Mode of estrogen action on cell proliferation in CAMA‐1 cells: II. Sensitivity of G1 phase population

Abstract

The mammary cancer cell line CAMA-1 synchronized at the G1/S boundary by thymidine block or at the G1/M boundary by nocodazole was used to evaluate (1) the sensitivity of a specific cell cycle phase or phases to 17β-estradiol (E₂), (2) the effect of E₂ on cell cycle kinetics, and (3) the resultant E₂ effect on cell proliferation. In synchronized G1/S cells, E₂-induced ³H-thymidine uptake, which indicated a newly formed S population, was observed only when E₂ was added during, but not after, thymidine synchronization. Synchronized G2/M cells, enriched by Percoll gradient centrifugation to approximately 90% mitotic cells, responded to E₂ added immediately following selection; the total E₂-treated population traversed the cycle faster and reached S phase approximately 4 hr earlier than cells not exposed to E₂. When E₂ was added during the last hour of synchronization (ie, at late G2 or G2/M), or for 1 hr during mitotic cell enrichment, a mixed response occurred: a small portion had an accelerated G1 exit, while the majority of cells behaved the same as controls not incubated with E₂. When E₂ addition was delayed until 2 hr, 7 hr, or 12 hr following cell selection, to allow many early G1 phase cells to miss E₂ exposure, the response to E₂ was again mixed. When E₂ was added during the 16 hr of nocodazole synchronization, when cells were largely at S or possibly at early G2, it inhibited entry into S phase. The E₂-induced increase or decrease of S phase cells in the nocodazole experiments also showed corresponding changes in mitotic index and cell number. These results showed that (1) the early G1 phase and possibly the G2/M phase are sensitive to E₂ stimulation, late G1, G1/S, or G2 are refractory; (2) the E₂ stimulation of cell proliferation is due primarily to an increased proportion of G1 cells that traverse the cell cycle and a shortened G1 period, (3) E₂ does not facilitate faster cell division; and (4) estrogen-induced cell proliferation or G1/S transition occurs only when very early G1 phase cells are exposed to estrogen. These results are consistent with the constant transition probability hypothesis, that is, E₂ alters the probability of cells entering into DNA synthesis without significantly affecting the duration of other cell cycle phases. Results from this study provide new information for further studies aimed at elucidating E₂-modulated Gl events related to tumor growth.