Figure 1.

Regulated tumor suppressor function of merlin expressed in the schwannoma cell line RT4-D6P2T. (A) At confluency, merlin expression is increased in primary rat Schwann cells but not in the RT4-D6P2T schwannoma cell line. Primary rat Schwann cells at early passage (2–3) were plated on poly-l-lysine-coated 100 mm dishes in DMEM + 10% FCS at high- and low-cell densities, RT4-D6P2T schwannoma cells on uncoated plates. After 24 h, lysates from density-matched cells were produced and resolved on a mini-gel followed by Western blotting using the antimerlin antibody C18 (αmerlin; merlin band at 70 kD). The equal loading of 25 μg of total protein was confirmed by an actin Western blot (αactin; apparent molecular mass 43 kD). (B) Doxycycline-inducible merlin expression clones. RT4-D6P2T cells were stably cotransfected with the r-tet regulator and doxycycline-inducible wild-type (clones 5₄ and 6₇) and mutant (L64P) merlin constructs. Cells at 70% confluency were harvested at 8 h after addition of doxycycline. Resolution of the 70 kD merlin band as in A. (C) Merlin reduces agar colony formation. Doxycycline-inducible clones expressing either wild-type merlin (clones 5₄ and 6₇), the mutant L64P, or vector control cells were placed in soft agar (see Material and Methods). Average number of colonies per well were plotted and standard errors indicated. (D) Merlin inhibits tumor growth in vivo. Subcutaneous tumor growth after injection of clone 5₄ cells into nude mice (see Material and Methods). Where indicated, doxycycline was added to the drinking water. The experiments also were performed with clone 6₇ with similar results.