Cytotoxic Effects Of Radiation Research Articles

Differential expression of small ubiquitin-like modifier family of proteins in patients with colorectal adenocarcinoma.

420 Background: The cytotoxic effects of radiation and chemotherapy are mediated in part by DNA damage. The small ubiquitin-like modifier (SUMO) family of proteins regulates DNA repair pathways by activating factors involved in homologous recombination (HR) or non- homologous end joining (NHEJ). Our objective was to determine the potential role of SUMO proteins in patients with colorectal cancer. Methods: We first assessed two established colorectal cancer cell lines (HCT116 and HT29) and two normal colonic mucosa cell populations utilizing ultra high-throughput expression analysis (Solexa) to examine differential expression of genes involved in SUMOylation (SAE1, SAE2, PIAS-1, and DNAPKcs). To verify the clinical relevance of SUMOylation in colorectal cancer, we obtained archived specimens from patients with colorectal cancer (n=51) and examined the expression of these proteins in both benign and malignant tissue by immunohistochemistry (IHC). Results: Ultra high-throughput expression analysis of cancer and normal cells revealed a 10 to 15-fold upregulation of SAE2 (heterodimer of the E1 activation enzyme in SUMOylation), a 10 to 11-fold upregulation of DNAPKcs (catalytic subunit in NHEJ repair pathway), and a 6-fold upregulation of SAE1 in cancer cells. In contrast, PIAS-1 showed similar expression levels between cancer and normal colonic cells. By IHC, SAE1 and SAE2 were highly expressed in 65% and 53% of tumor specimens, respectively; but only in 7% and 0% of normal tissues, respectively. DNAPKcs was also highly expressed in tumor tissues (63%) with corresponding low expression (0%) in normal tissues. Corroborating the cell line results, PIAS-1 was infrequently expressed in both tumor (10%) and normal tissues (0%). Conclusions: We are the first to demonstrate the differential expression of SUMO proteins in colorectal cancer cell lines and in clinical specimens. SUMO proteins have a role in DNA repair and their expression in colorectal cancer may modify tumor response to chemoradiotherapy. No significant financial relationships to disclose.

Morphology of Pararectal Lymph Nodes in Rectal Cancer after Neoadjuvant Therapy

Remodeling and cytograms of pararectal lymph nodes were studied in patients with rectal cancer after neoadjuvant therapy by different protocols. Radiotherapy and its combination with chemotherapy lead to an increase in the volume of connective tissue components and significant reduction of the volume density of lymphoid follicles without germinative centers. The counts of dividing cells, immunoblasts, and plasmoblasts in various compartments of the pararectal lymph nodes changed more significantly after radiotherapy, while changes in the count of monocytes and neutrophils were more pronounced after cytostatic therapy combined with exposure to ionizing radiation. These differences can be explained by the systemic toxic effect of chemotherapeutic drugs and primarily local cytotoxic effects of radiotherapy manifesting largely at the site of exposure.

Dendro[C60]fullerene DF-1 provides radioprotection to radiosensitive mammalian cells

In this study, the ability of the C(60) fullerene derivative DF-1 to protect radiosensitive cells from the effects of high doses of gamma irradiation was examined. Earlier reports of DF-1's lack of toxicity in these cells were confirmed, and DF-1 was also observed to protect both human lymphocytes and rat intestinal crypt cells against radiation-induced cell death. We determined that DF-1 protected both cell types against radiation-induced DNA damage, as measured by inhibition of micronucleus formation. DF-1 also reduced the levels of reactive oxygen species in the crypt cells, a unique capability of fullerenes because of their enhanced reactivity toward electron-rich species. The ability of DF-1 to protect against the cytotoxic effects of radiation was comparable to that of amifostine, another ROS-scavenging radioprotector. Interestingly, localization of fluorescently labeled DF-1 in fibroblast was observed throughout the cell. Taken together, these results suggest that DF-1 provides powerful protection against several deleterious cellular consequences of irradiation in mammalian systems including oxidative stress, DNA damage, and cell death.

The Impact of Intracellular Generation of Nitric Oxide on the Radiation Response of Human Tumor Cells

Nitric oxide (NO) is a potent radiosensitizer of hypoxic mammalian cells. There have been many reports demonstrating radiosensitization in vitro and in vivo by the use of NO donors to generate NO by chemical means or by producing agents that mimic the free radical mechanism(s) of NO for potentiating radiosensitivity. However, much of this work has been done without taking account of the endogenous NO that is generated in tumor cells by NO synthase (NOS) in vitro or in tumor cells and host cells in solid tumors in vivo. To evaluate the contribution of intracellular generated NO to cellular radiosensitivity, we exposed human HT1080 and MDA231 tumor cells to a cytokine cocktail that results in an increase in cellular NOS expression to a level that is seen in many human solid tumors. We also carried out parallel studies to determine the radiosensitivity of HT1080 and MDA231 cells engineered to constitutively overexpress the iNOS gene. When cells are treated with cytokines under anoxic conditions (<0.01% O(2)), there is up to a 9-15-fold increase in NOS expression, but no detectable NO is generated (since O(2) is required for the generation of NO via the NOS-mediated conversion of arginine to citrulline). As a consequence, when these cells are irradiated under hypoxic conditions, no radiosensitization is observed. However, as the oxygen tension was increased, the amount of NO generated also increased, and we show that this NO then contributes to an overall increase in the radiosensitivity of cells. For example, at 1% O(2) in control HT1080 cells, with little measurable NOS activity, the dose of radiation required to reduce survival by 90% was 6 Gy compared to 10 Gy in anoxic conditions. After cytokine treatment, the level of NO generated at 1% O(2) was significantly increased and the dose of radiation needed for 90% cell killing was reduced further to 4 Gy. The presence of the NOS inhibitor N(G)-methyl-l-arginine (NMLA) shortly before and during irradiation ablated this increase in radiosensitivity, confirming that the effect was due to the generation of NO. We conclude that cytokine-mediated up-regulation of the NOS expression in tumor cells can produce sufficient NO to significantly increase the cytotoxic effect of radiation and that this is particularly apparent at intermediate oxygen concentrations.

Antioxidant status in breast cancer patients of different ages after radiotherapy

In this study we investigated the effects of breast cancer radiotherapy on the antioxidant (AO) enzyme activities of copper, zinc superoxide dismutase (CuZnSOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR), as well as on the concentration of reduced glutathione (GSH) and lipid peroxides (LP) in blood of patients aged 45-58 years and older than 60 years. The results show that in blood of patients aged 45-58 years, radiotherapy increased the activities of CuZnSOD, CAT, and GR, as well as the concentration of GSH, without affecting the activity of GPx and concentration of LP. In patients older than 60 years, radiotherapy increased the activities of CuZnSOD and CAT, lowered the activity of GPx and concentration of GSH, and increased the concentration of LP. Our results indicate that the response to radiotherapy involves age-related impairment of AO capacity for elimination of H2O2, causing oxidative damage to blood cells. This suggests that cytotoxic effects of radiation on healthy tissues might be more pronounced during the aging of breast cancer patients, and should be considered in the further development of individualization protocols in cancer radiotherapy.

Protective Role of Hsp27 Protein Against Gamma Radiation–Induced Apoptosis and Radiosensitization Effects of Hsp27 Gene Silencing in Different Human Tumor Cells

The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines. We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to gamma-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.

M02-04: Genetic markers of tumor response

Recently, individualized and tailored molecular targeted therapy was investigated for overcoming the limit of the present modalities of cancer treatment. Detoxification enzymes (NQO1, GSTP1, MPO and MnSOD), DNA repair gene, XRCC1/2, ERCC1 and the others are studied, and the expression or mutations of these genes in human tissues are reported world-widely as a factor related to risk of cancer in lung or other sites. Furthermore, some reports investigated the difference of treatment results by expression or mutations of these genes with chemotherapy or radiation therapy. Firstly, most studies have focused on the DNA repair gene, because DNA repair enzyme is critical for the protection of the genome from carcinogenic exposure and the prevention of some types of cancer. It is possible that the DNA repair mechanism plays a key role in the response of cancer cells to ionizing radiation, because the cytotoxic effect of radiation is mediated by the single or double strand breaks in the DNA. There are 4 major DNA repair mechanisms for maintaining the genomic integrity against damage caused by mutagens: mismatch repair, base excision repair, nucleotide excision repair, and double strand break repair. The X-ray repair cross-complementing Group 1 (XRCC1) plays an important role in the DNA base excision repair pathway, giving the possibility that this enzyme has some relationship with the response to radiotherapy. Secondly, a few studies have revealed that NAD(P)H:quinine oxidoreductase (NQO1, previously called as DT-diaphorase) is manifested within epithelium and endothelium in human tissues, and is also at many tumors, particularly NSCLC, originated from epithelial cell. NQO1 is two-electron reductases which catalyze more toxic quinines to less toxic hydroquinones, so it was revealed that NQO1 may play a major role in cytoprotection and chemoprevention. NQO1 also prevents the generation of reactive oxygen (hydrogen peroxide, superoxide) to human tissue, and is revealed as scavenger of supreoxide. Radiation therapy gives cytotoxic damage to human tissue via pathway of direct or indirect DNA breakage and indirect pathway breaks DNA strands by secondary effect of free radical produced by radiation particle that a potent inducer of NQO1 expression in human tissues. β-Lapachone (3,4-dihydro-2,2-dimethyl1-2H-naphthol[1,2-b] pyran-5,6-dione) (β-Lap) was originally isolated from the bark of Lapacho tree (Tabebuia avellanedae) growing in South America. This drug has attracted considerable interest in recent years because of its potent cytotoxicity against various cancer cell lines through a mechanism that works independent of the cell cycle or p53 status. β-Lap induced cell death in vitro has been previously attributed to activation or inhibition of Topoisomerase II-α, inhibition of Topoisomerase II-α, and suppression of NF-ϰB activity. However, more recent studies have clearly indicated that none of these changes were the key determinant of cell death caused by β-Lap, particularly in vivo. Recently, NQO1 has been reported to be a key player in β-Lap induced cell death. NQO1 catalyzes a two-electron reduction of β-Lap to the hydroquinone form of β-lap, i.e., β-Lap(HQ), using NADH or NAD(P)H as electron donors. The resulting β-Lap(HQ) is unstable and consequently reoxidizes in the presence of O2 to original oxidized β-Lap, causing a futile cycling between the quinone and hydroquinone forms. This futile cycling of the drug causes a progressive depletion of NADH or NAD(P)H levels. We observed that IR sensitizes cancer cells to β-Lap by causing a long-lasting elevation of NQO1 activity. It thus appeared that the synergistic interaction of ionizing radiation (IR) and β-Lap in killing cancer cells was due to an increase in cellular susceptibility to β-Lap, probably in addition to β-lap induced radiosensitization. Importantly, the NQO1 level in many human tumors is markedly greater than this in normal tissues. The fact that NQO1 level in tumors can be increased further by IR suggests that the combination of β-Lap and radiotherapy may be a potentially effective approach to selectively damage tumors relative to normal tissue. The combination of β-Lap and radiotherapy is a potentially effective regimen for the treatment of human cancer. It is possible that genetic variations in genes mentioned above can affect the treatment result after radiation therapy, because its expression or mechanism is related to ionizing radiation. Single nucleotide polymorphisms (SNPs) make up about 90% of human genetic variations. We tried to investigated that the difference in enzymatic activity by genetic polymorphisms could affect the treatment response and might be used as genetic marker of tumor response after radiation therapy.

Impact of erythropoietin (EPO) on irradiation effect on the EPO receptor–positive head and neck cancer

6058 Background: Anemia frequently occurs in Head and Neck Squamous Cell Carcinoma (HNSCC) and has been associated with decreased quality of life, impaired treatment outcomes and shortened survival. Furthermore, anemia is a causative factor of tumor hypoxia, which compromises the efficacy of radiotherapy (RT). In order to circumvent the negative effects of anemia, several attempts have been made including the use of EPO, particularly in HNSCC. However, recent clinical data suggest a detrimental effect of EPO on survival for HNSCC patients treated with RT plus EPO. Methods: The HNSCC CAL-166 cell line, which is wild type p53 and expresses the EPO receptor (EPOR), was investigated in the present study; a MTT test was used to examine the effects of EPO on the cellular response to RT. Extended preliminary experiments were performed to standardize the conditions (time of exposure to hypoxia and time to return of normal culture conditions) and the administration of radiation (fractionated or in single dose) to optimize the effects of hypoxia on radiation response. Cells were exposed to a X-rays source at escalating doses from 2–20 Gy and showed classical dose- response curves. Hypoxia was mimed using a ‘Bug-Box‘ anaerobic workstation for 24–48 hours. Results: After a single RT dose of 10 Gy, the data confirmed the negative effect of hypoxia, directly related to the duration of hypoxia (% surviving cells when irradiated in normoxia: 43.95 ± 4.24; when irradiated after 24 hours of hypoxia, 62.4 ± 8.3 or after 48 hours, % surviving cells 70.8 ± 7.4; p = 0.001 versus RT in normoxia). The addition of EPO, from 24 hours before RT to 72 hours after, until MTT test, did not modify the cytotoxic effect of RT in hypoxic, but also in oxic conditions. Conclusions: These preliminary data do not support a direct negative effect of EPO when treating EPOR positive tumor cells with irradiation. Additional data concern the molecular impact of EPO on tumor cell lines with examination of EPOR, HIF-1 alpha and EGFR. No significant financial relationships to disclose.

Analysis of the Incidence of Treatment-Related Myelodysplastic Syndrome and Acute Myelogenous Leukemia in Registration and Compassionate-Use Trials of Ibritumomab Tiuxetan Radioimmunotherapy (RIT).

Radioimmunotherapy (RIT) is a therapeutic modality indicated for the treatment of patients with relapsed or refractory low-grade, follicular, or transformed non-Hodgkin's lymphoma (NHL), where the mechanism of action involves the intrinsic activity of the monoclonal antibody and the cytotoxic effects of radiation. The primary toxicity is a late-occurring, transient, and reversible myelosuppression. Because of concerns regarding the long-term effects of radiation on the bone marrow, we investigated the incidence of treatment-related myelodysplastic syndrome (t-MDS) and treatment-related acute myelogenous leukemia (t-AML) after ibritumomab tiuxetan RIT. A total of 746 patients with NHL were treated with the ibritumomab tiuxetan regimen in registration and compassionate-use trials between 1996 and 2002. Patients had a median age of 61 years (range, 24–87) and had received a median of 3 prior therapies (range, 0–9+). The crude incidence of t-MDS or t-AML was 2.3% (17/746), with an incidence of 4.7% (10/211) in patients enrolled in registration trials and of only 1.3% (7/535) in patients included in the compassionate-use trial, with a median follow-up of 5.7 and 3.5 years, respectively. These malignancies were documented at a median of 5.6 years (range, 1.2–13.9) after the diagnosis of NHL and 1.5 years (range, 0.1–5.8) after RIT. The annualized rates were 0.3% (95% CI, 0.2%–0.4%) a year after the diagnosis of NHL and 0.7% (95% CI, 0.4%–1.0%) a year after RIT. Cox multivariate regression analysis found that previous treatment with a purine nucleoside analog was a significant risk factor for t-MDS or t-AML (hazard ratio, 3.9 [95% CI, 1.5–10.4]; P = .006). All patients in whom t-MDS or t-AML developed and in whom cytogenetic data were available (n = 15) had multiple cytogenetic aberrations, commonly of chromosomes 5 and 7, suggesting an association with previous exposure to chemotherapy. There were documented bone marrow cytogenetic abnormalities before treatment in 2 patients who developed t-MDS or t-AML. These data suggest that the annualized incidence of t-MDS or t-AML following ibritumomab tiuxetan RIT is consistent with that expected on the basis of the patients' history of treatment for NHL. Cytogenetic testing before administration of RIT may identify existing chromosomal abnormalities in previously treated patients, particularly in those who have been treated with alkylating agents and/or purine nucleoside analogs and who are thereby at a higher risk for t-MDS or t-AML.

High superoxide dismutase and low glutathione peroxidase activities in red blood cells predict susceptibility of lung cancer patients to radiation pneumonitis

Radiation pneumonitis is an unpredictable complication of radiotherapy for lung cancer and a condition which can cause significant morbidity. The ability to identify patients at a high risk of developing pneumonitis is critical, since it will enable the individualization of the treatment plan. Because the cytotoxic effect of radiation is propagated through reactive oxygen species (ROS) and ROS-driven oxidative stress, the role of antioxidant defense systems in radiation pneumonitis was investigated. Using the pneumonitis-sensitive C3H/HeN mice as a model, we demonstrated that the antioxidant response of the lung correlated well with that of red blood cells (RBC). We then proceeded to test whether differences of RBC antioxidant response would predict the pneumonitis development in patients. Superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT) activities and glutathione in RBC were measured at baseline and then weekly for 6 weeks of treatment in 15 eligible patients receiving concurrent chemo-radiotherapy for unresectable stage III NSCLC. Striking differences were found in the antioxidant activities of RBC with respect to the pneumonitis development. Those who developed pneumonitis showed higher SOD and lower GPX activities at baseline compared to those who did not (3.7 vs 6.8 units/mg for median SOD, 16.5 vs 10.7 nmol/min/mg for median GPX). The functional imbalance of SOD and GPX was displayed consistently throughout the treatment period. The sensitivity and specificity of pneumonitis prediction were further increased when the GPX/SOD ratio was analyzed (pretreatment P = 0.0046). Our results provide a strong rationale to monitor SOD and GPX activities of RBC to identify patients who are at risk of developing pneumonitis, and to implement a strategy of increasing the GPX/SOD ratio in order to lower the risk.

Design of Hypoxia-Targeting Drugs as New Cancer Chemotherapeutics

The tumor microenvironment is now recognized as a major factor that influences not only the response to conventional anti-cancer therapies but also helps define the potential for malignant progression and metastasis. In particular, hypoxia is now considered a fundamentally important characteristic of the tumor microenvironment. Furthermore, discovery of the hypoxia inducible factor 1alpha (HIF-1alpha) has led to a rapidly increasing understanding of the molecular mechanisms involved in tumor hypoxia. This in turn has led to the current extensive interest in the signal molecules related to tumor hypoxia as potential molecular targets for cancer therapeutics. In this paper we give an overview of recent advances in hypoxia research, including cancer treatments that target tumor hypoxia. Progress in the development of hypoxia-targeting drugs will be discussed, including antiangiogenic hypoxic cell radiosensitizers and hypoxic cytotoxins, hypoxia targeting boron carriers and p53-inhibiting bifunctional radiosensitizers. We will also review our own recent research results in these areas. For example, we have found that certain of the 2-nitroimidazole radiosensitizers and heterocycle-N-oxide hypoxic cytotoxins we developed have antiangiogenic activity and antimetastatic activity. We propose that these activities are based on the inhibition of signal transduction mediated by HIF-1alpha. The anti-tumor activities of hypoxia response are considered to be cytostatic (tumor dormancy-inducing) effects in contrast to cytotoxic DNA damaging effects. The combination of these cytostatic effects that are related to radiosensitization with the cytotoxic effects of radiation should improve the prognosis and QOL of patients receiving radiation and lead to an overall response to treatment. Based on these considerations, we developed the antiangiogenic hypoxic cell radiosensitizers, TX-1877, TX-1898 and the hypoxic cytotoxin TX-402 that inhibits the HIF-1alpha pathway We will also discuss our research involved with the development of other drugs to exploit tumor hypoxia, including a hypoxia-targeting boron carrier for boron neutron capture therapy (BNCT) and a p53 inhibiting radiosensitizer.

Radiation-induced suppression of the Bmp2 signal transduction pathway in the pluripotent mesenchymal cell line C2C12: an in vitro model for prevention of heterotopic ossification by radiotherapy.

Heterotopic ossification is a common complication after total hip replacement. Clinical studies showed the effectiveness of radiation for prevention of heterotopic ossification. The mechanism of radiotherapy responsible for the reduction of heterotopic ossification is unclear. The purpose of this study was to study an analogue model showing a time- and dose-dependent effect of radiation. Using cells of the defined embryonic mouse cell line C2C12, the influence of ionizing radiation on the Bmp-induced signal cascade leading to osteogenic differentiation was analyzed. Binding of iodinated Bmp2 to the receptors, Smad1 activation, and alkaline phosphatase (ALP) activity were determined in cells with or without irradiation. The cytotoxic effect of radiotherapy was evaluated using viability tests. Radiotherapy reduced formation of the Bmp2/Bmp receptor complex. This effect was dependent on dose. The phosphorylation (activation) of Smad1 decreased after irradiation in a time-dependent manner, whereas the level of total Smads was not influenced by radiotherapy. The ALP activity decreased after radiotherapy. A dose of 7 Gy delivered 6 h before or after incubation with Bmp resulted in about a 30% decrease in ALP activity. No signs of cytotoxic effects were observed within the time window studied using doses of 0 to 20 Gy. The time- and dose-dependent effect of radiotherapy for prevention of heterotopic ossification known from the results of clinical studies has an analogue in the C2C12 cell model. The primary mechanism of radiotherapy seems to be an influence on cellular responsiveness to the Bmp2-induced osteoblastic differentiation. The results suggest a down-regulation of the Bmp2/receptor complex.

Induced p21waf expression in H1299 cell line promotes cell senescence and protects against cytotoxic effect of radiation and doxorubicin.

The CDK inhibitor p21waf is a principal mediator of p53 function but can also be transactivated by many p53-independent stimuli leading to cell growth arrest or differentiation. In order to study the function of p21waf in a p53-deficient environment, we established an inducible expression of p21waf in the p53-null lung cancer cell line H1299, based on the muristerone-regulated system. Overexpression of p21waf led cells to growth arrest which after several days became irreversible and the arrested cells acquired a senescent phenotype as judged by cell shape, the senescence-associated beta-gal marker and inhibition of colony formation. The effect of p21waf overexpression, in the absence of p53, on the cytotoxicity caused by irradiation, doxorubicin and taxol was studied. Expression of p21waf provided protection against the cytotoxic effect of radiation and doxorubicin but not of taxol. These results are relevant to treatment of cancer when p53 is inactive.

Induction of Multilocus Mutations at the Tk1 Locus after X Irradiation of L5178Y Cells at Different Times in the Mitotic Cycle

TK1+/- L5178Y-R16 cells were separated into G1, S and G2/M-phase populations by centrifugal elutriation and were treated with 1.5 Gy X radiation. Cells irradiated in the G1 and G2/M phases were most sensitive to the cytotoxic effects of radiation, while cells irradiated in the G2/M phase showed the highest mutant frequency at the thymidine kinase (Tk1) locus. DNA isolated from independent TK1-/- mutants was analyzed for loss of heterozygosity (LOH) at the Tk1 locus and two microsatellites, D11Mit48 and D11Nds7. Homogenates of each mutant were assayed for activity of galactokinase (GLK), the product of the galactokinase (Glk) gene neighboring the Tk1 gene on chromosome 11. Irradiated G1-phase cells had the highest percentage of mutants showing no LOH. The frequency of mutants with LOH at both Tk1 and D11Nds7 with no loss of GLK activity was high in all cell populations: There was no significant difference in the observed frequency of these mutants between the populations. The frequency of mutants losing GLK activity was low, particularly in cells irradiated in the S or G2/M phases. The possibility that the loss of GLK activity is not indicative of LOH at the Glk gene under the conditions of the present experiments is discussed.

498 Evaluation of effect of the active metabolite of amifostine (AMI), WR-1065, on the cytotoxicity of anticancer drugs against human ovarian cancer

Ami protects normal but not tumor tissue from cytotoxic effects of radiation and chemotherapy. The effect of amifostine's active metabolite, the free thiol, WR-1065, on the cytotoxicity of standard anticancer drugs was tested against human A2780 ovarian cancer in vitro using the sulforhodamine B assay for viability. Dose response and IC 50 values were determined in triplicate for each drug in the presence and absence of the highest nontoxic dose of WR-1065. IC 50 values (molar cone) for the drug alone and following pretreatment with WR-I065 were: taxol 1.7 vs 1.3 × 10 −8 ; cisplatin 9.2 vs 3.1 × 10 −4 ; carboplatin 9.6 vs 8.9 × 10 −3 ; doxorubicin 5.6 vs 0.08 × 10 −4 ; mitoxantrone 7,9 vs 0.18 × 10 −6 ; 5FU 8.5 vs 8.5 × 10 −5 ; vinblastine 4.5 vs 3.3 × 10 −8 , respectively. The IC 50 differences ± pretreatment with WR-1065 were not statistically significantly different. These data expand upon previous reports showing that Ami or WR-1065 does not protect tumors from the cytotoxic effects of anticancer agents. Ami's ability to protect dose-limiting toxicity to normal tissues without protection of tumor should enhance the efficacy ratio of a variety of standard anticancer drugs.

Acidic and neutralized metoclopramide formulations sensitize ionizing radiation induced cytotoxicity in a human lung adenocarcinoma xenografted to scid mice.

A neutralized formulation (Neu-Sensamide) of metoclopramide (MCA) has been shown to possess reduced sedative side effects compared with the conventional acidic formulations (Primperan). The acidic formulation of MCA has also been shown to sensitize the effect of ionizing radiation (6-8 Gy) using human squamous cell carcinomas from the head and neck xenografted to nude mice. In the present study, 2 mg MCA/kg body weight 1-3 h before treatment with 1 Gy radiation (single dose) was evaluated in scid mice xenografted with a human lung adenocarcinoma. MCA given alone in acidic or neutralized formulations did not show any effect on tumor growth retardation. However, when combined with radiation, both acidic and neutralized formulations of MCA sensitized the cytotoxic effect of radiation directed against the tumors by increasing tumor doubling time, tumor quadrupling time and specific growth delay, and by decreasing area under growth curve measurements. In addition, there was no statistically significant difference between the two formulations of MCA in the efficacy of sensitizing the cytotoxicity of a single low dose (1 Gy) of radiation.

Reassessment of human peripheral T-lymphocyte lifespan deduced from cytogenetic and cytotoxic effects of radiation.

Estimates of the mean lifetime of human peripheral blood T-lymphocytes (PBTL) have ranged from 1.5 to 10 year. To derive an improved estimate, kinetic and dose parameters of four deterministic, two-compartment models of loss and replacement of human peripheral blood lymphocytes (PBL) were fitted to a previously published extensive data set on loss of dicentrics plus rings (DR) in PBL sampled from X-ray-treated ankylosing spondylitis patients for > 20-year-post-treatment, involving a fixed external-dose regimen (with dose to PBL unknown). The four models investigated all incorporated fixed dose-response submodels for radiation-induced cytogenetic damage and cell killing that were based entirely on in vitro data for X-ray-exposed human PBTL. Two of the models are shown to provide a reasonably good prediction not only of the data on DR loss to which the models were fit, but also of corresponding data on recovery of the mean of measured total PBL. The modelling results indicate that it is unlikely that long-lived human recirculating 'memory' PBTL are homogeneous with respect to lifespan, but rather that two major subpopulations, T1 and T2, are involved having mean lifespans (and approximately 95% confidence limits) estimated to be approximately 1.1 (0.76-1.9) and approximately 6.3 (5.8-6.9) year, respectively. One of the two models consistent with the data posits that T1 (possibly 'short-term antigen-memory') cells are approximately 60% of recirculating T-lymphocytes that convert at a rate of approximately 9% year-1 into T2 (possibly 'long-term antigen-memory') cells, and that the T1 cells are virtually all derived from a central-body source such as thymus and/or central lymphatic tissues.

Normal tissue reactions in mice after combined treatment with metoclopramide and ionizing radiation.

We have previously shown that metoclopramide potentiates the effect of ionizing radiation and cisplatin treatment of human squamous cell carcinomas from the head and neck region xenografted to nude mice. In the present tumor study, the dose scheduling of metoclopramide in combination with radiation was evaluated, and metoclopramide was shown to be most effective in potentiating the cytotoxic effect of radiation when administered one hour before radiation. The effect of radiation in combination with metoclopramide on normal tissue was also studied in two well-established models. Acute skin reactions to radiation exposure were studied in 129-type mice, and metoclopramide did not enhance the acute skin reaction in this in vivo model. Survival after whole body irradiation was studied in heterozygote Balb/c nu/+ mice as a measure of bone marrow toxicity. Metoclopramide was not found to affect the LD50/30 in this in vivo model. The absence of potentiation of radiation damage to normal tissue in these animal studies, makes metoclopramide an interesting possibility for future clinical evaluation.

In Vitro Radiosensitivity of Human Diploid Fibroblasts Derived from Women with Unusually Sensitive Clinical Responses to Definitive Radiation Therapy for Breast Cancer

Between January 1985 and December 1986, 811 patients were treated for carcinoma of the breast at the Joint Center for Radiation Therapy by an identical protocol. Of these 811 patients, five patients (0.6%) were identified as having an unusually sensitive clinical response to routine external beam irradiation. This unusual clinical response was characterized by severe skin erythema and edema during the first few weeks of treatment, requiring treatment breaks. Skin fibroblast cell strains were established from these five women as well as from six women with a normal clinical response to breast irradiation (chosen at random from the population of 811 patients). Radiation survival parameters were determined by a colony formation assay from complete survival curves in coded and blinded samples. Cells from the sensitive patients were significantly more sensitive to the cytotoxic effects of radiation in vitro as determined by the parameters D0, D, D10, and n, than were the strains derived from patients with a normal response. We conclude that an unusually severe response to standard fractionated radiotherapy may be associated with greater intrinsic radiation sensitivity of the individual's somatic cells.

Cytotoxicity, Radiosensitization, and DNA Interaction of Platinum Complexes of Thiazin and Xanthene Dyes

Complexes of the platinum(II) tetrachlorodianion with positively charged nuclear dyes have been prepared in an effort to produce neutral molecules which could gain ready access to the nuclear DNA where the platinum(II) tetrachlorodianion could function as a radiosensitizing and a bifunctional alkylating agent. The thiazin dyes Thionin, Azure B, and Methylene Blue, the aminoxanthene dye Pyronin Y, and the thiazole dye Thioflavin have each been complexed to the platinum(II) tetrachlorodianion(PtCl4) in a ratio of 2:1(dye:PtCl4). Studies of the interaction of these complexes and of the dyes with the pBR322 plasmid superhelical DNA demonstrated that while each complex and dye readily associated with the DNA in a dose-dependent manner, only Pt(Thioflavin)2 and Thioflavin produced irreversible DNA changes (single-strand breaks). In exponentially growing EMT6 cells the cytotoxicity of these drugs was assessed in normally oxygenated and hypoxic cells at both pH 7.4 and 6.45. At concentrations ranging from 1 to 500 microM, Pt(Methylene Blue)2 was significantly more cytotoxic than the other thiazin dye complexes Pt(Thionin)2 and Pt(Azure B)2. The cytotoxicity of Pt(Thionin)2 and Pt(Methylene Blue)2 was increased in normally oxygenated and hypoxic cells at low pH. Both Pt(Pyronin Y)2 and Pt(Thioflavin)2 were more toxic than the thiazin complexes. Pt(Pyronin Y)2 was most cytotoxic to normally oxygenated cells at normal pH and hypoxic cells at low pH, while Pt(Thioflavin)2 was most cytotoxic to cells at low pH under both oxygenation conditions. In vitro studies of the radiosensitizing properties of these agents in EMT6 cells demonstrated that exposure to 100 microM for 1 h before and during irradiation (except for Pt[Thioflavin]2, which was assayed at 25 microM) resulted in enhancement rations of 2.5, 1.9, 1.5, and 1.5 for Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y)2, and Pt(Thioflavin)2, respectively, in hypoxic cells. In contrast, Pt(Methylene Blue)2 (and Methylene Blue) proved to be a radioprotector of normally oxygenated cells and did not sensitize hypoxic cells to the cytotoxic effects of radiation. In the FSaIIC fibrosarcoma in vivo administration of each drug at 100 mg/kg intraperitoneally (ip) 15 min prior to irradiation (except for Pt[Thioflavin]2, which was given at 1 mg/kg ip) showed that, with single radiation fractions of 10 and 20 Gy, dose-modifying factors of 2.1, 1.8, 1.5, and 1.2 were produced by Pt(Azure B)2, Pt(Thionin)2, Pt(Pyronin Y), and Pt(Methylene Blue)2, respectively, after correcting for growth delays induced by the drug alone. In comparison, misonidazole at 1 g/kg ip produced a dose-modifying factor of 1.4.(ABSTRACT TRUNCATED AT 400 WORDS)