Hypotonic Ca2+ signaling and volume regulation in proliferating and quiescent cells from multicellular spheroids.
Hypotonicity-induced Ca2+ signals and volume regulation were studied in proliferating and quiescent subpopulations of multicellular prostate cancer spheroids. Enzymatic dissociation of multicellular spheroids 100+/-19 microm in diameter, which are entirely proliferative, yielded a population of cells with a mean cell diameter of 17.5+/-1.4 microm. After dissociation of spheroids in a size class of 200+/-30, 300+/-60, and 400+/-65 microm in diameter, two subpopulations of cells with mean cell diameters corresponding to 12.9+/-1.9 microm and 16.7+/-2 microm 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 microm, 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 [Ca2+]i transient. Both effects were absent in the quiescent cell population. The [Ca2+]i transient persisted in low (10 nM) Ca2+ solution and in the presence of 4 mM extracellular Ni2+ but was abolished in the presence of the endoplasmic reticulum Ca2+-ATPase blocker 2,5-di-tert-butyl-hydrochinone (t-BHQ). The t-BHQ likewise inhibited RVD, indicating that Ca2+ release from intracellular stores was necessary for RVD. Moreover, [Ca2+]i and RVD were dependent on an intact microfilament cytoskeleton because after 30 min of preincubation with cytochalasin B the [Ca2+]i transient was significantly reduced and RVD was abolished. The absence of RVD and [Ca2+]i transient in quiescent cells may be due to differences in the amount and the cytosolic arrangement of F-actin observed in quiescent cells.
Sauer
H
H
Institute for Neurophysiology, University of Cologne, Germany. hs@physiologie.uni-koeln.de
Ritgen
J
J
Hescheler
J
J
Wartenberg
M
M
D000199:Actins / Q000032:analysis; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002455:Cell Division / Q000502:physiology; D048429:Cell Size / Q000502:physiology*; D003571:Cytochalasin B / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology*; D007150:Immunohistochemistry; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D019394:Ki-67 Antigen / Q000276:immunology; D008297:Male; D009532:Nickel / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000199:Actins; D006873:Hydroquinones; D007038:Hypotonic Solutions; D019394:Ki-67 Antigen; D003571:Cytochalasin B; D009532:Nickel; D015544:Inositol 1,4,5-Trisphosphate; D000255:Adenosine Triphosphate; C018855:2-tert-butylhydroquinone; D000252:Calcium-Transporting ATPases; D002118:Calcium