Discrimination and evaluation of lactoferrin and delta-lactoferrin gene expression levels in cancer cells and under inflammatory stimuli using TaqMan real-time PCR

Abstract

The lactoferrin gene is known to be expressed either constitutively or under inducible conditions such as hormonal stimuli or inflammation. Its transcription from alternative promoters leads to two products, lactoferrin (Lf) and delta-lactoferrin (DeltaLf) mRNAs the expressions of which are altered during oncogenesis. The comparison of the two enhancer/promoter regions revealed that the two isoforms might be differentially trans-activated. Nevertheless, concomitant expression of both transcripts has been found in some normal tissues and in a subset of breast cancer cell lines and biopsies. Moreover, we found putative inflammatory response elements in both P1 and P2 promoter regions suggesting that both Lf and DeltaLf might be upregulated under inflammatory stimuli. Therefore, a duplex Taqman gene expression assay has been developed and used to profile mRNA expression of the Lf gene in the case of cancer and under inflammatory conditions. Discrimination between the two transcripts is achieved by using a primer pairs/probe set within exon 1beta for DeltaLf and a primer pairs/probe set within exon 1 and exon 2 for Lf. In this study, we confirmed that Lf/DeltaLf Taqman gene expression assay is a powerful tool to investigate the expression of both Lf and DeltaLf transcripts. We also showed that lymphocytes and leukocytes isolated from fresh human blood expressed an extremely high level of DeltaLf messengers. An extensive series of cancer cell lines has been studied confirming that both P1 and P2 promoter regions of the Lf gene are downregulated or silenced in the case of cancer. Furthermore, using stimulation by bacterial lipopolysaccharides (LPS), we showed that in MDA-MB-231 and HT-29 epithelial cells, Lf expression is strongly increased with a higher expression level in MDA-MB-231 whereas DeltaLf expression is not. These results suggest that the NF-kappaB/cRel response elements present in the P1 promoter region are functional whereas those present in the P2 promoter region are not and show that DeltaLf is not regulated in inflammatory conditions.

SILAC-Based Proteomic Profiling of the Human MDA-MB-231 Metastatic Breast Cancer Cell Line in Response to the Two Antitumoral Lactoferrin Isoforms: The Secreted Lactoferrin and the Intracellular Delta-Lactoferrin

“…Since this cancerous cell-line produces very low amounts of Lf isoform transcripts [4], [5], we established a stable and inducible MDA-MB-231 cell line expressing ΔLf under doxycycline induction. These cells were either induced by doxycycline to express ΔLf or treated with two different concentrations of hLf.…”

“…Isolated clones were expanded to obtain cells named MDA-MB-231 dox-. Expression of ΔLf was followed as described [5]. Clones used for the study did not produce any detectable ΔLf without induction.…”

“…Real-time PCR (qRT-PCR) were performed as described [5] using an Mx3005 thermal cycler system and Brilliant SYBER Green QPCR Master Mix (Stratagene, Agilent Technologies). DNA primer pairs and conditions used to amplify mRNA are compiled in Table S1.…”

“…The primer pairs were purchased from Eurogentec (Seraing, Belgium). TaqMan qRT-PCR was performed as described [5]. The ΔLf and Lf probes were 5′-FAM-labeled, the normalizing HPRT gene probe was 5′-VIC-labeled (Applied Biosystems, Life Technologies) and the 3′ non-fluorescent quencher (NFQ) (Applied Biosystems) was used for each probe.…”

“…The two main isoforms are secreted Lf (Lf) [1] and its nucleocytoplasmic counterpart, delta-lactoferrin (ΔLf) [2], [3]. Their expression is downregulated or silenced in cancer cells [2], [4], [5]. In some cancers, significantly lower levels of Lf and/or ΔLf correlated with more advanced disease and an unfavourable prognosis [4], [6].…”