Association between SNPs in defined functional pathways and risk of early or late toxicity as well as individual radiosensitivity.

BACKGROUND AND PURPOSE: The aim of this study was to determine the impact of functional single nucleotide polymorphism (SNP) pathways involved in the ROS pathway, DNA repair, or TGFB1 signaling on acute or late normal toxicity as well as individual radiosensitivity. MATERIALS AND METHODS: Patients receiving breast-conserving surgery and radiotherapy were examined either for erythema (n = 83), fibrosis (n = 123), or individual radiosensitivity (n = 123). The 17 SNPs analyzed are involved in the ROS pathway (GSTP1, SOD2, NQO1, NOS3, XDH), DNA repair (XRCC1, XRCC3, XRCC6, ERCC2, LIG4, ATM) or TGFB signaling (SKIL, EP300, APC, AXIN1, TGFB1). Associations with biological and clinical endpoints were studied for single SNPs but especially for combinations of SNPs assuming that a SNP is either beneficial or deleterious and needs to be weighted. RESULTS: With one exception, no significant association was seen between a single SNP and the three endpoints studied. No significant associations were also observed when applying a multi-SNP model assuming that each SNP was deleterious. In contrast, significant associations were obtained when SNPs were suggested to be either beneficial or deleterious. These associations increased, when each SNP was weighted individually. Detailed analysis revealed that both erythema and individual radiosensitivity especially depend on SNPs affecting DNA repair and TGFB1 signaling, while SNPs in ROS pathway were of minor importance. CONCLUSION: Functional pathways of SNPs may be used to form a risk score allowing to predict acute and late radiation-induced toxicity but also to unravel the underlying biological mechanisms.

Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation.

The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ΔΔCq/Normfinder and ΔΔCq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2 Gy, and changes in gene expression were analyzed 24 h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ΔΔCq/Genorm or ΔΔCq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested.

Liquid crystals on deformable surfaces.

Liquid crystals with molecules constrained to the tangent bundle of a curved surface show interesting phenomena resulting from the tight coupling of the elastic and bulk-free energies of the liquid crystal with geometric properties of the surface. We derive a thermodynamically consistent Landau-de Gennes-Helfrich model which considers the simultaneous relaxation of the Q-tensor field and the surface. The resulting system of tensor-valued surface partial differential equation and geometric evolution laws is numerically solved to tackle the rich dynamics of this system and to compute the resulting equilibrium shape. The results strongly depend on the intrinsic and extrinsic curvature contributions and lead to unexpected asymmetric shapes.

No effect of the transforming growth factor ß1 promoter polymorphism C-509T on TGFB1 gene expression, protein secretion, or cellular radiosensitivity.

PURPOSE: To study whether the promoter polymorphism (C-509T) affects transforming growth factor ß1 gene (TGFB1) expression, protein secretion, and/or cellular radiosensitivity for both human lymphocytes and fibroblasts. METHODS AND MATERIALS: Experiments were performed with lymphocytes taken either from 124 breast cancer patients or 59 pairs of normal monozygotic twins. We used 15 normal human primary fibroblast strains as controls. The C-509T genotype was determined by polymerase chain reaction-restriction fragment length polymorphism or TaqMan single nucleotide polymorphism (SNP) genotyping assay. The cellular radiosensitivity of lymphocytes was measured by G0/1 assay and that of fibroblasts by colony assay. The amount of extracellular TGFB1 protein was determined by enzyme-linked immunosorbent assay, and TGFB1 expression was assessed via microarray analysis or reverse transcription-polymerase chain reaction. RESULTS: The C-509T genotype was found not to be associated with cellular radiosensitivity, neither for lymphocytes (breast cancer patients, P=.811; healthy donors, P=.181) nor for fibroblasts (P=.589). Both TGFB1 expression and TGFB1 protein secretion showed considerable variation, which, however, did not depend on the C-509T genotype (protein secretion: P=.879; gene expression: lymphocytes, P=.134, fibroblasts, P=.605). There was also no general correlation between TGFB1 expression and cellular radiosensitivity (lymphocytes, P=.632; fibroblasts, P=.573). CONCLUSION: Our data indicate that any association between the SNP C-509T of TGFB1 and risk of normal tissue toxicity cannot be ascribed to a functional consequence of this SNP, either on the level of gene expression, protein secretion, or cellular radiosensitivity.

Association of single nucleotide polymorphisms in the genes ATM, GSTP1, SOD2, TGFB1, XPD and XRCC1 with risk of severe erythema after breast conserving radiotherapy.

PURPOSE: To examine the association of polymorphisms in ATM (codon 158), GSTP1 (codon 105), SOD2 (codon 16), TGFB1 (position -509), XPD (codon 751), and XRCC1 (codon 399) with the risk of severe erythema after breast conserving radiotherapy. METHODS AND MATERIALS: Retrospective analysis of 83 breast cancer patients treated with breast conserving radiotherapy. A total dose of 50.4 Gy was administered, applying 1.8 Gy/fraction within 42 days. Erythema was evaluated according to the Radiation Therapy Oncology Group (RTOG) score. DNA was extracted from blood samples and polymorphisms were determined using either the Polymerase Chain Reaction based Restriction-Fragment-Length-Polymorphism (PCR-RFL) technique or Matrix-Assisted-Laser-Desorption/Ionization -Time-Of-Flight-Mass-Spectrometry (MALDI-TOF). Relative excess heterozygosity (REH) was investigated to check compatibility of genotype frequencies with Hardy-Weinberg equilibrium (HWE). In addition, p-values from the standard exact HWE lack of fit test were calculated using 100,000 permutations. HWE analyses were performed using R. RESULTS: Fifty-six percent (46/83) of all patients developed erythema of grade 2 or 3, with this risk being higher for patients with large breast volume (odds ratio, OR = 2.55, 95% confidence interval, CI: 1.03-6.31, p = 0.041). No significant association between SNPs and risk of erythema was found when all patients were considered. However, in patients with small breast volume the TGFB1 SNP was associated with erythema (p = 0.028), whereas the SNP in XPD showed an association in patients with large breast volume (p = 0.046). A risk score based on all risk alleles was neither significant in all patients nor in patients with small or large breast volume. Risk alleles of most SNPs were different compared to a previously identified risk profile for fibrosis. CONCLUSIONS: The genetic risk profile for erythema appears to be different for patients with small and larger breast volume. This risk profile seems to be specific for erythema as compared to a risk profile for fibrosis.

The potential role of G2- but not of G0-radiosensitivity for predisposition of prostate cancer.

PURPOSE: Comparing the chromosomal radiosensitivity of prostate cancer patients with that of healthy donors. MATERIALS AND METHODS: The study was performed on 81 prostate cancer patients characterised by a clinical stage of predominantly pT2c or pT3a and a median age of 67 years. As healthy donors 60 male monozygotic twin pairs were recruited with a median age of 28 years. Chromosomal radiosensitivity was measured using both G0- and G2-assay. RESULTS: No difference between healthy donors and prostate cancer patients was detected concerning G0-radiosensitivity, since medians were similar (Hodges-Lehmann estimate: -0.05, 95% CI: -0.18-0.08, p=0.4167). However, a pronounced difference was determined for G2-radiosensitivity with prostate cancer patients showing a significantly higher sensitivity compared to healthy donors (Hodges-Lehmann estimate: -0.41, 95% CI: -0.53 to -0.30, p=1.75(-9)). Using the 90% quantile of G2-radiosensitivity in healthy donors as a threshold for discrimination the fraction of prostate cancer patients with elevated radiosensitivity increased to 49%. CONCLUSION: G2-, but not G0-radiosensitivity is a promising marker for predisposition of prostate cancer.

Organic anion transporting polypeptide 2B1 and breast cancer resistance protein interact in the transepithelial transport of steroid sulfates in human placenta.

The human placenta has both protective and nurturing functions for the fetal organism. Uptake and elimination of xenobiotics and endogenous substances are facilitated by various transport proteins from the solute carrier (SLC) and ABC families, respectively. A functional interaction of uptake and elimination, which is a prerequisite for vectorial transport across cellular barriers, has not been described for placenta. In this study, we examined expression of organic anion transporter (OAT) 4 (SLC22A11), organic anion transporting polypeptide (OATP) 2B1 (SLCO2B1, OATP-B), and breast cancer resistance protein (BCRP) (ABCG2) in human placenta (n = 71) because all three proteins are involved in transmembranal transfer of estrone 3 sulfate (E3S; metabolic product) and dehydroepiandrosterone sulfate (DHEAS; precursor molecule). On the mRNA level, we found a significant correlation of OATP2B1 and BCRP (R(2) = 0.534; p < 0.01) but not between OAT4 and BCRP (R(2) = -0.104; p > 0.05). Localization studies confirmed basal expression of OATP2B1 and apical expression of BCRP. To study functional interactions between OATP2B1 and BCRP, we developed a Madin-Darby canine kidney cell model expressing both transport proteins simultaneously (OATP2B1 and BCRP in the basal and apical membrane, respectively). Using this cell model in a transwell system resulted in a significantly increased basal to apical transport of both E3S and DHEAS, when both transporters were expressed with no change of transfer in the apical to basal direction. Taken together, these data show the potential for a functional interaction of OATP2B1 and BCRP in transepithelial transport of steroid sulfates in human placenta.

Modification of OATP2B1-mediated transport by steroid hormones.

The family of the organic anion transporting polypeptides forms an increasing group of uptake transport proteins with a wide substrate spectrum. Although the expression of some members of this group, such as organic anion transporting polypeptide (OATP)-A or C, is limited to special tissues (such as liver or brain), the organic anion transporting polypeptide 2B1 (OATPB/SLCO2B1) is expressed in many organs, including liver, placenta, mammary gland, brain, and intestine. However, little is known about its function in those tissues because only a limited number of compounds, such as dehydroepiandrosterone-sulfate (DHEAS) and estrone-3-sulfate (E3S), have been characterized as OATP2B1 substrates. To further elucidate the role of OATP2B1 on steroid transport, we examined the influence of steroid hormones on OATP2B1-mediated E3S and DHEAS uptake using OATP2B1-overexpressing Madin-Darby canine kidney II cells. We identified unconjugated androgens (e.g., testosterone) as potent inhibitors for OATP2B1. In contrast, gestagenes such as progesterone enhanced E3S uptake in a concentration-dependent manner to up to 300% of the control, accompanied by a significant decrease in the OATP2B1 K(m) value for E3S (control, K(m) = 14 microM; in the presence of 31.6 muM progesterone, K(m) = 3.6 microM). Moreover, we demonstrated that testosterone and progesterone are not substrates of OATP2B1, indicating an allosteric mechanism for the observed effects. Furthermore, we showed that progesterone enhances the OATP2B1-dependent pregnenolone sulfate transport. Taken together, the results indicate functional modification of OATP2B1-mediated E3S and DHEAS as well as pregnenolone sulfate transport through steroid hormones such as progesterone. These effects can have physiological consequences for the organ-specific uptake of steroids.