Hypotonic Ca2+ signaling and volume regulation in proliferating and quiescent cells from multicellular spheroids

✍ Scribed by Heinrich Sauer; Jochen Ritgen; Jürgen Hescheler; Maria Wartenberg

• Publisher: John Wiley and Sons
• Year: 1998
• Tongue: English
• Weight: 598 KB
• Volume: 175
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/(sici)1097-4652(199805)175:2<129::aid-jcp2>3.0.co;2-q

✦ Synopsis

Hypotonicity-induced Ca 2/ signals and volume regulation were studied in proliferating and quiescent subpopulations of multicellular prostate cancer spheroids. Enzymatic dissociation of multicellular spheroids 100 { 19 mm in diameter, which are entirely proliferative, yielded a population of cells with a mean cell diameter of 17.5 { 1.4 mm. After dissociation of spheroids in a size class of 200 { 30, 300 { 60, and 400 { 65 mm in diameter, two subpopulations of cells with mean cell diameters corresponding to 12.9 { 1.9 mm and 16.7 { 2 mm were discriminated. The subpopulation of large cells was shown to be proliferative by positive Ki-67 antibody staining; the subpopulation of small cells was Ki-67 negative, indicating cell quiescence. In a spheroid size class of 100 { 19 mm, a distinct subpopulation of quiescent cells was absent. Superfusion by hypotonic solutions revealed that only the proliferating cell fraction showed a regulatory volume decrease (RVD) and a [Ca 2/ ] i transient. Both effects were absent in the quiescent cell population. The [Ca 2/ ] i transient persisted in low (10 nM) Ca 2/ solution and in the presence of 4 mM extracellular Ni 2/ but was abolished in the presence of the endoplasmic reticulum Ca 2/ -ATPase blocker 2,5-di-tert-butylhydrochinone (t-BHQ). The t-BHQ likewise inhibited RVD, indicating that Ca 2/ release from intracellular stores was necessary for RVD. Moreover, [Ca 2/ ] i and RVD were dependent on an intact microfilament cytoskeleton because after 30 min of preincubation with cytochalasin B the [Ca 2/ ] i transient was significantly reduced and RVD was abolished. The absence of RVD and [Ca 2/ ] i transient in quiescent cells may be due to differences in the amount and the cytosolic arrangement of F-actin observed in quiescent cells.