MCF-7 human breast cancer cells purchased from the American Tissue Culture Collection (Rockville, MD, USA) were maintained as monolayer cultures in 75 cm² plastic culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Gibco), penicillin (20 units/ml) and streptomycin (20 µg/ml) (Sigma), at 37°C in a humid atmosphere containing 5% CO₂. Cells were subcultured every 3-4 days by suspension in 5mM Na₂-EDTA in PBS (pH 7.4) at 37°C for 5 min.

Before each experiment, stock subconfluent monolayers (80%) of MCF-7 cells were incubated with 5mM Na₂-EDTA in PBS (pH 7.4) at 37 °C for 5 min, resuspended in DMEM supplemented with 10% FBS and passed repeatedly through a 25-gauge needle to produce a single cell suspension. Cell number and viability were determined by staining a small volume of cell suspension with 0.4% trypan blue saline solution and examining the cells in a hemocytometer. Cells were plated into 60 x 15 mm culture dishes (3.0 x 10⁵ cells/dish) containing DMEM plus 10% FBS and antibiotics. After the cells had firmly attached to the dishes (24h) melatonin (1 nM) or the diluent (ethanol, final concentration 0.01%) were added to the culture media. Twenty-four hour later cells were harvested from plates to determine apoptosis as well as p53 and p21WAF1 concentrations.

Apoptosis was determine by fluorescence microscopy by using an in situ cell death detection kit (TdT-mediated dUTP nick end labeling, TUNEL, Boehringer Mannheim). The expression of p53 and p21WAFF1 was quantified with ELISA kits from Oncogene Research.

Melatonin, at the physiological doses used in the experiment (1 nM), increased the percentage of dead cells (evaluated by the trypan blues exclusion test). In situ labeling of DNA strand breaks by the TUNEL reaction showed a significative increase of the number of apoptotic cells in those incubated with melatonin, in relation to the controls. The concentration of both p53 and p21WAF1 proteins was higher (p < .001) in MCF-7 cells incubated with melatonin than in control cells.

Recent studies have demonstrated that the treatment of DMBA-induced rat mammary cancer with antiestrogens causes some cancer cells to fall into apoptosis with a subsequent regression of the tumor (Watanabe et al. 1995). The treatment of MCF-7 cells with tamoxifen (a nonsteroidal antiestrogen) also induces apoptosis (Welsh 1994). On this basis, we considered it worthwhile studying whether melatonin may play a role in the control of programmed cell death in MCF 7 human breast cancer cells.

We found that melatonin decreases the viability of MCF-7 cells and increases the percentage of dead cells. In previous morphological studies (Hill and Blask 1988, Crespo et al. 1994) it had been demonstrated that some melatonin-treated MCF-7 cells display degenerative features such as mitochondrial swelling with disruption of cristae, cytoplasmic vacuolation, nuclear chromatin disgregation and cell lysis, all signs being suggestive of apoptosis. The in situ labeling of apoptosis-induced DNA strand breaks by the TUNEL reaction shows that melatonin is able to activate apoptotic cell death in this tumor cell line.

The p53 tumor suppressor gene delays cell cycle progression in response to DNA damage and arrests the cell cycle in G₁ phase before the initiation of DNA synthesis. This function is executed by accumulation of p53 followed by induction of WAF1 gene. Interestingly, the expression of both p53 and WAF1 genes in MCF-7 cells is increased by physiological doses of melatonin. These findings support the hypothesis that melatonin treatment increases the number of MCF 7 cells which fall into apoptosis, probably by interacting with p53 mediated mechanisms.

* Supported by grants from the Spanish DGICYT (PM97-0042) and Foundation "Marques de Valdecilla (6/98).

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