Ben Floot

Radiation-Induced Activation of TGF-β Signaling Pathways in Relation to Vascular Damage in Mouse Kidneys

Abstract Kruse, J. J. C. M., Floot, B. G. J., Te Poele, J. A. M., Russell, N. S. and Stewart, F. A. Radiation-Induced Activation of TGF-β Signaling Pathways in Relation to Vascular Damage in Mouse Kidneys. Radiat. Res. 171, 188–197 (2009). The purpose of this study was to investigate the long-term effects of radiation-induced alterations in TGF-β signaling pathways with respect to the development of vascular damage in the irradiated kidney. Total RNA was isolated from mouse kidneys at 1–30 weeks after irradiation, and quantitative real-time PCR analyses were performed for TGF-β receptors (ALK1, ALK5, endoglin), downstream mediators (Smad7, CTGF), and downstream targets (PAI-1 and Id-1). Expression of endoglin and Smad7 protein as well as nucleo-cytoplasmic distribution of phospho Smad 2/3 and phospho Smad 1/5 was analyzed by immunohistochemistry. Radiation caused a rapid and persistent increase in expression of TGF-β receptors and mediators from 1–30 weeks after treatment. Expression of Id-1, a downstream target of endothelial cell specific receptor ALK1, was transiently increased (1–10 weeks after irradiation) but returned to control levels at later times. Expression of PAI-1, a downstream target of ALK5, increased progressively from 10–30 weeks after irradiation. These results show that radiation activated TGF-β signaling pathways in the kidney and shifted the balance in favor of ALK5 signaling, which generally inhibits endothelial cell proliferation and migration. We hypothesize that prolonged activation of ALK5 signaling and relative suppression of ALK1 signaling may provide an explanation for the telangiectatic phenotype observed in irradiated kidneys.

Endoglin haploinsufficiency reduces radiation-induced fibrosis and telangiectasia formation in mouse kidneys

BACKGROUND AND PURPOSE Endoglin is a transforming growth factor beta (TGF-beta) co-receptor mainly expressed in dividing endothelial cells. It regulates cell proliferation and survival and is upregulated at sites of vessel repair. Mutations in endoglin have been linked to the vascular disease hereditary hemorrhagic telangiectasia (HHT). HHT patients display dilated capillaries (telangiectasia) that are prone to rupture. Cancer patients receiving radiotherapy develop similar vascular damage in normal tissues lying in the irradiation field. If located in the mucosa, irradiation-induced telangiectasia can lead to severe bleeding. Therefore, this study was aimed at investigating the role of endoglin in radiation-induced telangiectasia formation. MATERIALS AND METHODS Kidneys of endoglin heterozygous (Eng(+/-)) or wild type mice were irradiated with 16 Gy. Mice were sacrificed after 20 weeks and changes in gene expression and protein levels were analysed. RESULTS Expression of TGF-beta target genes involved in radiation-induced fibrosis and fibrosis development in the kidney decreased in Eng(+/-) compared to wild type mice. Unexpectedly, Eng(+/-) mice also displayed reduced telangiectasia formation in the irradiated kidney. CONCLUSIONS Endoglin plays an important role in the development of irradiation-induced normal tissue damage. Future studies will show whether interfering with endoglin functions protects tissues from late radiation toxicity.

Cellular distribution of cis-diamminedichloroplatinum(II)-DNA binding in rat dorsal root spinal ganglia: effect of the neuroprotecting peptide ORG.2766

The in situ binding of the anticancer drug cis-diamminedichloroplatinum(II) (cisDDP) to DNA was studied in the rat dorsal root spinal ganglion (DRG), using an antiserum against cisDDP-modified calf thymus DNA in a quantitative immunocytochemical assay. Rats received a dose of cisDDP (1 mg/kg), two times a week, up to a cumulative dose of 15 mg/kg (group I) or 34 mg/kg (group II). Rats of group III were given a single dose of 15 mg/kg. Rats were killed 48 hr (groups I and II) or 6 hr (group III) after the last injection. In groups I and II cisDDP-induced neurological damage was assessed by measuring both motor and sensory nerve conduction velocities (MNCV and SNCV). Whereas the MNCV was not influenced by the treatment with cisDDP, the SNCV decreased significantly. The level of cis-DDP-DNA binding in DRG satellite cells equalled that in liver cells, but binding could not be shown in DRG neuron nuclei. The level of cisDDP-DNA binding in spinal cord and brain was very low. The neuroprotecting peptide ORG.2766, an ACTH4-9 analog, was given sc (10 micrograms/rat) four times a week concomitantly with cisDDP to some rats of groups I and II. ORG.2766 prevented the decrease of the SNCV, but did not change the extent of cisDDP-DNA binding in satellite or liver cells. It is concluded that the amelioration of cisDDP toxicity by ORG.2766 is not directly related to the cisDDP-DNA binding in satellite cells.

Antibodies against cisplatin-modified DNA and cisplatin-modified (di) nucleotides

SummaryCytotoxic effects ofcis-diamminedichloroplatinum-(II) (cis-DDP) are thought to be mediated by binding to DNA. Studies on binding ofcis-DDP to cellular DNA rely heavily on the availability of specific antibodies. We therefore, raised and characterized four rabbit antisera: one againstcis-DDP-modified DNA (antiserum NKI-A59) and three others against thecis-DDP-modified (di)nucleotidescis-Pt(NH3)2d(pApG) (NKI-A68),cis-Pt(NH3)2d(GMP)2(NKI-A10), and Pt(NH3)3dGMP (NKI-A39). Reactivities to platinum compounds were determined in an enzyme-linked immunosorbent assay (ELISA) and in a quantitative immunocytochemical assay. In the ELISA, NKI-A59 showed a high affinity for DNA heavily substituted with eithercis-DDP or CBDCA [cis-diammine (1,1-cyclobutanedicarboxylato)platinum(II)]; amounts of platinum per well giving 50% inhibition (IA50) were as low as 15 and 76 fmol, respectively. NKI-A59 also showed affinity tocis-DDP-modified poly[d(G-C)] poly[d(G-C)], poly(dC), and poly(dG). No affinity was found fortrans-DDP [trans-diamminedichloro-platinum(II)]-modified DNA, enzymatically digestedcis-DDP-DNA, orcis-DDP-modified poly(dA)., poly(dT), oligo(dA)15−, oligo(dT)15−, oligo(dG)21, oligo(dG)42, or oligo(dAAAG)10. The efficiency of binding tocis-DDP-DNA decreased with decreasing DNA modification levels. Although othercis-DDP-DNA- andcis-DDP-(di)nuclcotide-specific antisera have been identified, NKI-A59 is the first antiserum described that is suitable for the in situ detection ofcis-DDP-DNA adducts at clinically relevant platinum levels. Adduct-specific immunostaining signals in cultured RIF-1 cells or rat liver paralleled platinum-DNA binding as measured by atomic absorption spectroscopy. The antisera NKI-A68, NKI-A10, and NKI-A39 showed high affinity for their corresponding haptens and varying affinity for non-haptencis-DDP-DNA adducts. Their affinity for digestedcis-DDP-modified DNA was up to 30 times that for intactcis-DDP-DNA. Neither NKI-A68 nor NKI-A10 resulted in specific immunocytochemical staining ofcis-DDP-DNA adducts. We conclude that NKI-A68, NKI-A10, and NKI-A39 are suitable for platinum-DNA adduct analysis of digested DNA in ELISA and that NKI-A59 is suitable for platinum-DNA adduct detection at the single-cell level using immunocytochemical methods.

The TGF-β co-receptor endoglin regulates macrophage infiltration and cytokine production in the irradiated mouse kidney

BACKGROUND AND PURPOSE We previously showed that mice with reduced levels of the transforming growth factor-beta (TGF-β) co-receptor endoglin (Eng(+/-) mice) develop less fibrosis and vascular damage after kidney irradiation than their wild type (Eng(+/+) mice) littermates; however, the underlying mechanism was unclear. Results from current studies suggest that this occurs via modulation of the radiation-induced inflammatory response. MATERIALS AND METHODS Kidneys of Eng(+/+) and Eng(+/-) mice were irradiated with 16Gy. Mice were sacrificed at 20weeks after irradiation and gene expression and protein levels were analyzed. RESULTS Kidney irradiation triggered the infiltration of macrophages in both Eng(+/+) and Eng(+/-) mice, however, levels of macrophage-produced cytokines interleukin 1 beta (Il1b) and interleukin 6 (Il6) were reduced in irradiated Eng(+/-) compared to Eng(+/+) mice. Double immuno-stainings confirmed that IL-6 was produced by macrophages, whereas IL-1β was mainly detected in other cell types. Accordingly, inflammatory cell precursors derived from the bone marrow of Eng(+/-) mice showed impaired ability to express Il1b and Il6 compared to wild type mice. CONCLUSIONS Endoglin promotes kidney inflammation after irradiation by regulating macrophage infiltration and interleukin production, thereby promoting pathogenic changes after radiation exposure.

Comparison of 32P-postlabeling and HPLC-FD analysis of DNA adducts in rats acutely exposed to benzo(a)pyrene

DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373-1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72, P = 0.006). In samples in which both tetrol I-1 and II-2 were detected by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.

Persistence and accumulation of (potential) single strand breaks in liver DNA of rats treated with diethylnitrosamine or dimethylnitrosamine: Correlation with hepatocarcinogenicity

Effects of diethylnitrosamine (DEN) and dimethylnitrosamine (DMN) on the sedimentation pattern of [3H]thymidine-labelled Sprague-Dawley female rat liver DNA in alkaline sucrose gradients were studied with regard to time and dose dependency. In experiments at 1--56 days after a single injection it was observed that (potential) single strand breaks induced by DEN were repaired at a low rate. At 56 days the sedimentation pattern was still grossly abnormal. Half-life values of 27 and 46 days were observed after 134 mg/kg DEN (approx. 45% of the LD50) and 13.4 mg/kg DEN, respectively. Identical experiments after DMN (10 mg/kg, corresponding to about 35% of the LD50) showed return to (almost) completely control sedimentation patterns within 56 days after injection (t 1/2 = 8 days). Experiments at 6 or 56 days after the last of a series of 5 or 10 weekly injections of DEN (13.4 mg/kg) showed that a major part of DEN-induced damage (measured as single strand breaks) is of a persistent and accumulating character. No accumulation of DMN-induced rat liver lesions was observed. It is concluded that DNA fragmentation and lack of DNA repair is not a consequence of hepatotoxicity. Since at equimolar doses DEN gives appreciably less DNA alkylation (including O6-alkylguanine) but is much more effective both as an inducer of preneoplastic liver lesions and as a hepatocarcinogen when compared with DMN, we believe that the formation of persistent (and accumulating) DNA damage after DEN administration might be relevant in the process of liver tumour formation.

ALK1 heterozygosity delays development of late normal tissue damage in the irradiated mouse kidney

BACKGROUND AND PURPOSE Activin receptor-like kinase 1 (ALK1) is a transforming growth factor β (TGF-β) receptor, which is mainly expressed in endothelial cells regulating proliferation and migration in vitro and angiogenesis in vivo. Endothelial cells also express the co-receptor endoglin, which modulates ALK1 effects on endothelial cells. Our previous studies showed that mice with reduced endoglin levels develop less irradiation-induced vascular damage and fibrosis, caused by an impaired inflammatory response. This study was aimed at investigating the role of ALK1 in late radiation toxicity. MATERIAL AND METHODS Kidneys of ALK(+/+) and ALK1(+/-) mice were irradiated with 14 Gy. Mice were sacrificed at 10, 20, and 30 weeks after irradiation and gene expression and protein levels were analyzed. RESULTS Compared to wild type littermates, ALK1(+/-) mice developed less inflammation and fibrosis at 20 weeks after irradiation, but displayed an increase in pro-inflammatory and pro-fibrotic gene expression at 30 weeks. In addition, ALK1(+/-) mice showed superior vascular integrity at 10 and 20 weeks after irradiation which deteriorated at 30 weeks coinciding with changes in the VEGF pathway. CONCLUSIONS ALK1(+/-) mice develop a delayed normal tissue response by modulating the inflammatory response and growth factor expression after irradiation.

The induction of chromosomal damage in rat hepatocytes and lymphocytes II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine, dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O6-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.

NAD+ depletion by APO866 in combination with radiation in a prostate cancer model, results from an in vitro and in vivo study

BACKGROUND APO866 is a highly specific inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), inhibition of which reduces cellular NAD(+) levels. In this study we addressed the potential of NAD(+) depletion as an anti-cancer strategy and assessed the combination with radiation. METHODS The anticipated radiosensitizing property of APO866 was investigated in prostate cancer cell lines PC3 and LNCaP in vitro and in PC3 xenografts in vivo. RESULTS We show that APO866 treatment leads to NAD(+) depletion. Combination experiments with radiation lead to a substantial decrease in clonogenic cell survival in PC3 and LNCaP cells. In PC3 xenografts, treatment with APO866 resulted in reduced intratumoral NAD(+) levels and induced significant tumor growth delay. Combined treatment of APO866 and fractionated radiation was more effective than the single modalities. Compared with untreated tumors, APO866 and radiation alone resulted in tumor growth delays of 14 days and 33 days, respectively, whereas the combination showed a significantly increased tumor growth delay of 65 days. CONCLUSIONS Our studies show that APO866-induced NAD(+) depletion enhances radiation responses in tumor cell survival in prostate cancer. However, the in vitro data do not reveal a solid cellular mechanism to exploit further clinical development at this moment.