RASSF1A variants isolated from tumor cell lines are unable to block proliferation. (a) H1299 cells were transfected with RASSF1A (A), RASSF1A(S131F) (B), or RASSF1A(A133S) (C) and processed as in Fig. 1b. Overlay images are shown with rhodamine red X-conjugated anti-rabbit IgG to detect the anti-Myc polyclonal antibody, and FITC-conjugated anti-mouse IgG to detect the anti-BrdU monoclonal antibody. Quantitation by microscopic observation of at least three independent experiments is shown in Table 1. (b) H1299 cells expressing the carboxy-terminal halves of the indicated RASSF1 variants were labeled with ³²P_i for 4 h. RASSF1 variants were immunoprecipitated, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and exposed to PhosphorImager plates to detect incorporation of ³²P_i (top panels). Total RASSF1 was detected by Western blotting with anti-Myc antibody (bottom panels). w.t., wild type.

Ras12V expression does not bypass RASSF1A-mediated growth arrest. HME50-hTERT cells were transfected with the indicated constructs and processed as described in the legend to Fig. 3b. The percentage of RASSF1A-expressing cells and RASSF1A+Ras12V-expressing cells incorporating BrdU is shown normalized to the BrdU incorporation frequency of cells expressing Ras12V alone (approximately 80% of total cells).

Enhanced accumulation of cylin D1 in the absence of RASSF1A expression. (a) BrdU incorporation in RASSF1A-expressing HeLa cells was assayed as described in Fig. 1. (b) HeLa cells were exposed to siRNA specifically targeting the RASSF1A transcript for 72 h. siRNA directed against caveolin 1 was used as a negative control. Cyclin D1 levels in cells exposed to caveolin 1 siRNA are identical to those in untreated cells (data not shown). Whole-cell lysates were assayed for expression of the indicated proteins. Ten micrograms of total protein was loaded for each sample. (c) RPAs were performed to examine the relative amounts of cyclin D1 mRNA present in cells treated as described for panel b.

RASSF1A blocks proliferation but does not induce apoptosis in human lung carcinoma cells. (a) H1299 cells were transiently transfected with Myc-tagged RASSF1A and placed in suspension cultures 24 h later. After 48 h of incubation in suspension cultures, cells were spun onto glass coverslips, fixed, stained with anti-Myc antibody to detect RASSF1A expression, and labeled by TUNEL to detect fragmented DNA. Cells were treated with 1 μM staurosporine for 4 h in suspension as a positive control for TUNEL (ST/TUNEL). (b) Twenty-four hours after transfection with the indicated constructs, H1299 cells were incubated for an additional 24 h in the presence of BrdU. RASSF1A expression was detected as in panel A. BrdU incorporation was detected with anti-BrdU antibody. An overlay image is shown. Quantitation by microscopic observation of three independent experiments is shown in Table 1. (c) Asynchronous H1299 cells were transfected with GFP or myc-RASSF1A. Forty-eight hours posttransfection, cells were trypsinized, fixed, and stained with propidium iodide. RASSF1A-transfected cultures were additionally stained with anti-Myc and fluorescein-conjugated anti-mouse secondary antibodies. Two-color FACS was used to determine the DNA content of GFP- or RASSF1A-expressing cells. Over 2,000 cells are scored for each analysis. The results shown are representative of three independent experiments. Quantitation of the population of cells in the indicated peaks is shown as a percentage of total events from the three experiments.

Bypass of the Rb family cell cycle restriction point allows proliferation of RASSF1A-expressing cells. (a) H1299 cells expressing the indicated constructs together with RASSF1A were assayed for expression and BrdU incorporation. The percentage of cells expressing RASSF1A and incorporating BrdU was quantitated by microscopic observation. Bars represent the standard error from the mean of values obtained from three independent experiments. (b) H1299 cells were transiently transfected with RASSF1A together with cyclin A and assayed for expression of RASSF1A (A), cyclin A (B), and BrdU incorporation (C). Panel D is an overlay of images A to C.

RASSF1A prevents accumulation of cyclin D1 protein. (a) H1299 cells were transfected with RASSF1A or RASSF1C and stained with anti-cyclin D1 and anti-Myc antibodies. Overlays are shown with rhodamine red X-conjugated anti-rabbit IgG antibodies to detect rabbit anti-cyclin D and FITC-conjugated anti-mouse IgG to detect the 9E10 anti-Myc antibody. Quantitation by microscopic observation of at least three independent experiments is shown in Table1. (b) H1299 cells or HME cells were transfected with the indicated constructs together with a luciferase reporter construct driven by the human cyclin D1 promoter (−1745 CD1-Luc). Relative luciferase values are shown normalized to activities obtained with empty vector (H1299) or Ras12V alone (HME). Ras12V expression resulted in fourfold induction of luciferase activity in HME cells over baseline levels observed in serum-starved cells. Bars indicate the standard error from the mean of average values from three independent experiments performed in duplicate. (c) MEFs derived from wild-type and cyclin D1^−/− mice were transiently transfected with the indicated constructs and assayed for BrdU incorporation as described in the legend to Fig. 1.