Our first approach was based on insertional mutagenesis by targeting cells with defective retroviruses.
The integration of virus can affect an individual gene and thereby altering the cell phenotype ( Dorssers . In view of the complexity and the workload of this approach, we have recently tested retroviral transduction of c DNA expression libraries ( Brummelkamp & Bernards 2003) as an alternative strategy.
Patients were evaluated every 3 months for the first 2 years, every 6 months for the next 3 years, and once a year thereafter. For this study, only specimen with at least 30% tumor nuclei, distributed uniformly over at least 70% of the section area, was included. Real-time quantitative RT-PCR (q RT-PCR) was performed using an ABI Prism 7700 Sequence detection system (Applied Biosystems) and a Stratagene Mx3000P QPCR System (Agilent Technologies, Waldbronn, Germany).
The prominent novel genes were selected for the development of primer sets designed to detect the most abundant splice variants, quality controls, and pilot analyses in a small group of tumor specimens.
By applying stringent selection criteria, we identified 15 breast cancer anti-estrogen resistance ( m RNA levels were significantly associated with tumor aggressiveness in lymph node-negative patients who had not received adjuvant systemic therapy.
Cells that acquired a gene product enabling growth in the presence of drugs can be recovered and the gene conferring resistance can be identified through PCR.To reduce the skewness, variables were log transformed.All transformed data were normally distributed and analyzed as continuous variables or in quartiles.Estrogen-dependent human breast cancer cells were transduced with different retroviral c DNA expression libraries and subjected to selective cultures with various anti-estrogens.From a total of 264 resistant cell clones, 132 different genes were recovered by PCR.Some gene quantifications were performed using inventoried Taq Man Gene Expression Assays from Applied Biosystems in combination with Taq Man Universal PCR Master Mix (Applied Biosystems) and in accordance with the protocol recommended by the manufacturer (Supplementary Table S1).test or Kruskal–Wallis test, including a Wilcoxon-type test for trend, when appropriate.The mechanistic basis for the resistant phenotype is thought to originate from the various aspects of estrogen signaling, the interaction with co-regulators, and the interplay with growth factor signaling ( Dorssers . In spite of the considerable progress made in the last decades, we still do not comprehend the complete spectrum of resistance mechanisms, and detailed study of different models may help to resolve these options.In recent years, we have used functional genetic screens to identify individual genes that contribute to or are responsible for resistance to anti-estrogens.Specific gene primer sets not meeting the stringent quality criteria (i.e.detection of genomic DNA or poor amplification efficiency) were excluded ( Sieuwerts . The primer sets were used in combination with SYBR green PCR Master Mix (Applied Biosystems) and are described in Supplementary Table S1, see section on supplementary data given at the end of this article.