen receptor (DeltaRaf-1:ER), which can be activated with estradiol. DeltaRaf-1:ER activation resulted in phosphorylation of MAPK. Activation of this pathway caused a dramatic loss of soft agar cloning ability, suppression of growth capacity, associated with cell accumulation in G1 and G2, and S phase depletion. Raf activation in these SCLC cells was accompanied by a marked induction of the cyclin-dependent kinase (cdk) inhibitor p27(kip1), and a decrease in cdk2 protein kinase activities. Each of these events can be inhibited by pretreatment with the MEK inhibitor PD098059. These data demonstrate that MAPK activation by DeltaRaf-1:ER can activate growth inhibitory pathways leading to cell cycle arrest. These data suggest that raf/MEK/ MAPK pathway activation, rather than inhibition, may be a therapeutic target in SCLC and other neuroendocrine tumors.

Hypoxic cell cytotoxin tirapazamine induces acute changes in tumor energy metabolism and pH: a 31P magnetic resonance spectroscopy study.

Tirapazamine is a hypoxic cell cytotoxin in phase II/III trials. To further understand its mechanism of action in vivo, we examined the effect of tirapazamine on tumor energy metabolism and pH. RIF-1 and SCCVII tumors were grown subcutaneously in the flanks of C3H mice. Tumor energy metabolism, expressed as the ratio of inorganic phosphate to nucleotide triphosphate (Pi/NTP), and intracellular pH (pHi), were measured by 31P magnetic resonance spectroscopy (MRS). In RIF-1 and SCCVII tumors, tirapazamine increased the Pi/NTP ratio by 2.6-fold and 3-fold, respectively, within the first hour after an intraperitoneal dose of 0.3 mmol/kg. A corresponding decrease in pHi from 7.05+/-0.07 to 6.48+/-0.06, and 7.21+/-0.09 to 6.45+/-0.02 in RIF-1 and SCCVII tumors, respectively, was observed. The decrease in tumor 31P bioenergetics and pH was reversible, as exemplified by RIF-1 tumors, which showed a further increase in Pi/NTP ratio of 3.5-fold by 5-8 hr, returning to normal range at 24 hr. Corresponding pHi of RIF-1 tumors was 6.88+/-0.05 at 5-8 hr and 7.16+/-0.05 at 24 hr. We concluded that tirapazamine induces acute changes in tumor energy metabolism and pHi. These findings are relevant to the rational selection and optimal timing of coadministered therapy.

Protracted exposure radiosensitization of experimental human malignant glioma.

Clinical modulation of radiosensitivity via combined fractionated high dose rate and continuous ultra-low dose rate irradiation (ULDR) holds promise for the radiosensitization of human malignant gliomas. We measured both the in vitro and in vivo responses of a human malignant glioma cell line to combined continuous ULDR and high dose rate treatments. For in vitro ULDR treatments, U251 human malignant glioma cells were cultured in media containing tritiated water to yield a continuous dose rate of 0.03 Gy/hr. After exposures of 24, 48, or 72 hr, cells were acutely (1.1 Gy/min) irradiated, replated, and scored for colony formation. In vivo, U251 flank xenografts in nude mice had 125-iodine (125-I) seed brachytherapy at a dose rate of 0.05 Gy/hr. For whole-body continuous ULDR (0.03 Gy/hr), a 137-Cs source was mounted a fixed distance above the cages of animals bearing xenografts. After 3 days' continuous exposure, xenografts were acutely irradiated (2 Gy x 8 vs. 5 Gy x 2 daily fractions), and the regrowth delay in tumors was measured. In vitro, exposure to ULDR (0.03 Gy/hr) alone caused only modest killing and reduced the surviving fraction by approximately 0.2 logs after 72 hr exposure. The highest (10 Gy) dose of acute irradiation alone reduced survival by 1 log. However, U251 cell killing increased to 2.5 logs after combined HDR and ULDR treatments. Linear-quadratic modeling showed comparatively greater increase in the beta than the alpha coefficients of the linear-quadratic model for cell killing. In vivo, the 125-I seed brachytherapy treatments delayed tumor growth but resulted in no regression. The HDR treatments (5 Gy x 2 or 2 Gy x 8 daily fractions) caused growth delays (in days) of 17+/-2 or 16+/-2 (P=NS) days, respectively. The combined seed and 5 Gy x 2 or 2 Gy x 8 daily fractions regimen resulted in striking prolongation of regrowth delay (52.3+/-8.7 vs. 59.5+/-7.7 days) (P < 0.001 vs. HDR treatments alone). External ULDR alone caused no regression and minimal growth delay. Combined continuous external ULDR and the 5 Gy x 2 vs. 2 Gy x 8 daily fraction regimens resulted in prolongation of growth delay (33+/-0.9 (P=0.01 vs. 5 Gy x 2 daily fractions alone) vs. 35+/-0.7 (P=0.049 vs. 2 Gy x 8 daily fractions alone). We conclude that continuous ULDR increases the effect of HDR treatments of experimental malignant glioma. This increased effect may prove clinically important in the treatment of human malignant brain tumors.

Implantable biodegradable polymers for IUdR radiosensitization of experimental human malignant glioma.

PURPOSE: The potential of halogenated pyrimidines for the radiosensitization of human malignant gliomas remains unrealized. To assess the role of local delivery for radiosensitization, we tested a synthetic, implantable biodegradable polymer for the controlled release of 5-iodo-2'-deoxyuridine (IUdR) both in vitro and in vivo and the resultant radiosensitization of human malignant glioma xenografts in vivo. MATERIALS AND METHODS: In vitro: To measure release, increasing (10%, 30%, 50%) proportions (weight/weight) of IUdR in the polyanhydride [(poly(bis(p-carboxyphenoxy)-propane) (PCPP): sebacic acid (SA) (PCPP : SA ratio 20:80)] polymer discs were incubated (1 ml phosphate-buffered saline, 37 degrees C). The supernatant fractions were serially assayed using high performance liquid chromatography. To measure modulation of release, polymer discs were co-loaded with 20 microCi 5-125-iodo-2'-deoxyuridine (125-IUdR) and increasing (10%, 30%, or 50%) proportions of D-glucose. To test radiosensitization, cells (U251 human malignant glioma) were sequentially exposed to increasing (0 or 10 microM) concentrations of IUdR and increasing (0, 2.5, 5.0, or 10 Gy) doses of acute radiation. In vivo. To measure release, PCPP : SA polymer discs having 200 microCi 125-IUdR were surgically placed in U251 xenografts (0.1-0.2 cc) growing in the flanks of nude mice. The flanks were reproducibly positioned over a collimated scintillation detector and counted. To measure radiosensitization, PCPP : SA polymer discs having 0% (empty) or 50% IUdR were placed in the tumor or contralateral flank. After five days, the tumors were acutely irradiated (500 cGy x 2 daily fractions). RESULTS: In vitro: Intact IUdR was released from the PCPP : SA polymer discs in proportion to the percentage loading. After 4 days the cumulative percentages of loaded IUdR that were released were 43.7 +/- 0.1, 70.0 +/- 0.2, and 90.2 +/- 0.2 (p < 0.001 ANOVA) for the 10, 30, and 50% loadings. With 0, 10, 30, or 50% D-glucose co-loading, the cumulative release of 125-IUdR from PCPP : SA polymers was 21, 70, 92, or 97% (p < 0.001), respectively, measured 26 days after incubation. IUdR radiosensitized U251 cells in vitro. Cell survival (log10) was -2.02 +/- 0.02 and -3.68 +/- 0.11 (p < 0.001) after the 10 Gy treatment and no (control) or 10 microM IUdR exposures, respectively. In vivo: 125-IUdR Release: The average counts (log10 cpm +/- SEM) (hours after implant) were 5.2 +/- 0.05 (0.5), 4.3 +/- 0.07 (17), 3.9 +/- 0.08 (64), and 2.8 +/- 0.06 (284). Radiosensitization: After intratumoral implantation of empty polymer or intratumoral 50% IUdR polymer, or implantation of 50% IUdR polymers contralateral to tumors the average growth delays of tumors to 4 times the initial volumes were 15.4 +/- 1.8, 20.1 + 0.1, and 20.3 + 3.6 (mean + SEM) days, respectively (p = 0.488 one-way ANOVA). After empty polymer and radiation treatments, no tumors regressed and the growth delay was 31.1 + 2.1 (p = 0.046 vs. empty polymer alone) days. After implantation of 50% IUdR polymers either contralateral to the tumors or inside the tumors, followed by radiation, tumors regressed; growth delays to return to the initial average volumes of 14.0 + 3.6 or 24.2 + 0.2 (p < 0.01) days, respectively. CONCLUSIONS: Synthetic, implantable biodegradable polymers hold promise for the controlled release and local delivery of IUdR for radiosensitization of gliomas.

Decreasing resistance during fast infusion of a subcutaneous tumor.

High interstitial pressure limits the uptake of systemically-administered macromolecular agents. Fast infusion, which has been found to spread by convection macromolecules in the brain, was examined in a xenograft tumor system. Subcutaneous human U251 glioblastoma tumors (0.3-1.3 cm3) were infused for up to 40 minutes starting at 20 microliters/minutes while line pressure was recorded. The spread of blue dextran (molecular weight 2 x 10(6) was examined in excised tumors. Resistance to infusion decreased with time so that the infusion rate could be increased without an increase in line pressure. Blue dextran was spread up to the length of the tumor (maximum of 1.5 cm), but the spreading appeared to be asymmetric. The results suggest the pressures produced by the infusion dilated the tumor tissue, thus producing increased hydraulic conductivity. Although this produces rapid convective spread, the spread is asymmetric. Possible methods for obtaining a more uniform or controlled convective spread of macromolecular agents are discussed.

Human papillomavirus E6 and E7 oncoproteins alter cell cycle progression but not radiosensitivity of carcinoma cells treated with low-dose-rate radiation.

PURPOSE: Low-dose-rate radiation therapy has been widely used in the treatment of urogenital malignancies. When continuously exposed to low-dose-rate ionizing radiation, target cancer cells typically exhibit abnormalities in replicative cell-cycle progression. Cancer cells that arrest in the G2 phase of the cell cycle when irradiated may become exquisitely sensitive to killing by further low-dose-rate radiation treatment. Oncogenic human papillomaviruses (HPVs), which play a major role in the pathogenesis of uterine cervix cancers and other urogenital cancers, encode E6 and E7 transforming proteins known to abrogate a p53-dependent G1 cell-cycle checkpoint activated by conventional acute-dose radiation exposure. This study examined whether expression of HPV E6 and E7 oncoproteins by cancer cells alters the cell-cycle redistribution patterns accompanying low-dose-rate radiation treatment, and whether such alterations in cell-cycle redistribution affect cancer cell killing. METHODS AND MATERIALS: RKO carcinoma cells, which contain wild-type P53 alleles, and RKO cell sublines genetically engineered to express HPV E6 and E7 oncoproteins, were treated with low-dose-rate (0.25-Gy/h) radiation and then assessed for p53 and p21WAF1/CIP1 polypeptide induction by immunoblot analysis, for cell-cycle redistribution by flow cytometry, and for cytotoxicity by clonogenic survival assay. RESULTS: Low-dose-rate radiation of RKO carcinoma cells triggered p53 polypeptide elevations, p21WAF1/CIP1 induction, and arrest in the G1 and G2 phases of the cell cycle. In contrast, RKO cells expressing E6 and E7 transforming proteins from high-risk oncogenic HPVs (HPV 16) arrested in G2, but failed to arrest in G1, when treated with low-dose-rate ionizing radiation. Abrogation of the G1 cell-cycle checkpoint activated by low-dose-rate radiation exposure appeared to be a characteristic feature of transforming proteins from high-risk oncogenic HPVs: RKO cells expressing E6 from a low-risk nononcogenic HPV (HPV 11) exposed to low-dose-rate radiation arrested in both G1 and G2. Surprisingly, despite differences in cell-cycle redistribution accompanying low-dose-rate radiation treatment associated with high-risk HPV transforming protein expression, no consistent differences in clonogenic survival following low-dose-rate radiation treatment were found for RKO cell sublines expressing high-risk HPV oncoproteins and arresting only in G2 during low-dose-rate radiation exposure vs. RKO cell sublines exhibiting both G1 and G2 cell-cycle arrest when irradiated. CONCLUSION: The results of this study demonstrate that neither HPV oncoprotein expression nor loss of the radiation-activated G1 cell-cycle checkpoint alter the sensitivity of RKO carcinoma cell lines to low-dose-rate radiation exposure in vitro. Perhaps for urogenital malignancies associated with oncogenic HPVs in vivo, HPV oncoprotein-mediated abrogation of the G1 cell-cycle checkpoint may not limit the potential efficacy of low-dose-rate radiation therapy.

Prediction of tumor response to experimental radioimmunotherapy with 90Y in nude mice.

PURPOSE: To identify those factors that predict variability in tumor response to 90Y-radioimmunotherapy based on measurement of incorporated activity and physical dimensions of individual tumors and to apply the concept of effective dose to radioimmunotherapy. METHODS AND MATERIALS: Human colon carcinoma xenografts growing in nude mice were treated with anti-CEA antibodies labeled with 90Y directly or through a bispecific antibody/labeled hapten system. Tumor response was measured as the delay in growth to eight times the treatment volume. Noninvasive activity (based on bremsstrahlung radiation) and dimension measurements were made in these animals at several times after label injection. The following parameters were compared for their ability to predict individual tumor response: (a) injected activity, (b) injected activity times a factor based on average uptake as a function of volume, (c) in vivo activity per volume measured in each animal at a single time, (d) the integral over time of in vivo activity per volume in each animal, and (e) the minimum dose for each animal in a uniformly active ellipsoid whose total activity and dimensions varied over time the same as the tumor. RESULTS AND CONCLUSION: After correcting for differences in injected activity, two parameters account for much of the variability in tumor response. One of these is the general trend of larger tumors to take up less activity per volume. Additional variability can be accounted for by the in vivo activity per volume measurements. The minimum dose as introduced here is likely to be useful in estimating the biologically effective dose delivered by each treatment.

Direct measurement of intratumor dose-rate distributions in experimental xenografts treated with 90Y-labeled radioimmunotherapy.

PURPOSE: To measure, quantify, and evaluate the planar dose-rate distribution for human tumor xenografts implanted into mice that are treated with 90Y-labeled monoclonal antibodies or bispecific antibodies and 90Y-labeled haptens. METHODS AND MATERIALS: Twenty-five LS174T human colon carcinoma tumors grown subcutaneously in nude mice were treated with 90Y by either directly labeled ZCE025 or bispecific ECA001-DBX antibody systems. A simple, quick technique using GAF radiochromic medium determined the dose-rate distribution in a plane passing through the tumor center. The dose-rate distribution is generated from exposure to activity situated in one-half of the tumor (0.045 to 0.83 g). RESULTS: Planar dose-rate distributions were obtained from the tumor xenografts. Planar dose-rate histograms were computed along with the coefficients of variance and skewness of the distributions. The observed dose-rate distributions were quantitatively compared to those calculated for a uniformly distributed activity in a half-ellipsoid of the same volume and approximate shape as the tumor half. The observed dose-rate distributions were usually broader with a more positive coefficient of skewness than the dose-rate distributions calculated from the uniformly active half-ellipsoids. For 90Y, tumor shape plays an important role in determining the minimum tumor dose. For these tumors, the tumor minimum dose-rate is always observed along the edge, usually where the edge curvature is most convex. Larger tumors tended to have broader dose-rate distributions and more positive coefficients of skewness. Exceptions to this trend were associated with dose-rate maxima displaced from the central regions due to activity heterogeneity or tumor size greatly exceeding the range of emission. Calculations for dose rate from the conventional Medical Internal Radiation Dose (MIRD) formulation exceeded the average and minimum dose rate derived from radiochromic media. The coefficient of skewness became more positive for increasing time between injection and tumor excision, consistent with the activity evolving into a more uniform activity distribution. CONCLUSION: Using radiochromic media to measure the spatial dose-rate distribution is a valuable method for comparing the dose-rate heterogeneity among experimental tumor xenografts in animals treated with radiolabeled antibodies. Tumor size (relative to the particle range) and changes in activity distribution radiolabeled antibodies. Tumor size (relative to the particle range) and changes in activity distribution affect the dose-rate distribution that are reflected by changes in the coefficients of skewness and variation of the dose-rate area histogram. The increase in coefficients of variation and skewness with tumor size and time results from the size of the 90Y beta particle penetration range that either exceeds or is comparable to the tumor dimensions. The minimum dose rate is more dependent, relative to the average and the maximum dose rates, on the curvature of the tumor surface.

The effect of polyamine depletion on the cytotoxic response to PUVA, gamma rays and UVC in V79 cells in vitro.

Difluoromethylornithine (DFMO) depletes cells of polyamines, sensitizing cells against the action of antineoplastic drugs and altering ability to repair radiation-induced DNA strand breaks. Others have hypothesized that the mechanism by which polyamine depletion sensitizes cells is through inhibition of DNA strand break repair or through altering the spectrum of initial DNA damage. We have compared the effect of polyamine depletion on cytotoxic effects in V79 cells for three agents that damage DNA: PUVA (8-methoxypsoralen and ultraviolet light, 365 nm), gamma-rays and UVC (ultraviolet light, 254 nm) in polyamine depleted V79 cells. DFMO pretreatment sensitizes cells to PUVA and gamma-ray toxicity but not to UVC. Unlike UVC photoinduction of DNA lesions, PUVA- and gamma-ray-induced DNA damage is modulated by chromatin structure. Our results suggest that polyamine depletion sensitizes cells to the action of PUVA and gamma-rays by mechanisms disparate from those for UVC, suggesting that the level or type of initial damage, rather than DNA repair, per se, may be the more important determinant of enhanced cytotoxicity.

Use of bremsstrahlung radiation to monitor Y-90 tumor and whole body activities during experimental radioimmunotherapy in mice.

BACKGROUND: Large differences in uptake between tumors, even for the same size, frequently observed in clinical and experimental radioimmunotherapy (RAIT), make monitoring of uptake in individual tumors imperative in comparing protocols. 90Y, widely-used for RAIT, emits no gamma radiation and absorption of the beta particle in tissue makes its detection unsuitable for in vivo monitoring. We tested whether bremsstrahlung radiation, produced when betas are decelerated by nuclei, could be used to monitor tumor uptake. METHODS: Subcutaneous human LS174T colon carcinoma tumors were grown in the upper thigh of nude mice and labeled antibody injected intracardially. With the tumor placed in the 2 cm-diameter aperture in a lead collimator, photons with energies from 100 to 200 keV transmitted through plastic, which absorbed the beta particles, were counted to maximize shielding from the rest of the body. The contribution of the normal tissues was subtracted by counting the non-tumor-bearing leg in the same position. Excretion was calculated from whole body activity determined by removing the collimator, placing the mouse in a syringe surrounded by tissue-equivalent material 10 cm from the detector, and counting photons between 200 and 740 keV to minimize the effect of tissue attenuation. RESULTS: For tumors larger than 0.14 gm, a good correlation was obtained between the in vivo bremsstrahlung measurements and the measurements on excised tumors in a calibrated well counter. Similar excretion rates observed in all the animals suggested that the whole body counting was accurate. CONCLUSIONS: Bremstrahlung detection appears feasible and reliable for monitoring both tumor and whole body activities.