Growth suppression by Lkb1 is mediated by a G(1) cell cycle arrest

Abstract

Germ-line mutations of LKB1 (STK11) lead to Peutz-Jeghers syndrome characterized by gastrointestinal polyps and cancer of different organ systems. The mutations lead to loss or severe impairment of Lkb1 serine/threonine kinase activity. Therefore LKB1 has been implicated as a tumor suppressor gene, but only a few mutations in the coding exons of LKB1 have been detected in sporadic tumors. Here, we have identified tumor cell lines with severely reduced mRNA levels and impaired Lkb1 kinase activity. Reintroducing Lkb1 into these cells suppressed cell growth. The Lkb1-mediated growth inhibition was caused by a G(1) cell cycle block and was not detected with several naturally occurring Lkb1 mutants. These results indicate that LKB1 has functional and specific growth-suppressing activity.

Figure 1

Lkb1 expression and activity in human tumor cell lines. (A) LKB1 mRNA expression in indicated cell lines (Upper). β-Actin mRNA is shown as a control (Lower). (B) Autocatalytic kinase activity of Lkb1 in indicated cell lines. Immunoprecipitates from cell lysates containing 400 μg of total protein with anti-Lkb1 antiserum or antiserum blocked with the antigenic peptide (+ block) were subjected to an in vitro kinase reaction. Subsequently, samples were analyzed by SDS/PAGE, and radioactivity was detected with a PhosphorImager.

Figure 2

Ectopic expression of Lkb1 in cells with impaired endogenous Lkb1 activity. (A) Subcellular localization of ectopic Lkb1 in G361 cells. Double immunofluorescence with anti-Lkb1 (Left) and anti-β-galactosidase (Center), indicating transfected cells. Nuclei were visualized by Hoechst staining (Right). (B) Lkb1 kinase activity in untransfected NIH 3T3 cells and G361 cells transfected with a Lkb1 encoding plasmid (G361 + Lkb1) or an empty vector (G361 + vector). Immunoprecipitation from cell lysates containing 400 μg of total protein and kinase activity assays were as performed as described in Fig. 1, except that samples were visualized by autoradiography. (C) G418-resistant colony formation by NIH 3T3, G361, and HeLa S3 cells after transfection with a vector control (solid bars) or with an Lkb1-expressing plasmid (open bars). For each cell line, the colony numbers obtained in the Lkb1 transfection were normalized to the vector transfection. A representative experiment is shown.

Figure 3

Growth effects of wild-type and naturally occurring mutant alleles of LKB1 in G361 melanoma cells. (A) Western blotting analysis of Lkb1 in cells transfected with indicated expression vectors. Detection was performed with anti-Lkb1, except for Lkb1-SL8, which was detected by antihemagglutinin because of a C-terminal truncation. (B) Photographs of Giemsa-stained G418-resistant colonies of G361 cells from plates transfected with indicated expression vectors. (C) Relative numbers of G418-resistant colonies after transfections with the indicated expression vectors shown in B. Standard deviations are from nine (vector, Lkb1) or six (G163D, SL26, SL8) independent experiments.

Figure 4

Cell cycle distribution of G361 cells after Lkb1 transfection. Cells cotransfected with the cell surface marker CD20 (CMV-CD20) together with vector control (vector), wild-type Lkb1 (Lkb1), or a naturally occurring kinase-deficient mutant (Lkb1-SL26) were treated with nocodazole (+ Noc) to induce a G₂/M arrest. Subsequently, transfected (CD20 positive) cells were analyzed by using flow cytometry as detailed in Materials and Methods. The cell cycle distribution of nontransfected, unsynchronized cells is shown as a control (− Noc).