Annette Steidler

Technical characterization of an ultrasound source for noninvasive thermoablation by high-intensity focused ultrasound

Objective To develop a generator for high‐intensity focused ultrasound (HIFU, a method of delivering ultrasonic energy with resultant heat and tissue destruction to a tight focus at a selected depth within the body), designed for extracorporeal coupling to allow various parenchymal organs to be treated.

Lymphangiogenesis occurs in upper tract urothelial carcinoma and correlates with lymphatic tumour dissemination and poor prognosis

To describe the lymphatic vessel density and to determine the functional and prognostic significance of tumoral lymphatic vessels in upper tract urothelial carcinoma (UTUC).

Cisplatin-resistant bladder carcinoma cells: enhanced expression of metallothioneins.

Abstract Cisplatin is one of the most potent cytotoxic drugs and in chemotherapy has ameliorated numerous tumors. Nevertheless, resistance to cisplatin is a problem that is encountered in the chemotherapy of urologic tumors, especially transitional cell carcinomas. In order to improve definition of the mechanisms of cisplatin-resistance we established a series of cisplatin-resistant sublines from the cell line RT 112 in increasing concentrations of cisplatin. The most resistant subline CP3 is approximately 10 times more resistant than the parental line and shows a 10-fold cross-resistance against methotrexate, whereas vinblastine and doxorubicin are equally effective in the parental and sublines. Combined treatment of CP3 cells with cisplatin and buthionine sulfoximine (BSO) does not result in enhanced cell kill, thereby ruling out glutathione as a resistance mechanism. However, in comparison with parental cells, CP3 cells are about 1.5 times more resistant against cadmium. On the protein level, the cisplatin-resistant cells reveal an enhanced expression of metallothionein II (MTII), but not MTI, suggesting that the cisplatin resistance we observed in these sublines is at least partly mediated by MTII. These sublines will in the future serve as valuable tools for the analysis of cisplatin resistance, especially in view of metallothionein-mediated resistance mechanisms.

HERV-E-Mediated Modulation of PLA2G4A Transcription in Urothelial Carcinoma

Human endogenous retroviruses (HERV) and related elements account for more than 8% of the human genome and significantly contribute to the human transcriptome by long terminal repeat (LTR) promoter activity. In this context, HERVs are thought to intervene in the expression of adjacent genes by providing regulatory sequences (cis-effect) or via noncoding RNA including natural antisense transcripts. To address the potential impact of HERV activity in urothelial carcinoma, we comparatively analyzed the HERV transcription profiles in paired samples of non-malignant urothelium and urothelial carcinoma derived from 13 patients with bladder cancer by means of a retrovirus-specific microarray (RetroArray). We established a characteristic HERV signature consisting of six ubiquitously active HERV subgroups (E4-1, HERV-Rb, ERV9, HERV-K-T47D, NMWV3, HERV-KC4). The transcription pattern is largely identical in human urothelial carcinoma, non-malignant urothelial tissue, four tumor-derived cell lines and in a non-malignant urothelial cell line (UROtsa). Quantitative reverse transcriptase PCR (qRT-PCR) of HERV-E4-1, HERV-K(HML-6) and HERV-T(S71-TK1) revealed a bias to lower HERV activity in carcinoma samples compared to non-malignant tissue. Determination of active HERV-E4-1 loci by cloning and sequencing revealed six HERV-E4-1 proviral loci that are differentially regulated in urothelial carcinoma cells and normal tissue. Two full-length HERV-E4-1 proviruses, HERV-Ec1 and HERV-Ec6, are located in antisense orientation in introns of the genes PLA2G4A and RNGTT, respectively. PLA2G4A encodes a cytosolic phospholipase A2 (cPLA2) that is dysregulated in many human tumors. PLA2G4A and HERV-Ec1 displayed reciprocal transcript levels in 7 of 11 urothelial carcinoma patients. Moreover, reciprocal shifts were observed after treatment of UROtsa cells with HERV-Ec1 and PLA2G4A-directed siRNAs or 5-aza-2′-deoxycytidine (aza-dC) pointing to an antagonistic regulation of PLA2G4A and HERV-Ec1 transcription in human urothelial cells. We suggest that transcription of HERV-Ec1 contributes to fine tuning of cPLA2 expression, thereby facilitating tumorigenesis.

Multidrug resistance in androgen-independent growing rat prostate carcinoma cells is mediated by P-glycoprotein

Prostate carcinomas are in general resistant against virtually all cytotoxic drugs. Up to now it has not been thoroughly evaluated whether specific resistance factors, such as the expression of theMDR1 gene, play a role in this multi-agent resistance and whether there is a link between drug resistance and hormone-independent growth. We investigated the resistance patterns of a hormone-sensitive and four hormone-independent Dunning rat carcinoma sublines against four drugs which are substrates of P-glycoprotein (vinblastine, taxol, doxorubicin, and etoposide) and two agents (methotrexate and cis-platinum) which are not transported by this efflux pump. All hormone-insensitive sublines, AT.1, AT. 3.1., MatLu and Mat LyLu, continuously showed a clearly enhanced resistance (3- to 26-fold) against the P-glycoprotein substrates, compared to the hormone-sensitive subline G. Only two of the androgen-independent sublines displayed enhanced resistance against methotrexate, whereas all of them were more sensitive against cisplatin than the androgen-sensitive G cells. By addition of verapamil the resistance against vinblastine (9- to 10-fold) and taxol (6.7- to 26.7-fold) in the hormone-insensitive cells could be almost totally reversed. Furthermore, the fluorescent P-glycoprotein substrate rhodamine-123 was effectively pumped out of the four tested hormone-independent cell lines, whereas the hormone-sensitive G cells were unable to extrude the dye. By reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for the ratmdr1b gene, the homologue to the humanMDR1 gene, we could easily detectmdr1b expression in the androgen independent cell lines, but not in the G cells. Our results suggest that the product of the ratmdr1b gene is involved in the multidrug resistance of androgen-independent Dunning prostate carcinoma cells.

New insights into the influence of cigarette smoking on urothelial carcinogenesis: smoking-induced gene expression in tumor-free urothelium might discriminate muscle-invasive from nonmuscle-invasive urothelial bladder cancer.

Smoking is the main risk factor for urothelial bladder cancer. In former smokers the risk decreases but does not reach the low level of never smokers. This indicates reversible and permanent smoking‐derived genetic alterations. Transcriptional changes may point to mechanisms, how smoking promotes urothelial bladder cancer. To identify smoking‐derived transcriptional changes we performed gene expression profiling in current, former, and never smokers, using tumor and tumor‐free urothelium from patients with nonmuscle‐invasive urothelial bladder cancer (NMIBC) or muscle‐invasive urothelial bladder cancer (MIBC). Smoking turned out to influence gene expression much less than tumor stage (NMIBC or MIBC) and tumor transformation (tumor‐free or tumor). Smoking seemed to influence gene expression in patients with MIBC more strongly compared to those with NMIBC. The least irreversible changes after smoking cessation were proposed in tumor‐free urothelium from patients with NMIBC. Growth factors and oncogenes were up‐regulated in tumor‐free urothelium from smokers with MIBC but not from smokers with NMIBC. A panel of genes up‐regulated in smokers have potential for early detection and distinction of MIBC from NMIBC using tumor‐free tissue.

Smoking Increases Transcription of Human Endogenous Retroviruses in a Newly Established in Vitro Cell Model and in Normal Urothelium

Human endogenous retroviruses (HERVs) accounting for 9% of the human genome are considered as surrogate markers for genetic instability and as a driving force of genetic variation. Moreover, they modulate regular gene activities and give rise to expression of disease-associated peptides that may serve as diagnostic markers or even targets for T cell-based immune responses. To date, no data are available on the potential link between urothelial carcinogenesis, HERV activity, and tobacco smoking, the main risk for bladder cancer. Here, we report on potential alterations in HERV transcription induced by smoking in a newly established in vitro model and in human urothelium. Normal human dermal fibroblasts were cultivated with urine from never (n = 6) and current smokers (n = 6) and transcription levels for the HERV subfamilies HERV-E 4-1, HERV-T S71-TK1, and HERV-K HML-6 were measured by quantitative real-time PCR. Tendencies toward increased mean transcript levels were detected for cells treated with urine from current smokers. Equally, activity measured in human urothelium supported an increase of HERV transcription in current smokers (n = 9) compared to never smokers (n = 4).

Expression and predictive value of lymph-specific markers in urothelial carcinoma of the bladder.

OBJECTIVE To evaluate the expression of multiple lymph-specific markers and to test its association with histopathological characteristics and clinical outcomes in patients with urothelial carcinoma of the bladder (UCB) treated by radical cystectomy (RC). PATIENTS AND METHODS Vascular endothelial growth factor-C and -D (VEGF-C/-D), its receptor VEGF receptor-3 (VEGFR-3), and chemokine receptor type 7 (CCR7) expressions were assessed by immunohistochemistry in RC specimens of 119 patients. Semiquantitative analyses of marker expressions were correlated with clinical and pathological characteristics. Univariable and multivariable analyses were performed to identify predictors of disease-specific survival (DSS) and recurrence free survival (RFS). RESULTS VEGF-C, VEGF-D, VEGFR-3, and CCR7 were overexpressed in 37.8%, 26.2%, 50.4%, and 23.5% of UCB samples, respectively. VEGF-D overexpression was significantly associated with a positive lymph node status (pN+). On univariable analysis, a higher pT stage, pN+, the presence of lymphovascular invasion (LVI) and vascular invasion (VI) (all P<0.001), and overexpressions of VEGF-D (P = 0.049) and VEGFR-3 (P = 0.032) were significantly associated with reduced DSS. On multivariable analysis, pT stage (P = 0.002) and pN+status (P = 0.009) were identified as independent predictors of reduced DSS. In a subgroup of patients without lymph node metastasis (pN0; n = 75), pT stage (P = 0.043) and VEGFR-3 overexpression (P = 0.008) were independent predictors of reduced DSS. CONCLUSION Lymph-specific markers are frequently overexpressed in UCB. VEGF-D overexpression is associated with the presence of lymphatic metastasis. In patients without lymph node metastasis at the time of RC, an assessment of VEGFR-3 expression may improve the identification of high-risk patients. These findings require prospective validation to determine the potential benefit of more aggressive adjuvant treatment.

Lymph vessel density in seminomatous testicular cancer assessed with the specific lymphatic endothelium cell markers D2-40 and LYVE-1: Correlation with pathologic parameters and clinical outcome

OBJECTIVES To evaluate the role of lymph vessel density (LVD) and lymphangiogenesis in seminomatous testicular cancer (STC) by using the lymphatic endothelial cell (LEC) markers LYVE-1 and D2-40. METHODS AND MATERIALS Paraffin embedded tumor specimens from 40 patients with STC were stained by specific D2-40 and Lyve-1 antibodies. LVD was measured in different representative and standardized areas. Fluorescence double immunostaining for Lyve-1 and Ki-67 was performed and results were correlated with clinicopathologic data. The median follow-up period was 55 (range 10-135) months. RESULTS Mean intratumoral LVD (D2-40: 1.30 ± 1.99; Lyve-1: 1.82 ± 2.34) was significantly lower than peritumoral LVD (D2-40: 4.94 ± 2.58; Lyve-1: 4.62 ± 2.73) and LVD in nontumoral areas (D2-40: 4.81 ± 3.79; Lyve-1: 4.22 ± 3.19). There was no significant difference between LVD measures when using D2-40 or LYVE-1. Detection rates of lymphatic vascular invasion (LVI) were significantly higher than in conventional HE-stained sections (77.5% vs. 52.5%). No proliferating lymphatic vessels were found. CONCLUSIONS We found that LVD is decreased within tumor areas of STC. Despite a higher peritumoral LVD, no signs of proliferating endothelial cells were observed, suggesting a lack of lymphangiogenesis in STC. Detection of LVI can be optimized by specific D2-40 or LYVE-1 staining.

The lymphatic system and its specific growth factor vascular endothelial growth factor C in kidney tissue and in renal cell carcinoma

To determine the role of vascular endothelial growth factor C (VEGF‐C) and the quantitative extent of the lymphatic system in renal cell carcinoma (RCC) to analyse a possible correlation with the metastatic spread of cancer cells.