Elsevier

Experimental Cell Research

Volume 315, Issue 18, 1 November 2009, Pages 3125-3132
Experimental Cell Research

Research Article
Folate receptor α regulates cell proliferation in mouse gonadotroph αT3-1 cells

https://doi.org/10.1016/j.yexcr.2009.05.006Get rights and content

Abstract

We have previously found that the mRNA and protein levels of the folate receptor alpha (FRα) are uniquely over-expressed in clinically human nonfunctional (NF) pituitary adenomas, but the mechanistic role of FRα has not fully been determined. We investigated the effect of FRα over-expression in the mouse gonadotroph αT3-1 cell line as a model for NF pituitary adenomas. We found that the expression and function of FRα were strongly up-regulated, by Western blotting and folic acid binding assay. Furthermore, we found a higher cell growth rate, an enhanced percentage of cells in S-phase by BrdU assay, and a higher PCNA staining. These observations indicate that over-expression of FRα promotes cell proliferation. These effects were abrogated in the same αT3-1 cells when transfected with a mutant FRα cDNA that confers a dominant-negative phenotype by inhibiting folic acid binding. Finally, by real-time quantitative PCR, we found that mRNA expression of NOTCH3 was up-regulated in FRα over-expressing cells. In summary, our data suggests that FRα regulates pituitary tumor cell proliferation and mechanistically may involve the NOTCH pathway. Potentially, this finding could be exploited to develop new, innovative molecular targeted treatment for human NF pituitary adenomas.

Introduction

Human pituitary tumors are benign adenomas arising from adenohypophyseal cells in the anterior pituitary. They comprise 10% of all brain tumors and occur in ∼ 20% of the population [1], [2]. Approximately 30% of all anterior pituitary adenomas are termed nonfunctional pituitary adenomas due to their lack of clinical hormone hypersecretion [3]. Clinically, nonfunctional pituitary adenomas manifest as hypopituitarism or visual field defects due to regional compression of the optic chiasm [4], [5], [6]. Another challenge of these tumors is that they are usually large (macroadenomas) at the time of diagnosis because clinical features are inapparent until tumor mass effects occur. Despite the lack of clinical hormone hypersecretion, immunocytochemical staining for hormones reveals evidence for at least one glycoprotein hormone subunit expression in up to 86% of these clinically nonfunctional adenomas [6]. Nonfunctional pituitary adenomas are not homogeneous; they include null cell (17%), oncocytoma (6%), silent corticotroph (8%), silent somatotroph (3%), and gonadotrophs (40–79%). Unlike functional tumors, currently there is no available medical treatment or specific imaging technique for these nonfunctional pituitary adenomas. Therefore, it is important to identify potential biological markers as diagnostic and therapeutic targets of nonfunctional pituitary adenomas.

Using microarray techniques, our laboratory first reported that both mRNA and protein level of folate receptor α (FRα) are robustly up-regulated in nonfunctional pituitary adenomas, but not in functional adenomas [7], [8], [9]. The FR genes are located on chromosome 11q13.3–13.5, a region frequently deleted in pituitary adenomas [10], [11] and commonly amplified in carcinomas of the head and neck and breast [12]. There are three isoforms of FR (FRα, FRβ, and FRγ) that vary in sequence, ligand preference and tissue distribution [13], [14]. FRα (GenBank U20391) is the major isoform mediating folate transport. FRα has distinctive advantages in that it binds to folate with high affinity. Many studies suggest that FRα is absent or poorly expressed in most normal tissues but is vastly over-expressed in some tumors [8].

Previous studies indicate that elevated levels of FRα induce cell proliferation in breast cancer cells and malignant tissues [15], [16], but inhibit cell proliferation in cervical carcinoma cells [17]. Therefore, although we observed the up-regulation of FRα in nonfunctional pituitary adenomas, it was critical to further determine whether FRα promotes or reduces the proliferation of pituitary adenomas. Because it is difficult to transfect plasmids into primary pituitary adenoma cells, and we were not successful in transfecting the human gonadotroph-derived pituitary cell line HP75 [18], we decided to employ a mouse gonadotrope-derived αT3-1 cell line to address this critical question. We chose αT3-1 cell line because most clinically nonfunctional adenomas originate from gonadotrope cells based on immunohistochemistry. The αT3-1 cell line is a gonadotrope-derived cell line derived by targeted oncogenesis in transgenic mice. Thus, the αT3-1 cell line provides a model to study the role of human FRα in nonfunctional adenomas. This is the only cell line of clinically nonfunctional adenomas available for our experiments. In this study, we analyzed the biological properties of the αT3-1 cell line after over-expression of FRα. We also transfected a folate receptor α mutant cDNA (FR67) as a negative control, because this mutant significantly inhibits folate binding and uptake in a tumor cell line [19]. Moreover, we investigate the molecular mechanisms associated with the over-expression of FRα in transfected αT3-1 cell lines. We performed real-time quantitative PCR to investigate the expression of genes involved in the Wnt pathway including SFRP1, β-catenin, PITX2, cyclin D1, RB1, TLE2; as well as the Notch pathway including NOTCH3 and HES-1. We also examined PTTG1, EGFR, FGFR1, and ESR2 expression because these genes are involved in pathogenesis of human pituitary adenomas.

Section snippets

Materials

Mouse gonadotrope-derived cell lines (αT3-1) [20] were obtained from Dr. Pamela Mellon (University of California, San Diego, CA, USA). Fetal bovine serum was purchased from Atlanta Biologicals (Norcross, GA, USA). High-glucose DMEM, folate-free RPMI 1640 medium, Zeocin, Lipofectamine 2000 transfection reagent, Trizol reagent, and Superscript II were obtained from Invitrogen (Carlsbad, CA, USA). Vector pZeoSV2(+), vector pZeoSV2(+)-FRα, the mutant of FRα = FR67 or vector pZeoSV2(+)-FR67, rabbit

FRα is over-expressed in stably transfected clones of αT3-1 cells

To establish stable transfectants, αT3-1 cells were first transfected with an expression vector containing FRα cDNA [pZeoSV2(+)-FRα] or the mutant of FRα [FR67 = pZeoSV2(+)-FR67] or expression vector alone [pZeoSV2(+)]. After culture with 300 μg/ml Zeocin for 3 weeks, 30 clones from each of the transfected cells were selected and expanded. Three representative clones for pZeoSV2(+)-FRα or FRwt (named FRwt-3, FRwt-11, FRwt-14) and mutant FR67 (named FR67-9, FR67-11, FR67-27), and one clone from

Discussion

In the present study, we demonstrate that the stably transfected cells express the human folate receptor. We used over-expression of FRα in αT3-1 cells as a model for nonfunctional pituitary tumor cells in both physiological folic acid culture condition and soft agar. There are limitations to the model because the αT3-1 cells are immortalized while nonfunctional tumors are usually benign and are not fully transformed. The αT3-1 line represents a single cell type while tumors are often mixtures

Acknowledgments

The authors gratefully acknowledge financial assistance to Nelson M. Oyesiku, M.D., Ph.D., FACS, from the National Institutes of Health (R01-NS5143901). We thank Milton Brown, Ph.D., Emory University, for the assistance in writing this manuscript.

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