ABSTRACT
PURPOSE: To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other. METHODS AND MATERIALS: Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death. RESULTS: Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4-5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome. CONCLUSIONS: The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens.
Subject(s)
Apoptosis , Endothelial Cells/radiation effects , Intestinal Mucosa/radiation effects , Intestine, Small/radiation effects , bcl-2 Homologous Antagonist-Killer Protein/physiology , bcl-2-Associated X Protein/physiology , Animals , Biomarkers/metabolism , Colony-Forming Units Assay , Endothelial Cells/cytology , Endothelium, Vascular/cytology , Endothelium, Vascular/radiation effects , Female , In Situ Nick-End Labeling , Intestinal Mucosa/cytology , Intestine, Small/cytology , Jejunum/cytology , Jejunum/radiation effects , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Radiation Injuries/etiology , Radiation Injuries/pathology , Sphingomyelin Phosphodiesterase/deficiency , Sphingomyelin Phosphodiesterase/genetics , Sphingomyelin Phosphodiesterase/metabolism , Whole-Body Irradiation , bcl-2 Homologous Antagonist-Killer Protein/deficiency , bcl-2-Associated X Protein/deficiencyABSTRACT
Although stem cells succumbing to reproductive death are assumed to be the single relevant targets in radiation tissue damage, recent studies showed intestinal stem cell damage is conditionally linked to crypt endothelial apoptosis, defining a two-target model. Here we report that when mouse intestines were protected against microvascular apoptosis, radiation switched as the dose escalated to a previously unrecognized crypt stem cell target, activating ceramide synthase-mediated apoptosis to initiate intestinal damage. Whereas ataxia telangiectasia-mutated (ATM) kinase normally represses ceramide synthase, its derepression in Atm(-/-) mice increased crypt stem cell radiosensitivity 3.7-fold without sensitizing the microvascular response. Discovery of this intestinal radiosensitivity mechanism allowed design of an antisense Atm oligonucleotide treatment which phenocopied the Atm(-/-) mouse, reordering ceramide synthase-mediated stem cell death to become the first-line gastrointestinal response of wild-type littermates. These experiments indicate that tissues operate multiple potential targets activated consecutively according to their inherent radiosensitivities that may be reordered therapeutically to control radiation tissue responses.
Subject(s)
Apoptosis/radiation effects , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/metabolism , Neoplasms/radiotherapy , Oxidoreductases/metabolism , Protein Serine-Threonine Kinases/metabolism , Stem Cells/radiation effects , Tumor Stem Cell Assay , Tumor Suppressor Proteins/metabolism , Whole-Body Irradiation , Animals , Ataxia Telangiectasia Mutated Proteins , Cell Cycle Proteins/genetics , Ceramides/metabolism , DNA-Binding Proteins/genetics , Dose-Response Relationship, Radiation , Endothelial Cells/metabolism , Enzyme Activation/radiation effects , Histological Techniques , Jejunum/cytology , Jejunum/metabolism , Jejunum/radiation effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotides , Protein Serine-Threonine Kinases/genetics , Radiation Tolerance/genetics , Tumor Suppressor Proteins/geneticsABSTRACT
Adenosine deaminase (ADA) can localize to the cell surface through its interaction with CD26. Using CD26-transfected cells, we demonstrate that cell surface ADA (ecto-ADA) can regulate adenosine receptor engagement by degrading extracellular adenosine (Ado) to inosine. This ability was dependent upon CD26 expression, the extent of CD26 saturation with ecto-ADA, and the kinetics of the cAMP response. Thus, the cAMP response was markedly decreased when CD26-transfected cells were incubated with an exogenous source of ADA to increase ecto-ADA expression. The ability of ecto-ADA to inhibit the cAMP response was demonstrated by treatment with the specific ADA inhibitor 2'-deoxycoformycin. This inhibited the ability of ecto-ADA to degrade Ado and increased the cAMP response. Although CD26 expression on human thymocytes was low compared with that of CD26-transfected cells, it was saturated with ecto-ADA. When thymocytes incubated at high densities (to mimic the situation in tissues) were exposed to exogenous adenosine, the cAMP response was dramatically decreased by ecto-ADA. We conclude that ecto-ADA has the potential to regulate adenosine receptor-mediated cAMP responses in vivo in tissues with CD26+ cells and sufficient cell death caused by apoptosis or inflammation to provide a source of ADA to bind to CD26.
Subject(s)
Adenosine Deaminase/physiology , Receptors, Purinergic P1/metabolism , T-Lymphocytes/enzymology , Adenosine/antagonists & inhibitors , Adenosine/pharmacology , Cell Line , Cells, Cultured , Cyclic AMP/metabolism , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/metabolism , Humans , Jurkat Cells , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism , Thymus Gland/cytology , Thymus Gland/growth & development , Thymus Gland/metabolism , TransfectionABSTRACT
Recent evidence suggests that microvascular endothelial apoptosis represents the primary lesion in radiation damage to the gastrointestinal (GI) tract. Rescue of endothelium by depletion of acid sphingomyelinase or i.v. treatment with basic fibroblast growth factor (FGF) prevented the lethal GI syndrome in C(57)Bl/6 mice. Here we show that basic FGF increased crypt survival after irradiation by 2-3 fold, with a dose modification factor at D(10) of 1.15 (P < 0.01). Basic FGF inhibited initial crypt damage, assessed by crypt shrinkage at 18-24 h, but did not significantly affect the regeneration of surviving crypts at 3.5 days after irradiation. These data suggest that microvascular function regulates expression of radiation-induced crypt stem cell clonogen damage in the evolution of radiation injury to the GI mucosa.