Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress.

INTRODUCTION: The etiology of radiation-induced erectile dysfunction (ED) is complex and multifactorial, and it appears to be mainly atherogenic. AIM: To focus on vascular aspects of radiation-induced ED and to elucidate whether the protective effects of sildenafil are mediated by attenuation of oxidative stress and apoptosis in the endothelial cells. METHODS: Bovine aortic endothelial cells (BAECs), with or without pretreatment of sildenafil (5 µM at 5 minutes before radiation), were used to test endothelial dysfunction in response to external beam radiation at 10-15 Gy. Generation of reactive oxygen species (ROS) was studied. Extracellular hydrogen peroxide (H2O2) was measured using the Amplex Red assay and intracellular H2O2 using a fluorescent sensor. In addition, ROS superoxide (O2•-) was measured using a O2•- chemiluminescence enhancer. Both H2O2 and O2•- are known to reduce the bioavailability of nitric oxide, which is the most significant chemical mediator of penile erection. Generation of cellular peroxynitrite (ONOO-) was measured using a chemiluminescence assay with the PNCL probe. Subsequently, we measured the activation of acid sphingomyelinase (ASMase) enzyme by radioenzymatic assay using [14C-methylcholine] sphingomyelin as substrate, and the generation of the proapoptotic C16-ceramide was assessed using the diacylglycerol kinase assay. Endothelial cells apoptosis was measured as a readout of these cells' dysfunction. MAIN OUTCOME MEASURES: Single high-dose radiation therapy induced NADPH oxidases (NOXs) activation and ROS generation via the proapoptotic ASMase/ceramide pathway. The radio-protective effect of sildenafil on BAECs was due to inhibition of this pathway. RESULTS: Here, we demonstrate for the first time that radiation activated NOXs and induced generation of ROS in BAECs. In addition, we showed that sildenafil significantly reduced radiation-induced O2•- and as a result there was reduction in the generation of peroxynitrite in these cells. Subsequently, sildenafil protected the endothelial cells from radiation therapy-induced apoptosis. STRENGTHS AND LIMITATIONS: This is the first study demonstrating that single high-dose radiation therapy induced NOXs activation, resulting in the generation of O2•- and peroxynitrite in endothelial cells. Sildenafil reduced ROS generation by inhibiting the ASMase/ceramide pathway. These studies should be followed in an animal model of ED. CONCLUSIONS: This study demonstrated that sildenafil protects BAECs from radiation-induced oxidative stress by reducing NOX-induced ROS generation, thus resulting in decreased endothelial dysfunction. Therefore, it provides a potential mechanism to better understand the atherogenic etiology of postradiation ED. Wortel RC, Mizrachi A, Li H, et al. Sildenafil Protects Endothelial Cells From Radiation-Induced Oxidative Stress. J Sex Med 2019;16:1721-1733.

Anticancer therapy and apoptosis imaging.

Early response prediction is considered an essential tool to obtain a more customized anticancer treatment because it allows for the identification of patients who will benefit most from a particular therapy and prevents the exposure of those patients to toxic, non-effective regimens. Recent discoveries of novel markers in functional imaging have created exciting opportunities for in vivo visualization and quantification of cell death. This review will focus on in vivo apoptosis imaging with various radiotracers as predictive tools for tumor response after anticancer therapy. Particular focus will be on annexin V imaging, a technique with the largest clinical experience to date. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice.

Prostate cancer is one of the leading causes of cancer-related deaths in the United States and a leading diagnosed non-skin cancer in American men. Genetic mutations underlying prostate tumorigenesis include alterations of tumor suppressor genes. We tested the tumor suppressor hypothesis for ABI1/hSSH3BP1 by searching for gene mutations in primary prostate tumors from patients, and by analyzing the consequences of prostate-specific disruption of the mouse Abi1/Hssh3bp1 ortholog. We sequenced the ABI1/hSSH3BP1 gene and identified recurring mutations in 6 out of 35 prostate tumors. Moreover, complementation and anchorage-independent growth, proliferation, cellular adhesion and xenograft assays using the LNCaP cell line, which contains a loss-of-function Abi1 mutation, and a stably expressed wild-type or mutated ABI gene, were consistent with the tumor suppressor hypothesis. To test the hypothesis further, we disrupted the gene in the mouse prostate by breeding the Abi1 floxed strain with the probasin promoter-driven Cre recombinase strain. Histopathological evaluation of mice indicated development of prostatic intraepithelial neoplasia (PIN) in Abi1/Hssh3bp1 knockout mouse as early as the eighth month, but no progression beyond PIN was observed in mice as old as 12 months. Observed decreased levels of E-cadherin, ß-catenin and WAVE2 in mouse prostate suggest abnormal cellular adhesion as the mechanism underlying PIN development owing to Abi1 disruption. Analysis of syngeneic cell lines point to the possibility that upregulation of phospho-Akt underlies the enhanced cellular proliferation phenotype of cells lacking Abi1. This study provides proof-of-concept for the hypothesis that Abi1 downregulation has a role in the development of prostate cancer.

Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice.

Gastrointestinal (GI) tract damage by chemotherapy or radiation limits their efficacy in cancer treatment. Radiation has been postulated to target epithelial stem cells within the crypts of Lieberkühn to initiate the lethal GI syndrome. Here, we show in mouse models that microvascular endothelial apoptosis is the primary lesion leading to stem cell dysfunction. Radiation-induced crypt damage, organ failure, and death from the GI syndrome were prevented when endothelial apoptosis was inhibited pharmacologically by intravenous basic fibroblast growth factor (bFGF) or genetically by deletion of the acid sphingomyelinase gene. Endothelial, but not crypt, cells express FGF receptor transcripts, suggesting that the endothelial lesion occurs before crypt stem cell damage in the evolution of the GI syndrome. This study provides a basis for new approaches to prevent radiation damage to the bowel.

Cell autonomous apoptosis defects in acid sphingomyelinase knockout fibroblasts.

A body of evidence suggests that stress-induced sphingomyelin hydrolysis to the second messenger ceramide initiates apoptosis in some cells. Although studies using lymphoblasts from Niemann-Pick disease patients or acid sphingomyelinase (ASMase)-deficient mice have provided genetic support for this hypothesis, these models have not been universally accepted as definitive. Here, we show that mouse embryonic fibroblasts (MEFs) prepared from asmase mice manifest cell autonomous defects in apoptosis in response to several stresses. In particular, asmase(-/-) MEFs failed to generate ceramide and were totally resistant to radiation-induced apoptosis but remained sensitive to staurosporine, which did not induce ceramide. asmase(-/-) MEFs were also partially resistant to tumor necrosis factor alpha/ actinomycin D and serum withdrawal. Thus, resistance to apoptosis in asmase(-/-) MEFs was not global but rather stress type specific. Most importantly, the sensitivity to stress could be restored in the asmase(-/-) MEFs by administration of natural ceramide. Overcoming apoptosis resistance by natural ceramide is evidence that it is the lack of ceramide, not ASMase, that determines apoptosis sensitivity. The ability to rescue the apoptotic phenotype without reversing the genotype by the product of the enzymatic deficiency provides proof that ceramide is obligate for apoptosis induction in response to some stresses.

Natural ceramide reverses Fas resistance of acid sphingomyelinase(-/-) hepatocytes.

The role of the second messenger ceramide in Fas-mediated death requires clarification. To address this issue, we generated hepatocytes from paired acid sphingomyelinase (ASMase; asmase)(+/+) and asmase(-/-) mice. asmase(-/-) hepatocytes, derived from 8-week-old mice, manifested normal sphingomyelin content and normal morphological, biochemical, and biologic features. Nonetheless, ASMase-deficient hepatocytes did not display rapid ceramide elevation or apoptosis in response to Jo2 anti-Fas antibody. asmase(-/-) hepatocytes were not inherently resistant to apoptosis because staurosporine, which did not induce early ceramide elevation, stimulated a normal apoptotic response. The addition of low nanomolar quantities of natural C16-ceramide, which by itself did not induce apoptosis, completely restored the apoptotic response to anti-Fas in asmase(-/-) hepatocytes. Other sphingolipids did not replace natural ceramide and restore Fas sensitivity. Overcoming resistance to Fas in asmase(-/-) hepatocytes by natural ceramide is evidence that it is the lack of ceramide and not ASMase which determines the apoptotic phenotype. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for Fas induction of apoptosis in hepatocytes.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Reversal of radiation resistance in LNCaP cells by targeting apoptosis through ceramide synthase.

Cell lines derived from human prostate cancer are regarded as relatively resistant to both radiation-induced clonogenic death and apoptosis. Here we attempted to modulate the response of LNCaP prostate cancer cells to radiation therapy (XRT) by pretreatment with 12-O-tetradecanoylphorbol acetate (TPA), a known apoptogenic agent in LNCaP cells. Using plateau-phase cultures, we investigated the response of these cells to XRT, TPA, and a combination of XRT and TPA. LNCaP irradiation did not result in ceramide generation or apoptosis. However, pretreatment with TPA enabled XRT to generate ceramide via activation of the enzyme ceramide synthase and signal apoptosis. Apoptosis was abrogated by the competitive inhibitor of ceramide synthase, fumonisin B1. Furthermore, when transplanted orthotopically into the prostate of nude mice, LNCaP cells produced tumors that recapitulated the responses of LNCaP cells in vitro. XRT or TPA failed to signal apoptosis in LNCaP tumors, whereas a combination of the two resulted in substantial (20-25%) apoptosis within 24 h. There was an additional benefit associated with this regimen because TPA pretreatment protected the adjacent rectum from radiation-induced apoptosis. This represents the first description of signaling-based therapy designed to overcome one form of radiation resistance expressed preferentially in LNCaP human prostate cancer cells.

Ataxia telangiectasia-mutated gene product inhibits DNA damage-induced apoptosis via ceramide synthase.

DNA double-stranded breaks (dsb) activate surveillance systems that identify DNA damage and either initiate repair or signal cell death. Failure of cells to undergo appropriate death in response to DNA damage leads to misrepair, mutations, and neoplastic transformation. Pathways linking DNA dsb to reproductive or apoptotic death are virtually unknown. Here we report that metabolic incorporation of 125I-labeled 5-iodo-2'deoxyuridine, which produces DNA dsb, signaled de novo ceramide synthesis by post-translational activation of ceramide synthase (CS) and apoptosis. CS activation was obligatory, since fumonisin B1, a fungal pathogen that acts as a specific CS inhibitor, abrogated DNA damage-induced death. X-irradiation yielded similar results. Furthermore, inhibition of apoptosis using the peptide caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone did not affect CS activation, indicating this event is not a consequence of induction of apoptosis. ATM, the gene mutated in ataxia telangiectasia, is a member of the phosphatidylinositol 3-kinase family that constitutes the DNA damage surveillance/repair system. Epstein-Barr virus-immortalized B cell lines from six ataxia telangiectasia patients with different mutations exhibited radiation-induced CS activation, ceramide generation, and apoptosis, whereas three lines from normal patients failed to manifest these responses. Stable transfection of wild type ATM cDNA reversed these events, whereas antisense inactivation of ataxia telangiectasia-mutated gene product in normal B cells conferred the ataxia telangiectasia phenotype. We propose that one of the functions of ataxia telangiectasia-mutated gene product is to constrain activation of CS, thereby regulating DNA damage-induced apoptosis.

Niemann-Pick human lymphoblasts are resistant to phthalocyanine 4-photodynamic therapy-induced apoptosis.

Stress-induced activation of sphingomyelinase (SMase) leading to generation of ceramide, a lipid mediator, has been associated with apoptosis in several malignant and nonmalignant cell lines. Photodynamic therapy (PDT), with the phthalocyanine photosensitizer Pc 4 [HOSiPcOSi(CH3)2(CH2)3N(CH3)2], is an oxidative stress associated with increased ceramide generation and subsequent induction of apoptosis in various cell types. We assessed the role of SMase in photocytotoxicity. Normal human lymphoblasts accumulated ceramide and underwent apoptosis after Pc 4-PDT. In contrast, Niemann-Pick disease (NPD) lymphoblasts, which are deficient in acid sphingomyelinase (ASMase) activity, failed to respond to Pc 4-PDT with ceramide accumulation and apoptosis, suggesting that ASMase may be a Pc 4-PDT target. NPD lymphoblasts were exposed to exogenous bacterial sphingomyelinase (bSMase) to test whether these defects are reversible. Treatment of NPD cells with bSMase itself led to elevated ceramide formation, which did not translate into induction of apoptosis. However, a combination of Pc 4-PDT + bSMase induced a significant apoptotic response. Thus, the combined treatment of Pc 4-PDT + bSMase, rather than bSMase alone, was required to restore apoptosis in NPD cells. These data support the hypothesis that SMase is a proapoptotic factor determining responsiveness of cells to Pc 4-PDT.

Contribution of phosphatidylinositol 3-kinase to radiation resistance in human melanoma cells.

The activity of phosphatidylinositol 3-kinase (PI3K), a key component of multiple signal transduction pathways, was investigated in early- and late-stage melanoma cells that have varying degrees of radiation resistance. Analysis of PI3K biproducts (PI-3,4-P2 and PI-3,4,5-triphosphate) revealed a direct correlation between radiation resistance and levels of PI3K activity. Treating melanoma cells with wortmanin or LY294002, two different PI3K inhibitors, decreased PI3K activity and caused a dose-dependent decrease in resistance to ultraviolet radiation. Lower resistance to radiation elicited by LY294002 coincided with increased apoptosis. To further establish the role of PI3K in radiation resistance, we transfected early-stage melanoma cells with the cDNA of p85, the regulatory subunit of PI3K. Clones that constitutively overexpressed p85 exhibited a higher degree of PI-3,4-P2 synthesis and a corresponding increase in their resistance to ultraviolet radiation. The results of this study point to the role of PI3K and its biproducts in radiation resistance of human melanoma cells.

Radiation-induced signal transduction and stress response.

Radiation-induced DNA damage can induce death by apoptosis by activation of signal transduction pathways. One such pathway is the sphingomyelin/ceramide signal transduction pathway that is involved in initiation of stress-induced apoptosis in a variety of normal and neoplastic cells. This pathway is under regulation by the protein kinase C (PKC) pathway that constitutes an anti-apoptosis mechanism. DNA damage can also increase ceramide levels by activating the biosynthesis pathway, through the activation of the ceramide synthase enzyme. Both pathways could serve as potential targets for strategies that take advantage of signaling-based apoptosis to enhance cell killing in radiation therapy.

The role of the stress-activated protein kinase (SAPK/JNK) signaling pathway in radiation-induced apoptosis.

Ionizing radiation, like a variety of other cellular stress factors, initiates apoptosis, or programmed cell death, in many cell systems. This mode of radiation-induced cell kill should be distinguished from clonogenic cell death due to unrepaired DNA damage. Ionizing radiation not only exerts its effect on the nuclear DNA, but also at the plasma membrane level where it may activate multiple signal transduction pathways. One of these pathways is the stress-activated protein kinase (SAPK) cascade which transduces death signals from the cell membrane to the nucleus. This review discusses recent evidence on the critical role of this signaling system in radiation- and stress-induced apoptosis. An improved understanding of the mechanisms involved in radiation-induced apoptosis may ultimately provide novel strategies of intervention in specific signal transduction pathways to favorably alter the therapeutic ratio in the treatment of human malignancies.

Nitroxides tempol and tempo induce divergent signal transduction pathways in MDA-MB 231 breast cancer cells.

Tempol and tempo are stable free radical nitroxides that possess antioxidant properties. In this study, we examined the effects of these compounds on components of the mitogen-activated protein kinase signal transduction cascade. Tempo treatment (15 min) of MDA-MB 231 human breast cancer cells resulted in significant levels of tyrosine phosphorylation of several as yet unidentified proteins compared with equimolar concentration of tempol (10 mM). Both compounds caused tyrosine phosphorylation and activation of Raf-1 protein kinase (30 min, 2-3-fold). Interestingly, however, only tempol caused increased extracellular signal-regulated kinase 1 activity (2 h, approximately 3-fold). On the other hand, tempo, but not tempol, potently activated stress-activated protein kinase (2 h, >3-fold). Consistent with these data, tempol was found to be noncytotoxic, whereas tempo induced apoptotic cell death (2 h, >50%). Tempo treatment also resulted in significant elevation of ceramide levels at 30 min (54% over control) and 1 h (71% over control) posttreatment, preceding stress-activated protein kinase activation and apoptosis. These data suggest that in the absence of an environmental oxidative stress, tempol and tempo elicit distinct cellular signaling pathways. The recognition of the molecular mechanisms of nitroxide action may have important implications for biological effectiveness of these compounds.

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis in LNCaP cells is mediated through ceramide synthase.

Protein kinase C (PKC) activation is often antiapoptotic, although in a few cell types PKC initiates apoptosis by an unknown mechanism. Recent investigations showed that activation of PKC alpha by 12-O-tetradecanoylphorbol 13-acetate (TPA) induced apoptosis in LNCaP prostate cancer cells. The present studies examine the mechanism of this effect and show that de novo ceramide generation through the enzyme ceramide synthase is required. TPA induced rapid ceramide generation, which was detectable by 1 h and increased linearly for 12 h. TPA-induced apoptosis was measurable by 12 h and was progressive for 48 h. Investigations into the mechanism of TPA-induced ceramide generation revealed that acid and neutral sphingomyelinase activities were not enhanced. However, TPA induced an increase in ceramide synthase activity that persisted for at least 16 h. Treatment with fumonisin B1, a specific natural inhibitor of ceramide synthase, abrogated both ceramide production and TPA-induced apoptosis. Ceramide analogues bypassed fumonisin B1 inhibition to initiate apoptosis directly. Thus, ceramide appears to be a necessary signal for TPA-induced apoptosis in LNCaP cells. This represents the first description of a pathway by which PKC may signal apoptosis.

Ceramide signaling in apoptosis.

The sphingomyelin pathway is a ubiquitous, evolutionarily conserved signaling system initiated by hydrolysis of the plasma membrane phospholipid sphingomyelin to generate ceramide. Ceramide acts as a second messenger in activating the apoptotic cascade. Diverse cytokine receptors and environmental stresses utilize ceramide to signal apoptosis. In several cell systems ceramide links to the stress-activated protein kinase (SAPK)/c-jun kinase (JNK) cascade to signal apoptosis. The engagement of the sphingomyelin pathway in signaling apoptosis is tightly regulated by anti-apoptotic control mechanisms, and the balance between pro- and anti-apoptotic systems determines the magnitude of the apoptotic response in vitro and in vivo. This review describes the known elements and molecular ordering of ceramide-mediated apoptosis and the anti-apoptotic mechanisms that regulate its expression. Understanding of pro- and anti-apoptotic signaling involved in ceramide-mediated apoptosis and the modes of their co-ordinated function may yield opportunities for pharmacological interventions with potential for clinical applications.

Differential inhibition of radiation-induced apoptosis.

The most common mechanism by which radiation kills cells is the induction of DNA double-strand breaks that results in the loss of cell proliferation. Even though apoptosis is increasingly identified in experimental systems in vitro and in vivo, it is still generally regarded as a rare mode of radiation-induced cell kill with minor relevance for the clinical effects of radiation. This review will focus on pro- and antiapoptotic signaling that affects the apoptotic outcome in irradiated mammalian cells. In particular, we will concentrate on the sphingomyelin/ceramide signal transduction pathway which is involved in initiation of stress-induced apoptosis in a variety of normal and neoplastic cells. We will also discuss the crosstalk between the sphingomyelin/ceramide pathway and the protein kinase C pathway which constitutes an antiapoptotic pathway, and the potential for pharmacological modulation to increase the fraction of apoptotic cells undergoing apoptosis after radiation exposure.

Basic fibroblast growth factor confers growth inhibition and mitogen-activated protein kinase activation in human breast cancer cells.

The effect of basic fibroblast growth factor (bFGF) on human breast cancer cells was studied in vitro. Exposure to bFGF resulted in significant growth inhibition, decreased DNA synthesis, and accumulation of cells in G0-G1. The IC50 for growth inhibition in MCF-7 cells was 50 pg/ml, and it was abrogated by neutralizing antibodies against bFGF. Inhibition of growth by bFGF was predominant over the growth stimulatory effects of 17beta-estradiol, insulin, or epidermal growth factor. Binding and cross-linking studies of 125I-labeled bFGF in intact MCF-7 cells demonstrated 5.2 x 10(3) saturable bFGF binding sites per cell, a dissociation constant of 57 pm, and a Mr 142,000 (125)I-labeled bFGF cross-linked protein. Stimulation of MCF-7 cells with bFGF at concentrations which effected growth inhibition also resulted in activation of p42(mapk) (ERK2) and p44(mapk) (ERK1) mitogen-activated protein kinases. These data demonstrate that whereas bFGF inhibits the growth of several breast cancer cell lines, it concomitantly activates ERK1 and ERK2, generally considered to signal mitogenic rather than growth inhibitory responses. Whether there is association between these phenomena remains unknown.

Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.

The endotoxic shock syndrome is characterized by systemic inflammation, multiple organ damage, circulatory collapse and death. Systemic release of tumor necrosis factor (TNF)-alpha and other cytokines purportedly mediates this process. However, the primary tissue target remains unidentified. The present studies provide evidence that endotoxic shock results from disseminated endothelial apoptosis. Injection of lipopolysaccharide (LPS), and its putative effector TNF-alpha, into C57BL/6 mice induced apoptosis in endothelium of intestine, lung, fat and thymus after 6 h, preceding nonendothelial tissue damage. LPS or TNF-alpha injection was followed within 1 h by tissue generation of the pro-apoptotic lipid ceramide. TNF-binding protein, which protects against LPS-induced death, blocked LPS-induced ceramide generation and endothelial apoptosis, suggesting systemic TNF is required for both responses. Acid sphingomyelinase knockout mice displayed a normal increase in serum TNF-alpha in response to LPS, yet were protected against endothelial apoptosis and animal death, defining a role for ceramide in mediating the endotoxic response. Furthermore, intravenous injection of basic fibroblast growth factor, which acts as an intravascular survival factor for endothelial cells, blocked LPS-induced ceramide elevation, endothelial apoptosis and animal death, but did not affect LPS-induced elevation of serum TNF-alpha. These investigations demonstrate that LPS induces a disseminated form of endothelial apoptosis, mediated sequentially by TNF and ceramide generation, and suggest that this cascade is mandatory for evolution of the endotoxic syndrome.

Modulation of the Apoptotic Response: Potential for Improving the Outcome in Clinical Radiotherapy.

The most prevalent mechanism of cell kill by radiation is mitosis dependent and results from lethal DNA double-strand breaks and failure to maintain normal replication. Apoptosis is believed to represent a minor component of the clinical effects of radiation. Apoptosis is a preprogrammed death pathway that is constitutively expressed in many cells, albeit in an inactive form, regulated by antiapoptotic mechanisms. Data are presented to show that in irradiated cells the balance between proapoptotic and antiapoptotic signaling may determine the apoptotic outcome in vitro and in vivo. This balance can be modulated by pharmacological intervention to produce a more proapoptotic phenotype, increasing apoptotic cell kill by radiation. These studies establish the basic principles of signaling-based apoptosis therapy, designed to overcome the relative resistance to radiation-induced apoptosis and to improve the therapeutic ratio in the treatment of human tumors with fractionated radiation.