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Constitutive production of human interleukin for DA cells/leukemia inhibitory factor by human tumor cell lines
H Gascan1, I Anegón, V Praloran
1INSERM U211, U.E.R. de Médecine, CHU de Nantes, France.
Insights
Leukemia inhibitory factor (LIF) was purified from bladder carcinoma cells and detected in multiple human tumor cell lines. PMA treatment significantly increased LIF secretion, indicating its potential role in cancer progression.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Leukemia inhibitory factor (LIF) is a cytokine known to induce proliferation in certain cell lines.
- Previous studies suggested LIF's role in cell proliferation, but its presence and secretion in various human tumor types remained largely uncharacterized.
Purpose of the Study:
- To purify human IL for DA cells (HILDA)/LIF from a human bladder carcinoma cell line.
- To develop and validate assays for detecting LIF in human tumor cell lines.
- To investigate the prevalence and regulation of LIF secretion in human cancers.
Main Methods:
- Purification of HILDA/LIF using a multi-step chromatographic procedure (cation exchange, Con A affinity, reverse phase HPLC, gel filtration).
- Radioligand binding assay using M1 cells to quantify LIF.
- Proliferative assay using DA1.a cells.
- Analysis of LIF mRNA expression via RT-PCR.
Main Results:
- HILDA/LIF was purified to homogeneity from 5637 cell supernatant, yielding a 43 kDa protein.
- A sensitive radioreceptor assay detected LIF in 12 out of 17 human tumor cell line supernatants.
- Phorbol 12-myristate 13-acetate (PMA) treatment increased LIF secretion 10-20 fold in tested cell lines.
- LIF mRNA transcripts were detected in secreting cell lines, with a major species at 3.8 kb.
Conclusions:
- HILDA/LIF is secreted by a significant proportion of human tumor cell lines.
- LIF secretion can be upregulated by PMA, suggesting a potential regulatory mechanism in cancer.
- The developed assays provide tools for further investigation into LIF's role in tumorigenesis.
Abstract:
The cytokine HILDA (human IL for DA cells)/LIF (leukemia inhibitory factor), previously reported to trigger the proliferation of the DA1.a cell line was purified to homogeneity from the culture supernatant of the 5637 bladder carcinoma cell line by using a four step procedure, including cationic exchange chromatography at pH 6, Con A affinity chromatography, reverse phase HPLC, and gel filtration HPLC. The purified radiolabeled protein was analyzed in SDS-PAGE and a single band with a molecular mass of 43 kDa was revealed. The iodinated material was then tested in a radioreceptor assay using the M1 cell line as a second target cell line. The binding of the natural 125I hormone was abrogated in a dose dependent and specific fashion by either natural and rHILDA. The limit of signal detection of displacement for the radiolabeled ligand by unlabeled sample was found to be 0.05 ng/ml. The proliferative DA1.a assay and the M1 radioreceptor assay were used to analyze the HILDA content of 17 human tumor cell line culture supernatants, and 12 among them were spontaneously secreting a detectable level of LIF. The secreted amount of HILDA was largely enhanced by treating the cell line with 30 to 80 nM PMA; in these conditions a 10 to 20x increase in the detected amount was observed. The specificity of the detected signal was reinforced by analyzing the mRNA expression of HILDA in the tumor cell lines. The secreting cell lines were found to express various forms of LIF transcripts with a major specie at 3.8 kb.
