Changes in the Expression of the Tumor-Associated Antigen Recognized by Monoclonal Antibody 44–3A6 in A549 Cells due to Calcium

Abstract

Although there is extensive data available on Ca²⁺ effects in normal tissues, comparatively little is known about its effects or regulation in tumor cells. The present studies were undertaken to investigate whether various extracellular calcium concentrations could modulate the expression of the tumor-associated antigen (TAA) recognized by monoclonal antibody (MAb) 44–3A6. It is highly expressed by the human lung adenocarcinoma cell line A549 and has been shown to be a 40-kD integral plasma membrane protein. Treatment of the A549 cell line with various concentrations of exogenous calcium showed a dose-dependent rise in the internal free calcium levels up to 2.4–2.9 mM (external calcium treatment). At higher concentrations, the internal calcium level showed a decline, indicating a higher calcium efflux. The calmodulin-dependent Ca²⁺-ATPase enzyme involved in calcium homeostasis was assayed under these same conditions. The enzyme activity increased with increasing external calcium concentrations showing a 5-fold increase in cells treated with 4.05 mM calcium. These data suggest that as the internal calcium approaches toxic levels, the Ca²⁺-regulated ATPase activity increases to reduce the calcium overload within the cell. Employing Western blot analysis and immuno-peroxidase staining studies, this report shows that the antigen recognized by MAb 44–3A5 on A549 cells increased with an increase in calcium concentration. Evidence that this antigen is phosphorylated is presented using Western blot analysis of a radiolabeled antigen-enriched plasma membrane fraction. The previously reported subcellular localization, and now the phosphorylation and responsiveness to calcium by this TAA, gives it the properties predicted to be seen in a calcium ‘pump-like’ molecule. Thus, these studies support the hypothesis that this TAA may be important in intracellular calcium concentration control or that it is regulated via some calcium-mediated process.

Expression of the Epitope Recognized by the Monoclonal Antibody 44–3A6 during Human Fetal Development

“…Recently, we have reported that the extra cellular concentration of calcium can in fluence the intracellular free calcium concen trations within the human adenocarcinoma cell line, A549 [9], These studies demon strated that calcium can influence the ex pression of this antigen, in that increased concentrations of calcium resulted in the in creased expression of this antigen. It is not clear at the present time, whether or not this is a direct effect, or if it is an epiphenomenon.…”

Expression of the Adenocarcinoma‐Related Antigen Recognized by Monoclonal Antibody 44‐3A6 in Salivary Gland Neoplasias

“…It is known that the expression of the antigen recognized by Mab 44-3A6 in A549 can be modulated by changes in pH (manuscript in preparation, 1996) and intracellular Ca 2+ concentrations. 23 The antigen recognized by Mab 44-3A6 has been shown to be a useful marker for pulmonary adenocarci- nomas, where it is found to be more abundantly expressed in well-differentiated tumors. 15 Immunostaining for this antigen has also been reported to parallel the degree of differentiation in adenocarcinomas arising in the breast.…”

Monoclonal antibody 44‐3A6 as an adjunct in cytodiagnosis of adenocarcinomas in body fluids

“…However, it does appear that this 40-Kd transmembrane phosphoprotein may be linked to intracellular calcium concentrations, and/or the cellular components which regulate intracellular free calcium levels. 35 Considerable work remains before the function of this important AC antigen is known, as well as what role it plays in the growth of antigen positive cells.…”

“…21- 35 The present study was undertaken to determine the effectiveness of MCA 44-3A6 in detecting AC in body fluids (BF) arising in various tissues.…”

“…The monoclonal antibody (MCA) 44-3A6 was raised against the human pulmonary adenocarcinoma cell line A549, is well characterized, and has been shown to react with human pulmonary adenocarcinomas (ACs) in histologic sections. 21- 35 The present study was undertaken to determine the effectiveness of MCA 44-3A6 in detecting AC in body fluids (BF) arising in various tissues.…”