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1.  Identification of alternative transcripts of rat CD9 expressed by tumorigenic neural cell lines and in normal tissues 
Genetics and Molecular Biology  2013;36(2):276-281.
CD9 is the best-studied member of the tetraspanin family of transmembrane proteins. It is involved in various fundamental cellular processes and its altered expression is a characteristic of malignant cells of different origins. Despite numerous investigations confirming its fundamental role, the heterogeneity of CD9 or other tetraspanin proteins was considered only to be caused by posttranslational modification, rather than alternative splicing. Here we describe the first identification of CD9 transcript variants expressed by cell lines derived from fetal rat brain cells. Variant mRNA-B lacks a potential translation initiation codon in the alternative exon 1 and seems to be characteristic of the tumorigenic BT cell lines. In contrast, variant mRNA-C can be translated from a functional initiation codon located in its extended exon 2, and substantial amounts of this form detected in various tissues suggest a contribution to CD9 functions. From the alternative sequence of variant C, a different membrane topology (5 transmembrane domains) and a deviating spectrum of functions can be expected.
PMCID: PMC3715295  PMID: 23885211
CD9; tetraspanin; transcript; splice variant; membrane topology
2.  Ranibizumab efficiently blocks migration but not proliferation induced by growth factor combinations including VEGF in retinal endothelial cells 
Proliferation and migration of retinal endothelial cells (REC) are associated with the development of proliferative diabetic retinopathy. REC proliferation is stimulated by isoforms of vascular endothelial growth factor-A (i.e., VEGF121 and VEGF165), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1) of which VEGF165 also enhances migration of REC. Effects induced by VEGF-A can be blocked with ranibizumab, a VEGF-binding Fab fragment used in therapy of diabetic macular edema. In this study, we investigated potential angiogenic effects of placental growth factors (PlGF-1, PlGF-2) as other members of the VEGF family and whether the primary action of VEGF165 is modulated in the presence of bFGF, IGF-1 and PlGF-1/-2. We also studied how effects of growth factor combinations can be attenuated with ranibizumab.
Effects of single growth factors or their combinations on proliferation and migration of immortalized bovine retinal endothelial cells (iBREC) were studied with or without ranibizumab or the inhibitor of VEGF receptors KRN951.
Proliferation of iBREC was significantly stimulated by 1–100 ng/ml PlGF-1 or PlGF-2, but additive effects were not observed with various combinations of the tested growth factors. Ranibizumab neutralized VEGF’s effect on proliferation but was not effective when the other growth factors were used in combination with VEGF. bFGF and IGF-1 but not PlGF-1 or PlGF-2 stimulated iBREC migration as single agents, and they further enhanced VEGF-induced migration. The effects of such growth factor combinations including VEGF on migration were efficiently blocked by targeting only VEGF with ranibizumab. Migration induced by VEGF plus bFGF and IGF-1 was also almost completely inhibited by KRN951 interfering with VEGF receptor signalling.
Migration but not proliferation of iBREC induced by combinations of bFGF, IGF-1, PlGF-1 or PlGF-2 together with VEGF is efficiently suppressed by ranibizumab. VEGF-mediated signalling through VEGFR2 seems to control REC migration dominantly in the presence of other growth factors.
PMCID: PMC3777160  PMID: 23760670
Retinal endothelial cells; Growth factors; Ranibizumab; Migration; Proliferation; Proliferative diabetic retinopathy
3.  Actions of bevacizumab and ranibizumab on microvascular retinal endothelial cells: similarities and differences 
The British Journal of Ophthalmology  2012;96(7):1023-1028.
Retinal endothelial cells are crucially involved in the genesis of diabetic retinopathy which is treated with vascular endothelial growth factor (VEGF) inhibitors. Of these, ranibizumab can completely restore VEGF-induced effects on immortalised bovine retinal endothelial cells (iBREC). In most experiments supporting diabetic retinopathy therapy with bevacizumab, only non-retinal EC or retinal pigment epithelial cells have been used. Also, bevacizumab but not ranibizumab can accumulate in retinal pigment epithelial cells.
To investigate the effects of bevacizumab on VEGF-induced changes of iBREC properties and potential uptake and accumulation of both inhibitors.
Uptake of VEGF inhibitors by iBREC with or without pretreatment with VEGF165 was visualised by immunofluorescence staining and western blot analyses. Measured transendothelial resistance (TER) of iBREC (±VEGF165) showed effects on permeability, indicated also by the western blot-determined tight junction protein claudin-1. The influence of bevacizumab on proliferation and migration of iBREC was studied in the presence and absence of VEGF165.
Bevacizumab strongly inhibited VEGF-stimulated and basal migration, but was less efficient than ranibizumab in inhibiting VEGF-induced proliferation or restoring the VEGF-induced decrease of TER and claudin-1. This ability was completely lost after storage of bevacizumab for 4 weeks at 4°C. Ranibizumab and bevacizumab were detectable in whole cell extracts after treatment for at least 1 h; bevacizumab accumulated during prolonged treatment. Ranibizumab was found in the membrane/organelle fraction, whereas bevacizumab was associated with the cytoskeleton.
Both inhibitors had similar effects on retinal endothelial cells; however, some differences were recognised. Although barrier properties were not affected by internalised bevacizumab in vitro, potential adverse effects due to accumulation after repetitive intravitreal injections remain to be investigated.
PMCID: PMC3382447  PMID: 22539748
Retinal endothelial cells; VEGF inhibition; diabetic macular oedema; diabetic retinopathy; biochemistry; diagnostic tests/investigation; macula; neovascularisation; retina
4.  Medium-sized deletion in the BRCA1 gene: Limitations of Sanger sequencing and MLPA analyses 
Genetics and Molecular Biology  2012;35(1):53-56.
We describe a family with a history of breast and ovarian cancer in which MLPA analysis of the BRCA1 gene pointed to a deletion including a part of exon 11. Further characterization confirmed a loss of 374 bp in a region completely covered by conventional sequencing which had not revealed the deletion. Because this alteration was only detected serendipitously with an MLPA probe, we calculated the probabilities of detecting medium-sized deletions in large exons by methods including initial PCR amplification. This showed that a considerable fraction of medium-sized deletions are undetectable by currently used standard methods of mutation analyses. We conclude that long, widely overlapping amplicons should be used to minimize the risk of missing medium-sized deletions. Alternatively, large exons could be completely covered by narrow-spaced MLPA probes.
PMCID: PMC3313516  PMID: 22481874
mutation analysis; DNA sequencing; PCR; hereditary breast cancer; model calculations
5.  Association of the Variants CASP8 D302H and CASP10 V410I with Breast and Ovarian Cancer Risk in BRCA1 and BRCA2 Mutation Carriers 
The genes caspase-8 (CASP8) and caspase-10 (CASP10) functionally cooperate and play a key role in the initiation of apoptosis. Suppression of apoptosis is one of the major mechanisms underlying the origin and progression of cancer. Previous case-control studies have indicated that the polymorphisms CASP8 D302H and CASP10 V410I are associated with a reduced risk of breast cancer in the general population.
To evaluate whether the CASP8 D302H (CASP10 V410I) polymorphisms modify breast or ovarian cancer risk in BRCA1 and BRCA2 mutation carriers, we analyzed 7,353 (7,227) subjects of white European origin provided by 19 (18) study groups that participate in the Consortium of Investigators of Modifiers of BRCA1/2 (CIM-BA). A weighted cohort approach was used to estimate hazard ratios (HR) and 95% confidence intervals (95% CI).
The minor allele of CASP8 D302H was significantly associated with a reduced risk of breast cancer (per-allele HR, 0.85; 95% CI, 0.76–0.97; Ptrend = 0.011) and ovarian cancer (per-allele HR, 0.69; 95% CI, 0.53–0.89; Ptrend = 0.004) for BRCA1 but not for BRCA2 mutation carriers. The CASP10 V410I polymorphism was not associated with breast or ovarian cancer risk for BRCA1 or BRCA2 mutation carriers.
CASP8 D302H decreases breast and ovarian cancer risk for BRCA1 mutation carriers but not for BRCA2 mutation carriers.
The combined application of these and other recently identified genetic risk modifiers could in the future allow better individual risk calculation and could aid in the individualized counseling and decision making with respect to preventive options in BRCA1 mutation carriers.
PMCID: PMC3077716  PMID: 20978178
6.  Evidence for SMAD3 as a modifier of breast cancer risk in BRCA2 mutation carriers 
Walker, Logan C | Fredericksen, Zachary S | Wang, Xianshu | Tarrell, Robert | Pankratz, Vernon S | Lindor, Noralane M | Beesley, Jonathan | Healey, Sue | Chen, Xiaoqing | Stoppa-Lyonnet, Dominique | Tirapo, Carole | Giraud, Sophie | Mazoyer, Sylvie | Muller, Danièle | Fricker, Jean-Pierre | Delnatte, Capucine | Schmutzler, Rita K | Wappenschmidt, Barbara | Engel, Christoph | Schönbuchner, Ines | Deissler, Helmut | Meindl, Alfons | Hogervorst, Frans B | Verheus, Martijn | Hooning, Maartje J | van den Ouweland, Ans MW | Nelen, Marcel R | Ausems, Margreet GEM | Aalfs, Cora M | van Asperen, Christi J | Devilee, Peter | Gerrits, Monique M | Waisfisz, Quinten | Szabo, Csilla I | Easton, Douglas F | Peock, Susan | Cook, Margaret | Oliver, Clare T | Frost, Debra | Harrington, Patricia | Evans, D Gareth | Lalloo, Fiona | Eeles, Ros | Izatt, Louise | Chu, Carol | Davidson, Rosemarie | Eccles, Diana | Ong, Kai-Ren | Cook, Jackie | Rebbeck, Tim | Nathanson, Katherine L | Domchek, Susan M | Singer, Christian F | Gschwantler-Kaulich, Daphne | Dressler, Anne-Catharina | Pfeiler, Georg | Godwin, Andrew K | Heikkinen, Tuomas | Nevanlinna, Heli | Agnarsson, Bjarni A | Caligo, Maria Adelaide | Olsson, Håkan | Kristoffersson, Ulf | Liljegren, Annelie | Arver, Brita | Karlsson, Per | Melin, Beatrice | Sinilnikova, Olga M | McGuffog, Lesley | Antoniou, Antonis C | Chenevix-Trench, Georgia | Spurdle, Amanda B | Couch, Fergus J
Breast Cancer Research : BCR  2010;12(6):R102.
Current attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies.
We successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers.
SNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r2 = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, Ptrend = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, Ptrend = 0.018).
This study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.
PMCID: PMC3046447  PMID: 21114847
7.  Differential expression of tetraspanin CD9 in basal cell and squamous cell carcinomas of the skin and actinic keratosis 
Oncology Letters  2010;1(1):37-40.
Tetraspanins are potentially useful molecular markers that differentiate between tumour classes and subtypes, since members of this protein family were often found to be altered during malignant conversion and tumour progression. In this study, we analysed expression of the tetraspanin CD9 in the frequent cutaneous neoplasms basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and actinic keratosis (AK), which is considered a precursor lesion (carcinoma in situ) from which an invasive SCC can develop. A moderate to strong CD9-specific staining of the tumour cells’ plasma membranes was uniquely observed in all BCCs, SCCs and AKs. All SCCs showed additional intracellular CD9 which was rarely (20%) seen in AKs. Semi-quantitative assessment of CD9 present in the plasma membranes of tumour cells of BCCs (mean staining intensity 1.91) and SCCs (3.64) reflected the different CD9 expression of normal precursor cells from which these tumours most likely originate. Although considered an intermediate stage in the development of SCCs, AKs did not show intense staining of the plasma membranes typical of normal keratinocytes or invasive SCCs (p=0.011) but only moderate intensity (mean 1.63). In BCCs, significantly (p=0.0005, n=56) stronger CD9-specific immunoreactivity was seen in the inner regions of the tumours than at their sites of expansion. In summary, our results point to an important role of CD9 at the front of tumour expansion in BCCs and SCCs, and in the pathogenesis of invasive SCCs.
PMCID: PMC3436406  PMID: 22966252
tetraspanin; cutanous neoplasm; solar keratosis; immunohistochemistry

Results 1-7 (7)