LONG-TERM EXPOSURE TO CYCLIC AMP INHIBITS PROLIFERATION AND IN VITRO TUMORIGENIC POTENTIAL OF HUMAN THYROID ANAPLASTIC CARCINOMA CELLS.

Biological response modifiers, such as dibutyryl-cAMP (db-cAMP) and retinoic acid (RA) have been demonstrated to induce the reappearance of a differentiated phenotype in transformed cells (i.e.: neuroblastoma, myeloid leukaemia, teratoma cells, etc.), simultaneously decreasing cell proliferation rate. Aim of the study was to evaluate the influence of db-cAMP and RA treatment on the growth rate, potential re-induction of differentiation markers and phenotypic properties of a highly malignant human anaplastic thyroid carcinoma cell line (ARO). Continuous exposure of ARO cells to db-cAMP (1mM) for 72 hours induced a marked reduction (- 44%) of cell proliferation rate, as assessed by (3H)-thymidine uptake. On the contrary, no effects on cell growth were observed in the presence of RA (5?M), while the combination of RA and db-cAMP treatment showed the same growth-inhibiting effect obtained with db-cAMP alone. db-cAMP-dependent reduction of ARO cell proliferation was confirmed by direct assessment of the number of mitotic cells. Expression of cell cycle-related genes, such as those coding for cyclins and cyclin-dependent kinases, was not modified after ARO cell treatment with either db-cAMP or RA, as shown by indirect immunofluorescence, Northern and Western blot analyses. In order to study their effectiveness as tumor-suppressive agents, cAMP and RA ability to modulate ARO cell growth in a semi-solid medium was assayed. db-cAMP alone, but not RA, dramatically suppressed ARO cell clonogenic potential in the soft agar assay. Expression of thyroid-specific genes was evaluated by semi-quantitative RT-PCR measurement of TSH-receptor, thyroglobulin and thyroid peroxidase steady-state mRNA levels on ARO cells under different experimental conditions. No differences in the re-expression of all thyroid-specific markers were observed between ARO cells treated with cAMP or RA as compared to untreated controls. In conclusion, prolonged exposure to db-cAMP but not to RA treatment is able to inhibit the proliferation rate and to greatly reduce the tumorigenic potential of ARO cells in vitro.