Identification of Risk Loci for Radiotoxicity in Prostate Cancer by Comprehensive Genotyping of TGFB1 and TGFBR1.

Genetic variability in transforming growth factor beta pathway (TGFB) was suggested to affect adverse events of radiotherapy. We investigated comprehensive variability in TGFB1 (gene coding for TGFß1 ligand) and TGFBR1 (TGFß receptor-1) in relation to radiotoxicity. Prostate cancer patients treated with primary radiotherapy (n = 240) were surveyed for acute and late toxicity. Germline polymorphisms (n = 40) selected to cover the common genetic variability in TGFB1 and TGFBR1 were analyzed in peripheral blood cells. Human lymphoblastoid cell lines (LCLs) were used to evaluate a possible impact of TGFB1 and TGFBR1 genetic polymorphisms to DNA repair capacity following single irradiation with 3 Gy. Upon adjustment for multiplicity testing, rs10512263 in TGFBR1 showed a statistically significant association with acute radiation toxicity. Carriers of the Cytosine (C)-variant allele (n = 35) featured a risk ratio of 2.17 (95%-CI 1.41-3.31) for acute toxicity ≥ °2 compared to Thymine/Thymine (TT)-wild type individuals (n = 205). Reduced DNA repair capacity in the presence of the C-allele of rs10512263 might be a mechanistic explanation as demonstrated in LCLs following irradiation. The risk for late radiotoxicity was increased by carrying at least two risk genotypes at three polymorphic sites, including Leu10Pro in TGFB1. Via comprehensive genotyping of TGFB1 and TGFBR1, promising biomarkers for radiotoxicity in prostate cancer were identified.

Diversity of Clinically Relevant Outcomes Resulting from Hypofractionated Radiation in Human Glioma Stem Cells Mirrors Distinct Patterns of Transcriptomic Changes.

Hypofractionated radiotherapy is the mainstay of the current treatment for glioblastoma. However, the efficacy of radiotherapy is hindered by the high degree of radioresistance associated with glioma stem cells comprising a heterogeneous compartment of cell lineages differing in their phenotypic characteristics, molecular signatures, and biological responses to external signals. Reconstruction of radiation responses in glioma stem cells is necessary for understanding the biological and molecular determinants of glioblastoma radioresistance. To date, there is a paucity of information on the longitudinal outcomes of hypofractionated radiation in glioma stem cells. This study addresses long-term outcomes of hypofractionated radiation in human glioma stem cells by using a combinatorial approach integrating parallel assessments of the tumor-propagating capacity, stemness-associated properties, and array-based profiling of gene expression. The study reveals a broad spectrum of changes in the tumor-propagating capacity of glioma stem cells after radiation and finds association with proliferative changes at the onset of differentiation. Evidence is provided that parallel transcriptomic patterns and a cumulative impact of pathways involved in the regulation of apoptosis, neural differentiation, and cell proliferation underly similarities in tumorigenicity changes after radiation.

Regeneration competent satellite cell niches in rat engineered skeletal muscle.

Satellite cells reside in defined niches and are activated upon skeletal muscle injury to facilitate regeneration. Mechanistic studies of skeletal muscle regeneration are hampered by the inability to faithfully simulate satellite cell biology in vitro. We sought to overcome this limitation by developing tissue engineered skeletal muscle (ESM) with (1) satellite cell niches and (2) the capacity to regenerate after injury. ESMs contained quiescent Pax7-positive satellite cells in morphologically defined niches. Satellite cells could be activated to repair (i) cardiotoxin and (ii) mechanical crush injuries. Activation of the Wnt-pathway was essential for muscle regeneration. Finally, muscle progenitors from the engineered niche developed de novo ESM in vitro and regenerated skeletal muscle after cardiotoxin-induced injury in vivo. We conclude that ESM with functional progenitor niches reminiscent of the in vivo satellite cell niches can be engineered in vitro. ESM may ultimately be exploited in disease modeling, drug screening, or muscle regeneration.

Noninvasive imaging of liver repopulation following hepatocyte transplantation.

Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore Optix System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.

Noninvasive Imaging of Liver Repopulation following Hepatocyte Transplantation.

Near infrared fluorescence (NIRF) optical imaging is a technique particularly powerful when studying in vivo processes at the molecular level in preclinical animal models. We recently demonstrated liver irradiation under the additional stimulus of partial hepatectomy as being an effective primer in the rat liver repopulation model based on hepatocyte transplantation. The purpose of this study was to assess optical imaging and the feasibility of donor cell expansion tracking in vivo using a fluorescent probe. Livers of dipeptidylpeptidase IV (DPPIV)-deficient rats were preconditioned with irradiation. Four days later, a partial hepatectomy was performed and wild-type (DPPIV+) hepatocytes were transplanted into recipient livers via the spleen. Repopulation by transplanted DPPIV+ hepatocytes was detected in vivo with Cy5.5-conjugated DPPIV antibody using the eXplore Optix™ System (GE HealthCare). Results were compared with nontransplanted control animals and transplanted animals receiving nonspecific antibody. Optical imaging detected Cy5.5-specific fluorescence in the liver region of the transplanted animals, increasing in intensity with time, representing extensive host liver repopulation within 16 weeks following transplantation. A general pattern of donor cell multiplication emerged, with an initially accelerating growth curve and later plateau phase. In contrast, no specific fluorescence was detected in the control groups. Comparison with ex vivo immunofluorescence staining of liver sections confirmed the optical imaging results. Optical imaging constitutes a potent method of assessing the longitudinal kinetics of liver repopulation in the rat transplantation model. Our results provide a basis for the future development of clinical protocols for suitable fluorescent dyes and imaging technologies.

In vitro studies on the modification of low-dose hyper-radiosensitivity in prostate cancer cells by incubation with genistein and estradiol.

BACKGROUND: As the majority of prostate cancers (PC) express estrogen receptors, we evaluated the combination of radiation and estrogenic stimulation (estrogen and genistein) on the radiosensitivity of PC cells in vitro. METHODS: PC cells LNCaP (androgen-sensitive) and PC-3 (androgen-independent) were evaluated. Estrogen receptor (ER) expression was analyzed by means of immunostaining. Cells were incubated in FCS-free media with genistein 10 microM and estradiol 10 microM 24 h before irradiation and up to 24 h after irradiation. Clonogenic survival, cell cycle changes, and expression of p21 were assessed. RESULTS: LNCaP expressed both ER-alpha and ER-beta, PC-3 did not. Incubation of LNCaP and PC-3 with genistein resulted in a significant reduction of clonogenic survival. Incubation with estradiol exhibited in low concentrations (0.01 microM) stimulatory effects, while higher concentrations did not influence survival. Both genistein 10 microM and estradiol 10 microM increased low-dose hyper-radiosensitivity [HRS] in LNCaP, while hormonal incubation abolished HRS in PC-3. In LNCaP cells hormonal stimulation inhibited p21 induction after irradiation with 4 Gy. In PC-3 cells, the proportion of cells in G2/M was increased after irradiation with 4 Gy. CONCLUSION: We found an increased HRS to low irradiation doses after incubation with estradiol or genistein in ER-alpha and ER-beta positive LNCaP cells. This is of high clinical interest, as this tumor model reflects a locally advanced, androgen dependent PC. In contrast, in ER-alpha and ER-beta negative PC-3 cells we observed an abolishing of the HRS to low irradiation doses by hormonal stimulation. The effects of both tested compounds on survival were ER and p53 independent. Since genistein and estradiol effects in both cell lines were comparable, neither ER- nor p53-expression seemed to play a role in the linked signalling. Nevertheless both compounds targeted the same molecular switch. To identify the underlying molecular mechanisms, further studies are needed.

Irradiation as preparative regimen for hepatocyte transplantation causes prolonged cell cycle block.

PURPOSE: Hepatocyte transplantation following liver irradiation (IR) and partial hepatectomy (PH) leads to extensive liver repopulation. We investigated the changes in the liver induced by IR explaining the loss of reproductive integrity in endogenous hepatocytes. MATERIALS AND METHODS: Right lobules of rat liver underwent external beam IR (25 Gy). A second group was subjected to additional 33% PH of the untreated left liver lobule. Liver specimens and controls were analyzed for DNA damage, apoptosis, proliferation and cell cycle related genes (1 hour to up to 12 weeks). RESULTS: Double strand breaks (phosphorylated histone H2AX) induced by IR rapidly declined within hours and were no longer detectable after 4 days. No significant apoptosis was noted and steady mRNA levels (B-cell lymphoma 2-associated X protein (BAX), caspase 3 and 9) were in line with the lack of DNA fragmentation. However, gene expression of p53 and p21 in irradiated liver tissue increased. Transcripts of cyclin D1, proliferating cell nuclear antigen (PCNA), and cyclin B augmented progressively, whereas cyclin E was only affected moderately. Following PH, irradiated livers displayed persistently high protein levels of p21 and cyclin D1. However, cell divisions were infrequent, as reflected by low PCNA levels up to four weeks. CONCLUSION: IR leads to a major arrest in the G(1)/S phase and to a lesser extent in the G(2)/M transition of the cell cycle, resulting in reduced regenerative response following PH. The persistent block of at least four weeks may promote preferential proliferation of transplanted hepatocytes in this milieu.