Global gene expression analysis indicates that multiple pathways are affected during growth arrest of non-malignant human mammary epithelial cells in three dimensional cell cultures

5402 The growth arrest of non-malignant human mammary epithelial cells (HMEC) in Matrigel provides a three dimensional model system in which to study a normal cell process that is disrupted in cancer. To systematically probe the molecular changes that lead to growth arrest in this system, we have analyzed the expression profiles of 22,283 gene transcripts using Affymetrix HG-U133A microarrays. Microarray experiments were performed using RNA samples harvested from HMT3522 S1 cells at 3, 5, and 7 days post-seeding in Matrigel. Growth arrest in G0/G1 occurred between days 5 and 7. Transcripts exhibiting greater than 2-fold differences in 2 independent experiments were analyzed by 1-way ANOVA. By these criteria, 354 genes, including many involved in cell cycle transit, cytokinesis, and mitosis, were found to be down-regulated, and 105 genes, including many involved in epidermal differentiation, inflammatory processes, and negative regulation of cell proliferation, were found to be up-regulated. About 5% of the 459 identified genes would be expected to show such changes by chance alone. Interestingly, among the cell cycle regulatory genes examined, p21 transcripts showed no change, while p27 transcripts showed strong up-regulation. We used immunoblot analyses to validate and extend the results of the microarray analyses at the protein level. We initially confined these analyses to cell cycle regulators known to affect G1 checkpoints. Phosphorylation of several sites on Rb was found to gradually decrease between days 5 and 10 in the non-malignant cells, consistent with the growth suppressive role of the hypophosphorylated form. When normalized to total protein content, levels of cyclins E, A, and D1, as well as their binding partners - CDK2, 4, and 6, also decreased in the non-malignant cells during this period. Protein levels of the CDK inhibitors - p27 and p57, increased between days 5 and 10, while levels of p21 did not change. Additional analysis of the HMEC strain, 184, led to the identification of MAP2K1, TGF-beta, ATR, and SMARCA4, as key regulatory genes whose expression and/or function was altered in common in the two different HMEC specimens early in the time course. In current studies, we are determining which of the pathways identified are required for growth arrest in the three dimensional HMEC cultures.