Abstract
In serum-deprived human fibroblasts IMR-90 and WI-38 cells, the addition of fetal calf serum or basic fibroblast growth factor stimulates DNA synthesis in an extracellular Ca(2+)-dependent manner; the effect of serum on [3H]thymidine incorporation into DNA is 4-16-fold greater at 2.0 mM CaCl2 as compared with that at 0.03 mM CaCl2. By contrast, in SV40 virus-transformed WI-38 (SV-WI-38) cells DNA synthesis is essentially independent of the extracellular calcium concentration ([Ca]out) and serum growth factors. To explore the role of Ca2+ in mitogenic signal transduction through G1 to S phase cell cycle progression, we studied and compared the effect of [Ca]out on phosphorylation of RB protein, the product of a tumor suppressor retinoblastoma gene. In IMR-90 and WI-38 cells, serum or basic fibroblast growth factor induces an increase in the amount of hyperphosphorylated forms of RB protein in a manner strictly dependent on [Ca]out. In sharp contrast, in SV-WI-38 cells, the extent of RB phosphorylation is little affected by [Ca]out or the presence or absence of serum growth factors. In addition, potent calmodulin antagonists W-7 and calmidazolium, but not an inactive analogue W-12 or W-5, strongly inhibit serum-induced increases in DNA synthesis and RB phosphorylation in IMR-90 and WI-38 cells, whereas in SV-WI-38 cells, the inhibitory effect is much more limited. Under the same treatment conditions, we measured histone H1 kinase activity associated with anti-p34cdc2 immunoprecipitate and found that the serum-induced increase in p34cdc2 kinase activity is strongly dependent on [Ca]out and is potently inhibited by the active calmodulin antagonists in IMR-90 and WI-38 cells, but not in SV-WI-38 cells. In IMR-90 cells that have been incubated with serum in 0.03 mM [Ca]out for 24 h, restoration of [Ca]out to 2.0 mM results in initiation of DNA synthesis after 13 h and concomitant increases in RB phosphorylation and p34cdc2 histone H1 kinase activity. These results suggest that in human fibroblasts, Ca2+/calmodulin regulates the signaling cascade leading to cdc2 kinase activation, RB protein phosphorylation, and DNA synthesis and that this Ca(2+)-dependent regulation is abrogated in SV40-transformed cells.
Highlights
WI-38 cells, the addition of fetal calf serum o r basic pensable for mammalian cell growth and proliferation both fibroblast growth factor stimulates DNA synthesis in i n vivo and i n vitro [1,2,3,4,5,6,7,8,9,10]
In SV40 virus-trans- lated to tumorigenicity i n vivo [4, 11,12,13,14]. It is of formed WI-38 (SV-WI-38) cells DNA synthesis is es- vital importance to elucidate the site(s) and/or the mechasentially independentof the extracellularcalcium con- nism(s) of action of Ca2+ incell growth regulatory pathways
4) Controlled expression of sense or antiserum-induced increase in p34cdc2 kinase activity is sense calmodulin mRNA leads to ainncrease or a decrease in strongly dependent on [CaIoutand is potently inhibited cellular calmodulin content, respectively, and either transient by the active calmodulin antagonists in IMR-90 and acceleration of proliferation or transient cell cycle arrest
Summary
Mannbeim), 60 p~ ATP, 10 mM MgCI,, 1 mM dithiothreitol, 50 mM Tris-HC1(pH 7.4), and 2 pCi of [y3’P]ATP. The reaction was WI-38 and IMR-90 are humandiploid fibroblast cell lines derived terminated by adding 4 X sample buffer and boiling for 5 min. They were obtained from the Japanese PAGE, followed by autoradiography. Cells were maintained in Dulbec- corresponding to histone H1was determined by Fuji BAS 2000 Bio-. CO’S modified Eagle’s medium containing10%fetal bovine serum Image Analyzer. Cells at passage numbers 11-18 for Each experiment was performed a t least three times, and similar. IMR-90 and 15-21 for WI-38 were used in the present study.Before results were obtained. Mm dishes were serum deprivedfor at least 3 daysin Dulbecco’s modified Eagle’s medium containing 0.2% bovine serumalbumin
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
