Effect of the all trans retinoic acid (ATRA) on the differentiation, invasion and growth, in 3D culture and in vivo, of the murine mammary cell lines LM38. (FIRCA R03 TW007207) Free

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ATRA regulates growth, differentiation and apoptosis in different cellular types. Previously we demonstrated that ATRA treatment inhibited growth in monolayer of mammary lines LM38-HP (constituted by epithelioid cells), LM38-D2 (myoepithelial clone) and LM38-LP (containing both cellular types). Now we are evaluating the effect of ATRA in differentiation, survival and invasion of LM38 cells in three-dimensional matrigel cultures as well as in vivo growth and dissemination. For in vitro studies, cells grown for 3 days in matrigel were treated or not with 1uM ATRA for 2 additional days. Colonies were marked with different antibodies and analysed by confocal microscopy. The bicellular line LM38-LP developed polarized colonies (GM130, Golgi marker) with acinar structure. ATRA maintained the polarity and induced an increase in E-cadherin expression and reorganization. ATRA also induced a decrease of myoepithelial cells, indicated by the lower expression of the myoepithelial marker CK14 in the periphery of the colonies. Treatment with ATRA also increased the expression of activated Caspase 3 and promoted the nuclear translocation of p27 in the colony center, suggesting arrest of cellular cycle and induction of death. On the other hand, LM38-HP cells generated starry colonies without lumen and variable size, without polarized cells, even though ATRA induced an increase of activated Caspase 3. LM38-D2 cells organized in colonies with variable size and form without lumen or polarization. Treatment with ATRA showed a decrease in the expression of CK14 as well as and an increase of activated Caspase 3 together with the nuclear translocation of p27. In all cases ATRA treatment induced a reduction in colony size. In vivo studies were curried out employing LM38-LP cell line due to its ability to growth as invasive primary tumor and to metastasize lung. Cell were inoculated s.c. in Balb/c mice and when tumours were palpable, oral treatment with ATRA (2 mg/animal, twice a week) was administered for 15 days. A significant decrease in tumor growth, local invasion and the number and size of pulmonary metastasis was observed in ATRA treated mice 40 days after inoculation (p < 0,05). Besides, ATRA reduced invasion in matrigel (transwell) of all cellular lines, showing its most marked effect on the mixed LM38-LP (64% less than control, p < 0,05). Our results suggest that interactions between luminal and myoepithelial cells are necessary to accentuate the effect of ATRA in differentiation, death, invasion and metastatic dissemination.