Evidence for Differential Regulation of Transferrin Receptor 1 in Normal vs. Malignant B Cells.
Authors: Chiotoglou Ioanna , Smilevska Tatjana , Stamatopoulos Kostas , Samara Maria , Likousi Sophia , Belessi Chrysoula , Laoutaris Nikolaos , Fassas Athanasios , Stathakis Nikolaos , Kollia Panagoula 
Published: April 2020

Abstract
Transferrin receptor 1 (TfR1, CD71) expression is related to the proliferative state of the cells as well as the induction of differentiation; furthermore, CD71 is one of the “classical” activation markers up-regulated upon B-cell activation. As we have recently shown (Smilevska et al., Leuk Res 2005, in press), CLL is characterized by almost uniformly high CD71 expression, regardless of IGH mutational load; this is in keeping with the activated status of neoplastic cells. In the present study, we evaluated TfR1 expression at the mRNA and protein level in CD19+ B cells sorted from peripheral blood mononuclear cells of two healthy donors, 37 patients with typical chronic lymphocytic leukemia (CLL), as well as the BV173 (B cell acute lymphoblastic leukemia) and EHEB (human chronic B cell leukemia) cell lines. In all CLL cases, the tumor load was at least 70%. Twenty-two out of 37 CLL cases (59%) carried IGHV genes with &lt;98% homology to the closest germline gene (“mutated”); the remainder (15/37, 41%) carried “unmutated” IGHV genes. TfR1 cDNA sequences were detected by RT-PCR in all cell samples using primers specific for TfR1 gene exons 15–17. Real-time PCR analysis (using h-HPRT as a housekeeping gene) revealed low TfR1 mRNA transcripts in both cell lines (relative TfR1/h-HPRT ratios for BV173/EHEB cells: 0.05/0.08), contrasting normal peripheral blood CD19+ B cells, which on average had a 1-log higher concentration (relative TfR1/h-HPRT ratios: 0.995). TfR1 mRNA levels were widely divergent in CLL samples (relative TfR1/h-HPRT ratios: 0.23–13.3); no statistically significant associations were identified with IGH mutation status. Cell lysates were separated by SDS-polyacrylamide gel electrophoresis, electroblotted onto the nitrocellulose filters for probing with anti-TfR1-specific antibody and actin polyclonal antibody (control); quantitative gel banding densitometry was performed using with Epson GT-8000 Laser scanner. TfR1 protein levels were similar in CD19+ peripheral blood normal B cells as well as malignant cell lines (CD19: 1.16, BV173: 1.16, EHEB: 1.12), although CLL samples showed greater individual variation (0.4–2); median values were not different from either normal B cells or B cell lines. CD71 expression was also studied by flow cytometry in 20/37 CLL cases; all analyzed cases were CD71-positive with a generally high percentage of CD71-expressing neoplastic cells (median 91%, range: 28.3–100.0%); based on staining intensity, 18/20 CD71 (+) cases had bright fluorescence intensity, whereas the remainder (2/20) had low fluorescence intensity (dim). These results allude to differential regulation of TfR1 expression in normal vs. malignant B cells. In normal B cells, transcriptional mechanisms exert an important control over TfR1 expression. In malignant B cells (primary cells or established cell lines), similar levels of TfR1 (CD71) protein in the context of widely divergent TfR1 mRNA levels might be attributed to the operation of post-transcriptional mechanisms (perhaps increased mRNA stability).