Transkriptionales Targeting zur zielgerichteten Krebsgentherapie des Mammakarzinoms

Zusammenfassung

Fragestellung: Die Gentherapie ist ein Therapiekonzept der molekularen Medizin, welches durch ein Targeting der Vektoren die gerichtete Expression therapeutischer Gene für eine systemische und tumorspezifische Krebstherapie ermöglicht. Tumorspezifische Promotoren (TSPs) sind geeignet, um eine selektive transgene Expression in der Krebszelle zu erreichen. Heparanase (HPR) wird in Mammakarzinomzellen überexprimiert und spielt eine wichtige Rolle im Rahmen der Metastasenentwicklung. Ziel dieser Studie war es zu untersuchen, ob HPR ein geeigneter Promotor zur Krebsgentherapie des Mammakarzinoms ist.

Material und Methodik: Untersuchungen zur HPR-Expression erfolgten mittels quantitativer RT-PCR in etablierten Mammakarzinomzelllinien, primären aufgereinigten Mammakarzinomzellen von Patientinnen und gesunden Kontrollzellen. Zur Untersuchung der Promotoraktivität wurde ein Adenoviraler (Ad) Vektor (AdHPRluc) konstruiert. Dieser exprimiert Luziferase als Reportergen unter der transkriptionalen Kontrolle des HPR-Promotors. Zur Kontrolle diente ein Ad-Vector, welcher den ubiquitär exprimierten CMV-Promotor (AdCMVluc) enthält.

Ergebnisse: Die quantitative RT-PCR zeigte eine 4,5 - 44,6fach, (p < 0,05) erhöhte Expression des HPR-Gens in verschiedenen Mammakarzinomzelllinien im Vergleich zu gesunden Mammazelllinien. AdHPRluc zeigte eine hohe Aktivität in verschiedenen Mammakarzinomzelllinien (5,5 - 12,7 % im Vergleich zu CMV) und primären Mammakarzinomzellen (8,8 - 14,4 %).

Schlussfolgerung: Der Promotor des HPR-Gens ist geeignet für transkriptionale Targetingstrategien im Rahmen einer adenoviralen Krebsgentherapie des Mammakarzinoms.

Abstract

Purpose: Gene therapy with adenoviral (Ad) vectors is a promising new approach for different tumor types. Strategies to restrict adenoviral-mediated transgene expression are important to avoid side effects due to gene transfer into healthy cells. Heparanase (HPR) is highly overexpressed in human cancers including breast cancer but low or undetectable in differentiated, healthy tissue.

Material and Methods: To evaluate the utility of HPR as a TSP for breast cancer gene therapy, RT-PCR was performed to evaluate the expression of the HPR gene in various established breast cancer cell lines, primary human breast cancer tissue samples and normal breast cell lines. We constructed an Ad vector, AdHPR.luc, encoding luciferase under the control of the HPR promoter to determine relative activity in a variety of breast cancers, normal human breast cell lines and purified breast cancer tissue samples. An Ad vector containing the ubiquitously expressed CMV promoter (AdCMV.luc) was used as control.

Results: Quantitative RT-PCR revealed a 4.5 - 44.6fold, (p < 0.05) increased expression of the HPR gene in several breast cancer cell lines compared to a normal breast control cell line. When compared to the ubiquitous CMV promoter, the HPR promoter showed a high level of activity in four breast cancer cell lines (5.5 - 12.7 % compared to CMV) and primary breast cancer patient samples (8.8 - 14.4 %), whereas activity in normal breast cells was low.

Conclusion: These findings show that the HPR pathway is a target for the development of breast cancer directed gene therapy strategies.

Krebsgentherapie - Mammakarzinom - Heparanase - transkriptionales Targeting

Breast cancer - tissue-specific promoters - transcriptional targeting - cancer gene therapy

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