Connexin Expression by Human Primary AML Cells
Authors: Foss Brynjar , Sæterdal Lars Rune , Ryningen Anita , Øyan Anne M. , Bruserud Øystein 
Published: October 2019

Abstract
Abstract 2147

Introduction:
Normal hematopoiesis takes place in the bone marrow niches where the hematopoietic stem cells are surrounded by stromal cells and extracellular matrix, and were both soluble mediators as well as cell-cell interactions seem to regulate proliferation and initial maturation. Among the various molecules participating in hematopoietic regulation are gap junctions (GJs) that are formed by connexins (Cxs) and represent intercellular communication channels (reviewed in Foss B et al., Stem Cell Dev. 18(6): 807–812, 2009). Acute myelogenous leukemia (AML) is characterized by bone marrow accumulation of immature leukemic cells, and both disease development as well as chemosensitivity of the leukemic cells seem to be affected neighbouring stromal cells in the bone marrow microenvironment. Studies of leukemic cell lines and animal models suggest that especially Cx43 and possibly Cx32 are involved in regulation of AML cell proliferation and differentiation, but the functional potential of Cxs in AML seems to be wider (reviewed in Foss B, et al., Biochim Biophys Acta. 1798(1):1-8, 2010). Still, the expression profile of Cxs by primary human AML cells are not well characterized.

Methods:
We characterized the mRNA and protein expression of various Cxs in primary human AML cells. The mRNA expression was investigated in microarray assay (n = 47) and the protein expression in the cell surface membrane by flow cytometry (n = 38).

Results:
The mRNA levels of Cx32 (mean 0.29, stdv ± 0.16), Cx43 (0.09 ± 0.21) and Cx45 (0.56 ± 0.25) showed very low levels whereas Cx37 showed higher expression (1.66 ± 0.93). The membrane expression was classified as positive (i.e. > 20% of AML cells stained positive) only for a minority of patients especially when investigating Cx32 (5 out of 38 patients examined) but also for Cx37 (13/38) and Cx43 (16/38), whereas the leukemic cells were classified as Cx45 positive for a majority of the patients (21/38). The mean fluorescence intensities (MFI) of the membrane expression for Cx32, Cx37, Cx43 and Cx45 were all significantly correlated (P<0.001). The strongest correlation was observed between Cx43 and Cx45 (Pearson correlation coefficient, r=0.946). The corresponding regression analysis showed R2 = 0.896, clearly suggesting a linear relationship between the membrane expression of Cx43 and Cx45. The membrane expression of Cx43 and Cx45 correlates with the expression of CD14 and CD15, and for Cx45 also with CD11c (all with P<0.05). In addition, the membrane expression of Cx43 and Cx45 were also correlating with cell morphology; cells without signs of differentiation (FAB M0+M1 classification) show less Cx expression than cells with signs of monocytic differentiation (FAB M4+M5, P<0.05, see figure). On the other hand, there was no correlation between the expression of the various Cxs and Flt-3-internal tandem duplication mutation and expression of CD33 and CD34.

Conclusions:
These results show for the first time that primary AML cells express various Cxs on their cell membranes, but patients are heterogeneous and the expression is seen especially in AML cells with signs of monocytic differentiation.

Disclosures:
No relevant conflicts of interest to declare.