Identification of an alternative form of human lactoferrin mRNA that is expressed differentially in normal tissues and tumor-derived cell lines

Abstract

Lactoferrin (LF), traditionally known as an iron-binding protein present in high concentrations in milk and various secretions, has emerged as a multifunctional protein involved in many aspects of the host defense against infection. Recently, LF has been shown to inhibit the growth of solid tumors and reduce experimental metastasis in mice, suggesting that LF also may play a role in the defense against tumorigenesis. Here we provide the sequence of the cDNA and promoter region, the chromosome assignment, and tissue expression pattern of a novel form of LF mRNA (⌬LF). The sequence of ⌬LF mRNA is nearly identical to that of LF mRNA; however, at the 5 end, we find a novel sequence that replaces the N-terminal signal peptide sequence of LF mRNA. We map the ⌬LF mRNA to human chromosome 3 and find that both ⌬LF and LF sequences colocalize to the same cloned 90-to 150-kb genomic DNA fragment. We further show that the ⌬LF mRNA is the product of alternative splicing of the LF gene and likely is specified by use of an alternative promoter. Although we find ⌬LF mRNA at various levels in 20 of 20 adult and fetal human tissues, we do not find ⌬LF mRNA in any of 14 diverse tumor-derived cell lines.

Expression and prognostic value of lactoferrin mRNA isoforms in human breast cancer

“…Discrimination between the 2 isoforms at the mRNA level in the breast cancer cell lines showed that while a decrease in Lf expression was comparable to previously reported studies, ⌬Lf mRNA was not absent from these cells. 9,16,26 -28 In contrast to the data of Siebert and Huang, 9 in which ⌬Lf transcripts were absent after 42 PCR amplification cycles in 10 derived tumor cell lines, we found that ⌬Lf mRNA was in fact present in different breast cancer cell lines after 40 PCR amplification cycles. In fact, in selecting T47D as a representative breast cancer cell line, they unfortunately chose the worst Lf and ⌬Lf messenger-producing cell line according to our results.…”

“…14 The P2 promoter possesses upstream potential regulatory elements such as XRE, 1/2 HRE, AML-1a and Sp1. 9,10 Recently, it was shown that both promoters contain GGAA motifs for Ets binding and that Ets-1 is a strong regulator of the P2 promoter. 10 Most data in the literature concern the secreted protein Lf, well known as a cationic protein with iron-chelating activity belonging to the transferrin family.…”

“…It was first detected as a messenger present at various levels in all human tissues and absent in 10 derived tumor cell lines whose normal tissues express relatively high levels of this mes-senger. 9 Since ⌬Lf expression has been reported as being downregulated in cancers, we recently investigated ⌬Lf function using a stable HEK 293 cellular model, which expressed ⌬Lf under doxycycline induction. We were able to show that ⌬Lf expression, which could be observed in both the cytoplasm and the nucleus, led to cell cycle arrest in the S phase.…”

“…4 -8 Transcription of the Lf gene leads to 2 products, Lf and ⌬Lf mRNAs, which result from the use of alternative promoters: P1 for Lf and P2 for ⌬Lf. 9,10 Alternative splicing concerns exon 1, which characterizes the Lf mRNA and is replaced by exon 1␤ in the ⌬Lf messenger. Apart from the 5Ј end, the ⌬Lf messenger is identical to the Lf mRNA and translation from exon 1␤ leads to a protein devoid of a leader sequence and the first 25 amino acids.…”

Induction of lactoferrin gene expression by innate immune stimuli in mouse mammary epithelial HC-11 cells

“…To study the mechanism that regulates LF gene expression in epithelial cells faces a challenge, because the LF gene is permanently silenced in a majority of cancer cells [34][35][36]. Due to the lack of a suitable cell system for mechanistic study, most of the regulatory studies on LF were conducted in animals and in transiently transfected culture cell lines [7,17,37,38].…”