Abstract 3952: CALM deficiency in fit1 embryonic cells alters expression and rate of endocytosis of growth factor receptors
Authors: Scotland Paula B. , Lavau Catherine P. , Conway Amanda E. , Wechsler Daniel S. 
Published: February 2011

Background: Gene rearrangements involving the Clathrin Assembly Lymphoid Myeloid Leukemia (CALM) and Mixed-Lineage Leukemia (MLL) or AF10 genes have been identified in aggressive leukemias and lymphomas. Expression of CALM-containing fusion proteins immortalizes murine hematopoietic cells in vitro, correlating with leukemogenesis in vivo. Disruption of normal CALM, MLL or AF10 protein function as a result of these translocations likely contributes to transformation, although the precise mechanisms are unknown. The native CALM protein is involved in clathrin-mediated endocytosis (CME); it localizes to the cytoplasmic side of endocytic vesicles, interacts with both membrane elements and clathrin, and when over- or under-expressed, disrupts endocytosis. To better understand the effects of altered CALM activity, we have focused our studies on mutant fit1 mice that lack CALM expression. Previous studies have shown that these mice have iron deficiency, defective hematopoiesis and ultimately premature death. Here we examine CALM's role in endocytosis and cellular function using cells derived from fit1 mice.

Objective: To examine the effect of CALM deficiency on cell surface receptor expression and endocytosis using mouse embryonic fibroblasts (MEFs) and fetal liver cells derived from normal and CALM-deficient mice.

Design: Day 14 MEFs were generated from normal, heterozygous, and mutant fit1 embryos. Immortalized MEFs were compared in terms of quantity of cell surface and total cellular receptors (by flow cytometry and Western blotting), receptor mRNA levels, and rate of endocytosis. Receptors were also measured in fetal liver derived from the same embryos.

Results: When compared to their normal counterparts, cells lacking full length CALM showed altered cell surface receptor expression: transferrin receptor (TfR) protein levels measured by flow cytometry were increased two-fold, while EGF receptor (EGFR) levels were reduced two-fold. These results correlated with increased total cellular TfR protein and mRNA levels and decreased total EGFR protein levels. Whereas the rate of internalization of TfR was similar, the rate of endocytosis of EGFR was significantly reduced in CALM-deficient compared with wildtype cells. Elevated TfR levels were also identified in CALM-deficient fetal liver cells.

Conclusions: CALM deficiency in fit1-derived cells results in increased TfR surface expression; this is consistent with the iron deficiency phenotype of fit1 mice. The reduced surface expression of EGFR seen in fit1 cells may be a compensatory mechanism that counteracts the increased growth factor receptor signaling that is associated with impaired endocytosis. Since CALM haploinsufficiency is a feature of CALM-AF10 and MLL-CALM leukemias, our results suggest that the perturbation of normal growth factor biology may contribute to transformation in these malignancies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3952.