EXPERIMENTAL CELL RESEARCH 225, 399–410 (1996) ARTICLE NO. 0191 Inhibition of the Proliferative Cycle and Apoptotic Events in WiDr Cells after Down-Regulation of the Calcium-Binding Protein Calretinin Using Antisense Oligodeoxynucleotides JEAN-CHARLES GANDER, 1 VASILLIS GOTZOS, 1 BENOI ˆ T FELLAY, AND BEAT SCHWALLER 2 Institute of Histology and General Embryology, University of Fribourg, CH-1705 Fribourg, Switzerland corresponding protein has been detected in Drosophila The colon adenocarcinoma cell line WiDr expresses melanogaster [5]. By immunohistochemical analysis the calcium-binding protein calretinin (CR). In order calretinin has been detected mainly in the central ner- to deduce possible functions of calretinin in these vous system in a specific set of neurons [6, 7], in inter- cells we decreased its concentration by antisense stitial cells of rat ovary [8], avian thymus [9], and in techniques. Treatment of WiDr cells with phosphoro- colon carcinoma cells [10]. The specific function of this thioate antisense oligodeoxynucleotides (AS-ODNs) protein is still uncertain, but a role in intracellular led to a drop in calretinin expression, as evidenced calcium transport or Ca 2/ -buffering in nerve cells has by immunohistochemical staining of WiDr cells and been suggested [11]. The role played by calretinin in Western blot analysis of cytosolic cell extracts. The nonexcitable cells and particularly in human tumor morphology of these epithelial cells changed from cells is unknown. polygonal to spherical and they formed dense cell In the human colon adenocarcinoma cell line WiDr, clusters. Cells displaying morphological alterations a close correlation between the cell cycle and CR-immu- typical for apoptotic cells were observed after incuba- noreactivity has been observed [10]. Since among other tion with AS-ODNs, as evidenced by phase-contrast functions, the intracellular messenger Ca 2/ acts as a and electron microscopy. The mitotic rate of AS-ODN- regulator of cell division (for review, see [12]) and treated cells dropped significantly, as demonstrated CaBPs are involved in mediating the Ca 2/ signal, a by mitotic labeling and time-lapse microcinematogra- phy. Furthermore, an accumulation of cells in phase function for calretinin in the cell cycle of WiDr cells G1 and a reduction of [ 3 H]thymidine-labeled cells was has been suggested [10]. For calmodulin, another mem- observed in antisense-treated cells. The basal level of ber of the EF-hand family of Ca 2/ -binding proteins, an [Ca 2/ ] i was not influenced by the down-regulation of important role in the regulation of the cell cycle has calretinin. WiDr cells incubated with the nonsense, already been demonstrated [13]. In Aspergillus nidu- reverse-sense, or with an oligodeoxynucleotide with lans, by replacing the endogenous calmodulin gene by a totally unrelated sequence did not show any sig- one which is regulated by an inducible promoter, a di- nificant differences when compared to control cells. rect correlation between calmodulin levels and cell pro- We conclude that calretinin levels have an impact on liferation has been demonstrated [14]. The question of the progression of the cell cycle of WiDr cells.  1996 the involvement of other CaBPs in cell cycle regulation Academic Press, Inc. is still under debate [15, 16]. A tightly regulated balance among proliferation, dif- ferentiation, and cell death is required for normal tis- INTRODUCTION sue homeostasis. Although abnormal regulation of cell proliferation or differentiation is a hallmark of cancer Calretinin (CR) is a calcium-binding protein (CaBP) cells, a defect in the regulation of the programmed cell of the EF-hand family [1], most closely related to cal- death (apoptosis) has been recognized as an important bindin-D28k [2]. The amino acid sequence is highly factor underlying the pathogenesis of tumor growth conserved among the vertebrate species studied [3] [17]. Apoptosis, as opposed to necrosis, is best charac- (even more so than cytochrome c [4]), and recently the terized by distinct morphological alterations first de- scribed by Kerr et al. [1972] [18] (for reviews, see [19, 1 J.-Ch.G. and V.G. equally contributed to the work. 20]). Several lines of evidence suggest that the same 2 To whom correspondence and reprint requests should be ad- proteins can have a function in the regulation of the dressed at Institute of Histology and General Embryology, Univer- sity of Fribourg, CH-1705 Fribourg, Switzerland. Fax: 037 299 732. cell cycle as well as in apoptosis depending on the level 399 0014-4827/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved.