Combination of Cytosine Deaminase With Uracil Phosphoribosyl Transferase With 5-fluorocytosine...
2
Conclusions: The use of hormonally-based dietary supplement such as ProstateRx should be discouraged due to preferential radio- sensitization of normal prostate cells. This effect would be exaggerated by fractionated radiotherapy or prolonged low-dose rate treatments. Little impact would occur after high dose-rate brachytherapy. Author Disclosure: B. Marples, None; D. Schoenherr, None; S. Galoforo, None; Y. Hasan, None; A.A. Martinez, None; G.D. Wilson, None. 2755 Lethal and Non-Lethal Toxicity Incidence in Gamma Ray and Neutron Exposed Mice Treated With and Without Amifostine A. O. Wahl 1 , D. Paunesku 1 , T. Paunesku 1 , Y. Kataoka 2 , D. J. Grdina 2 , G. Woloschak 1 1 Department of Radiation Oncology, Northwestern University, Chicago, IL, 2 Department of Radiation Oncology, The University of Chicago, Chicago, IL Purpose/Objective(s): Amifostine (WR2721, Ethyol Ò Medimmune Inc.) is a cytoprotective prodrug shown to protect mucosal tissue and salivary glands from radiotoxicity in clinical trials. The purpose of this study was to determine the effects of amifostine or WR-151327 on a variety of non-lethal toxicities in a large cohort of mice exposed to g-ray or fission-spectrum neutron irradiation (RT). Materials/Methods: From October 1984 to October 1985, 4000 B6CF mice received total body irradiation with cobalt-60 gamma or fission-spectrum neutron RT at Argonne National Laboratory. In the neutron cohort, amifostine, WR-151327 or saline was in- jected intraperitoneally 30 minutes before RT and in cobalt-60 cohort, amifostine or saline was injected with a similar protocol. The RT dose levels for cobalt-60 were 206 cGy and 417 cGy and for neutrons were 10 cGy and 40 cGy. No significant differences were noted between the unirradiated mice that received saline or amifostine (p = 0.326) and therefore, they were combined to form the control group. Following RT, daily death checks were performed, with moribund animals removed, sacrificed and necropsies per- formed by a DVM pathologist. Histopathological examination was performed in approximately 80% of the animals. Non-lethal gross necropsy findings (NLGNF) and lethal gross necropsy findings (LGNF) were recorded in various organ systems, which included late tissue toxicities, and logged into a pathology database. Logistic linear regression analysis (STATA/SE 9) was performed to compare the effect of RT dose on the incidence of non-tumor NLGNF and LGNF in each group. Results: In the cobalt-60 206 cGy dose level, the addition of amifostine was associated with no increase in NLGNF incidence compared to controls in harderian gland, pneumonitis, infection, hydrothorax, and uterus (p \0.05) and no increase in LGNF com- pared to controls in chronic renal disease and cysts (p \0.05). In the cobalt-60 417 cGy dose level, amifostine was associated with no differences in NLGNF compared to controls in the following sites (p \ 0.05): harderian gland, pneumonitis, ascites, edema, hydrothorax, infection and uterus. In the cobalt-60 417 cGy dose level, amifostine was associated with no increase in LGNF com- pared to controls in chronic renal disease (p \ 0.05). In the 10 cGy fission-spectum neutron cohort, NLGNF were not increased compared to controls with the addition of amifostine in the testes, adrenal gland, harderian gland, uterus, ascites, chronic renal disease, and infection (p \ 0.05). NLGNF were not increased in the amifostine group given 40 cGy fission spectrum neutron RT versus controls in the adrenal gland, uterus, ascites, chronic renal disease, bladder, liver and infection (p \ 0.05). Conclusions: Amifostine protected both cobalt-60 gamma ray and neutron exposed mice from developing non-lethal and lethal toxicities, which is similar to the protection amifostine afforded against RT-induced carcinogenesis reported in prior studies. Experiments to examine gene mutation frequency in tumor and normal tissue are currently underway. Author Disclosure: A.O. Wahl, None; D. Paunesku, None; T. Paunesku, None; Y. Kataoka, None; D.J. Grdina, None; G. Woloschak, None. 2756 Combination of Cytosine Deaminase With Uracil Phosphoribosyl Transferase With 5-fluorocytosine Radiosensitizes Rat Prostate Cancer Cells L. Xing 1 , 2 , X. Deng 1 , T. P. F. Gade 1 , C. C. Ling 1 , J. A. Koutcher 1 , G. C. Li 1 1 Memorial Sloan-Kettering Cancer Center, New York, NY, 2 Shandong Cancer Hospital and Institute, Jinan, China Purpose/Objective(s): The enzyme/prodrug gene therapy strategy combining cytosine deaminase (CD) and uracil phosphoribosyl transferase (UPRT) with the 5-fluorocytosine (5-FC) can significantly enhance the anti-tumor effects of CD/5-FC. The radiosensi- tizing ability of CD/5-FC has been reported preclinically and clinically, but that of CD-UPRT/5-FC has not been investigated. In this study, we assessed the radiosensitizing ability of expression of a fusion gene consisting of the yeast S. cerevisiae CD and the H. influenzae UPRT. Materials/Methods: R3327-AT cells were transfected with plasmids containing the hygromycin resistance gene and the fusion genes encoding either cd fused with mDsRed, or cd fused with uprt and mDsRed; clones stably expressing CD (R3327-CD) or CD/UPRT (R3327-CDUPRT) were selected and established. Expression of the fusion proteins was determined by flowcytometry (for mDsRed) and Western Blotting using anti-CD antibody. The sensitivity of R3327-CD and R3327-CDUPRT cells to 5-FC (0– 100 mg/ml for 24 h) was assessed by colony formation assay. In radiosensitizing experiments, R3327-CD and R3327-CDUPRT cells were treated with 5-FC at 50 mg/ml (R3327-CD) or 2 mg/ml (R3327-CDUPRT) for 24 h before irradiation (0–12 Gy), the survival fraction was determined by the colony formation assay and normalized to that of non-irradiated cells treated with 5- FC. The AUCs (Area Under Curve of the survival curves) were calculated to compare the cytotoxicity of the treatment among the cell lines. Results: The results of our experiments showed that 5-FC killed transfected R3327 cells in a dose dependent manner, and that both R3327-CD and R3327-CDUPRT cells were more sensitive to 5-FC than the parental controls. In addition, R3327-CDUPRT cells were more sensitive to 5-FC than R3327-CD cells (AUC CD /AUC CDUPRT = 12). Whereas the radiosensitivity of untreated R3327- CD, R3327-CDUPRT and parental cells were similar, 5-FC treatment radiosensitized the R3327-CD and R3327-CDUPRT but not the parental cells. Of the two transfected cell lines, a much smaller concentration (by 20 fold) of 5-FC is required to radiosensitize R3327-CDUPRT cells relative to that needed for the R3327-CD cells. For example, the same radiosensitizing effect was observed in R3327-CD cells with 50 mg/ml of 5-FC as in R3327-CDUPRT cells with only 2 mg/ml of 5-FC(AUC CD without 5-FC/AUC CD with 5-FC = 1.61, AUC CDUPRT without 5-FC/AUC CDUPRT with 5-FC = 1.56). Proceedings of the 49th Annual ASTRO Meeting S617
Transcript of Combination of Cytosine Deaminase With Uracil Phosphoribosyl Transferase With 5-fluorocytosine...
Proceedings of the 49th Annual ASTRO Meeting S617
Conclusions: The use of hormonally-based dietary supplement such as ProstateRx should be discouraged due to preferential radio-sensitization of normal prostate cells. This effect would be exaggerated by fractionated radiotherapy or prolonged low-dose ratetreatments. Little impact would occur after high dose-rate brachytherapy.
Author Disclosure: B. Marples, None; D. Schoenherr, None; S. Galoforo, None; Y. Hasan, None; A.A. Martinez, None;G.D. Wilson, None.
2755 Lethal and Non-Lethal Toxicity Incidence in Gamma Ray and Neutron Exposed Mice Treated
With and Without AmifostineA. O. Wahl1, D. Paunesku1, T. Paunesku1, Y. Kataoka2, D. J. Grdina2, G. Woloschak1
1Department of Radiation Oncology, Northwestern University, Chicago, IL, 2Department of Radiation Oncology,The University of Chicago, Chicago, IL
Purpose/Objective(s): Amifostine (WR2721, Ethyol� Medimmune Inc.) is a cytoprotective prodrug shown to protect mucosaltissue and salivary glands from radiotoxicity in clinical trials. The purpose of this study was to determine the effects of amifostineor WR-151327 on a variety of non-lethal toxicities in a large cohort of mice exposed to g-ray or fission-spectrum neutron irradiation(RT).
Materials/Methods: From October 1984 to October 1985, 4000 B6CF mice received total body irradiation with cobalt-60 gammaor fission-spectrum neutron RT at Argonne National Laboratory. In the neutron cohort, amifostine, WR-151327 or saline was in-jected intraperitoneally 30 minutes before RT and in cobalt-60 cohort, amifostine or saline was injected with a similar protocol. TheRT dose levels for cobalt-60 were 206 cGy and 417 cGy and for neutrons were 10 cGy and 40 cGy. No significant differences werenoted between the unirradiated mice that received saline or amifostine (p = 0.326) and therefore, they were combined to form thecontrol group. Following RT, daily death checks were performed, with moribund animals removed, sacrificed and necropsies per-formed by a DVM pathologist. Histopathological examination was performed in approximately 80% of the animals. Non-lethalgross necropsy findings (NLGNF) and lethal gross necropsy findings (LGNF) were recorded in various organ systems, whichincluded late tissue toxicities, and logged into a pathology database. Logistic linear regression analysis (STATA/SE 9) wasperformed to compare the effect of RT dose on the incidence of non-tumor NLGNF and LGNF in each group.
Results: In the cobalt-60 206 cGy dose level, the addition of amifostine was associated with no increase in NLGNF incidencecompared to controls in harderian gland, pneumonitis, infection, hydrothorax, and uterus (p\0.05) and no increase in LGNF com-pared to controls in chronic renal disease and cysts (p\0.05). In the cobalt-60 417 cGy dose level, amifostine was associated withno differences in NLGNF compared to controls in the following sites (p \ 0.05): harderian gland, pneumonitis, ascites, edema,hydrothorax, infection and uterus. In the cobalt-60 417 cGy dose level, amifostine was associated with no increase in LGNF com-pared to controls in chronic renal disease (p \ 0.05). In the 10 cGy fission-spectum neutron cohort, NLGNF were not increasedcompared to controls with the addition of amifostine in the testes, adrenal gland, harderian gland, uterus, ascites, chronic renaldisease, and infection (p \ 0.05). NLGNF were not increased in the amifostine group given 40 cGy fission spectrum neutronRT versus controls in the adrenal gland, uterus, ascites, chronic renal disease, bladder, liver and infection (p \ 0.05).
Conclusions: Amifostine protected both cobalt-60 gamma ray and neutron exposed mice from developing non-lethal and lethaltoxicities, which is similar to the protection amifostine afforded against RT-induced carcinogenesis reported in prior studies.Experiments to examine gene mutation frequency in tumor and normal tissue are currently underway.
Author Disclosure: A.O. Wahl, None; D. Paunesku, None; T. Paunesku, None; Y. Kataoka, None; D.J. Grdina, None;G. Woloschak, None.
2756 Combination of Cytosine Deaminase With Uracil Phosphoribosyl Transferase With 5-fluorocytosine
Radiosensitizes Rat Prostate Cancer CellsL. Xing1,2, X. Deng1, T. P. F. Gade1, C. C. Ling1, J. A. Koutcher1, G. C. Li1
1Memorial Sloan-Kettering Cancer Center, New York, NY, 2Shandong Cancer Hospital and Institute, Jinan, China
Purpose/Objective(s): The enzyme/prodrug gene therapy strategy combining cytosine deaminase (CD) and uracil phosphoribosyltransferase (UPRT) with the 5-fluorocytosine (5-FC) can significantly enhance the anti-tumor effects of CD/5-FC. The radiosensi-tizing ability of CD/5-FC has been reported preclinically and clinically, but that of CD-UPRT/5-FC has not been investigated.In this study, we assessed the radiosensitizing ability of expression of a fusion gene consisting of the yeast S. cerevisiae CD andthe H. influenzae UPRT.
Materials/Methods: R3327-AT cells were transfected with plasmids containing the hygromycin resistance gene and the fusiongenes encoding either cd fused with mDsRed, or cd fused with uprt and mDsRed; clones stably expressing CD (R3327-CD) orCD/UPRT (R3327-CDUPRT) were selected and established. Expression of the fusion proteins was determined by flowcytometry(for mDsRed) and Western Blotting using anti-CD antibody. The sensitivity of R3327-CD and R3327-CDUPRT cells to 5-FC (0–100 mg/ml for 24 h) was assessed by colony formation assay. In radiosensitizing experiments, R3327-CD and R3327-CDUPRTcells were treated with 5-FC at 50 mg/ml (R3327-CD) or 2 mg/ml (R3327-CDUPRT) for 24 h before irradiation (0–12 Gy), thesurvival fraction was determined by the colony formation assay and normalized to that of non-irradiated cells treated with 5-FC. The AUCs (Area Under Curve of the survival curves) were calculated to compare the cytotoxicity of the treatment amongthe cell lines.
Results: The results of our experiments showed that 5-FC killed transfected R3327 cells in a dose dependent manner, and that bothR3327-CD and R3327-CDUPRT cells were more sensitive to 5-FC than the parental controls. In addition, R3327-CDUPRT cellswere more sensitive to 5-FC than R3327-CD cells (AUCCD/AUCCDUPRT = 12). Whereas the radiosensitivity of untreated R3327-CD, R3327-CDUPRT and parental cells were similar, 5-FC treatment radiosensitized the R3327-CD and R3327-CDUPRT but notthe parental cells. Of the two transfected cell lines, a much smaller concentration (by�20 fold) of 5-FC is required to radiosensitizeR3327-CDUPRT cells relative to that needed for the R3327-CD cells. For example, the same radiosensitizing effect was observedin R3327-CD cells with 50 mg/ml of 5-FC as in R3327-CDUPRT cells with only 2 mg/ml of 5-FC(AUCCD without 5-FC/AUCCD
with 5-FC = 1.61, AUCCDUPRT without 5-FC/AUCCDUPRT with 5-FC = 1.56).
S618 I. J. Radiation Oncology d Biology d Physics Volume 69, Number 3, Supplement, 2007
Conclusions: Our study indicated that CD/UPRT expression, and to a less extent CD expression, significantly enhanced theefficacy of combined 5-FC and radiation treatment in R3327 prostate cancer cells. This suggests that CD/UPRT/5-FC strategymay have potential as an adjuvant to improve the outcome of radiotherapy.
Author Disclosure: L. Xing, None; X. Deng, None; T.P.F. Gade, None; C.C. Ling, None; J.A. Koutcher, None; G.C. Li, None.
2757 Effect of Ionizing Radiation on Rat Lung Tissue: A Proteomic Analysis
P. R. Graves1, H. Sankala1, Z. Vujaskovic2
1Virginia Commonwealth University, Richmond, VA, 2Duke University Medical School, Durham, NC
Purpose/Objective(s): The objective of this study is to examine the short and long-term effects of radiation on the rat lung pro-teome. Our goal is to determine proteomic changes associated with radiation-induced lung injury in order to identify early predic-tors of radiation response and potential therapeutic targets.
Materials/Methods: Fischer 344 rats were irradiated with 150 kV x-rays at a single dose of 28 Gy delivered to the right hemi-thorax. Control animals were sham irradiated. Rats were sacrificed and lung tissue harvested post radiation at the followingtime points: 1, 3, 7, 10 days, 2, 4, 6, 8,12,14, and 20 wks. As an initial analysis of these complex samples, total lung cell lysatesfrom each time point were resolved by one-dimensional gel electrophoresis and proteins visualized by silver staining. The proteinsthat showed differential levels of silver staining were excised and identified by mass spectrometry.
Results: Significant changes in protein expression were observed at different times post irradiation (Fig. 1). The most significantones are listed in Table 1. The process of validation of identified proteins by Western blotting is underway. Furthermore, a com-prehensive analysis of irradiated lung tissue using PF 2D, a non-gel based method for 2-dimensional fractionation of complex pro-tein samples is being conducted.
Conclusions: Proteins upregulated after radiation are most commonly associated with cellular proliferation and adhesion, hypoxia,oxidative stress, and hemodynamic changes. In comparison, preliminary results show two antioxidants with important roles in pul-monary protection against oxidative stress, glutathione-s-transferase and peroxiredoxin-2, were downregulated post-radiation.
Table 1
Proteins Down-regulated by radiation
1. Filamin
2. Alpha-1 proteinase inhibitor III
3. 14-3-3
4. Carbonic Anhydrase and Glutathione-S-transferase
5. Rho-GDI alpha
6. Peroxiredoxin-2
Proteins Up-regulated by radition
7. Xanthine dehyrogenase/oxidase
8. Alpha spectrin-2 (Fodrin)
9. Protein disulfide isomerase
10. Cyclophilin B
11. Annexin II
Author Disclosure: P.R. Graves, None; H. Sankala, None; Z. Vujaskovic, None.
2758 Detection of Circulating Tumor Cells in the Peripheral Blood of Patients With Androgen-Independent,
Advanced or Metastatic Prostate CancerD. T. Marshall, M. C. Mitas, U. B. Chaudhary, S. Gattoni-Celli
Medical University of South Carolina, Charleston, SC
Purpose/Objective(s): Accurate biochemical monitoring of late stage, androgen-independent prostate cancer (AIPC) is difficultand the correlation of clinical symptoms and disease progression with blood levels of prostate-specific antigen (PSA) is often poor.An increasing body of experimental evidence indicates that several types of cancer ‘‘leak’’ tumor cells into the bloodstream. Thepresence of circulating tumor cells (CTC) has been observed in colorectal and prostate cancer. Furthermore, CTC have been as-sociated with poor prognosis in patients with metastatic breast cancer. This suggests that the monitoring of CTC could provide
