Effects of 1,25‐dihydroxyvitamin D3 on cell‐cycle kinetics of T 47D human breast cancer cells

Publisher Website
Google Scholar
Abstract
The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25‐(OH)2D3, which inhibit cellular replication, on the cell‐cycle kinetics of a 1,25‐(OH)2D3‐responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25‐(OH)2D3 in the range 10−9 M to 10−6 M caused a time‐ and concentration‐dependent decrease in cell numbers. Treatment of cells growing in charcoal‐treated fetal calf serum with 10−8 M 1,25‐(OH)2D3 for 6 days reduced cell numbers to 49% ± 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% ± 0.5% (n = 11) to 19.6% ± 2.3% (n = 9), significant by paired analysis (P > 0.002). At higher concentrations of 1,25‐(OH)2D3 (10−7 −10−6 M), there was a concentration‐dependent decline in S phase and increases in both Go/G1 and G2 + M phase cells.Detailed analysis of the temporal changes in cell‐cycle phase distribution following treatment with 2.5 × 10−8 and 10−7 M 1,25‐(OH)2D3 showed an initial accumulation of cells in Go/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in Go/G1 phase and an increase in G2 + M. In contrast cells treated with 10−7 M 1,25‐(OH)2D3 had significantly elevated % Go/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from Go/G1 following 4 days of exposure to 10−7 M 1,25‐(OH)2D3. This accumulation of cells in Go/G1 was accompanied by a fall in % S phase cells. After 3 days of treatment the % Go/G1 phase cells began to decline, due presumably to a reduced rate of entry into this phase caused by the transition delay through G2 + M which was present at early times but increased rapidly between days 2 and 4 to reach a maximal effect between 4 and 6 days. These studies confirm the 1,25‐(OH)2D3 effect on progression through G1 phase previously reported in hemopoietic cells. Furthermore they document an additional transition delay in G2 + M which is a major contributor to the known growth inhibitory effects of this hormone in T 47D human breast cancer cells. Further study of these effects may lead to a better understanding of hormonal regulation of cellular replication in human breast cancer.