Article Figures & Data
Figures
- FIG. 1.
Expression of Cul1 mutants increases protein levels of SCF complex substrates. (A) Cul1-N252 and Cul1-N385 mutant proteins associate with Skp1 and the F-box protein Skp2 but not with Rbx1/Roc1. 293T cells were transfected with 3′-Flag-tagged Cul1 mutants encoding N-terminal amino acid peptides residues 1 to 252 (Cul1-N252) or residues 1 to 385 (Cul1-N385) or with wt Cul1. Transfected cells were selected for 5 days after the addition of puromycin (1 μg/ml). Lysates were first immunoprecipitated with mouse anti-Flag antibody and then immunoblotted with rabbit anti-Flag antibody (lanes 1 through 4) or the Skp1, Skp2, or Rbx1/Roc1 antibody (lanes 5 through 8). (B) Expression of Cul1-N252 mutants increases protein levels of p27, cyclin E, β-catenin, and p21. 293T cells transfected with empty vector (Pallino) (lanes 1 and 5), Cul1-N252 (lanes 2 and 6), Cul1-N385 (lanes 3 and 7), or wt Cul1 (lanes 4 and 8) were selected with puromycin for 5 days. Lysates from transfected cells were then immunoblotted with the indicated antibodies. (C) Coexpression of Skp1 attenuates the effects of the Cul1-N252 mutant on p27 accumulation. 293T cells were transfected with vector (pcDNA3) alone (2 μg) (lane 1), with a combination of empty vector and His-tagged Skp1 (0.4 and 1.6 μg) (lane 2), with empty vector and the Cul1-N252 mutant (1.6 and 0.4 μg) (lane 3), or with both His-Skp1 and the Cul1-N252 mutant (1.6 and 0.4 μg) (lane 4). Cells were lysed 48 h posttransfection and immunoblotted with the anti-Flag, p27, and Skp1 antibodies.
- FIG. 2.
Generation of CD4-Cul1-N252 Tg mice. (A) Expression of the Flag-tagged Cul1-N252 mutant and other cell cycle regulatory proteins in CD4-Cul1-N252 Tg mice. Extracts (30 μg of protein) from the thymocytes of two control non-Tg mice (wt), Cul1-N252 Tg line 10 mice, and Tg line 20 mice were immunoblotted with the antibodies to the indicated proteins. (B) Association of the human Flag-tagged Cul1-N252 mutant with endogenous murine Skp1. Thymocytes of a wt (middle lane) and a CD4-Cul1-N252 Tg mouse (right lane) were immunoprecipitated with a rabbit anti-Flag antibody and then immunoblotted with antibodies to Flag or Skp1. 293T cells transfected with Cul1-N252 were used as a positive control (left lane). The bottom panel shows the corresponding total lysates immunoblotted with anti-Flag antibodies. (C) Expression of Skp1 in CD4-Skp1 Tg mice. Thymocytes of wt and Tg mice were immunoblotted with the indicated antibodies.
- FIG. 3.
Characterization of Cul1-N252 Tg mice. (A through I) Cellular depletion in lymphoid organs of Cul1-N252 Tg mice. Histological analysis of thymi and peripheral lymphoid organs from wt, Cul1-N252 Tg (line 20), and Cul1-N252, Skp1 double-Tg mice (6 to 8 weeks old). Sections were stained with hematoxylin and eosin stain. (J) Cul1-N252 Tg mice showing a decreased number of lymphoid cells. Total numbers of thymocytes in wt, Cul1-N252, and Cul1-N252, Skp1 double-Tg mice are shown. Data are the means ± SD representative of six animals for each group. (K) Decreased sensitivity to mitogenic stimulation of Cul1-N252 thymocytes. Thymocytes from wt, Cul1-N252, and Cul1-N252, Skp1 Tg mice were cultured with PHA and IL-1, IL-4, and IL-7 for 72 h. [3H]Thymidine was added to the medium during the last 24 h. The results are shown as the means of [3H]thymidine incorporation representative of three independent experiments with three animals for each group. (L and M) Increased expression of p27 in subcortical thymocytes of Cul1-N252 Tg mice. Anti-p27 was used in immunohistochemistry of wt (L), Cul1-N252 Tg (M), or Cul1-N252, Skp1 Tg mouse thymi. Magnification, ×100 for thymus and lymph node, ×400 for spleen and p27 in thymus.
- FIG. 4.
Lymphomas in Cul1-N252 Tg mice. (A) Percent survival in the progeny of Cul1-N252 Tg mice (lines 10 and 20). Non-Tg control mice are indicated as wt. The percentage of survivors is given against the age in weeks. (B) Increased survival in CD4-Cul1-N252 and CD4-Skp1 double-Tg mice. (C) Hematoxylin-stained section of a thymic lymphoma showing a normal (upper right) and a neoplastic (lower left) thymic lobe. (D) Advanced stage of thymic lymphoma with a high percentage of apoptotic and mitotic cells (inset). (E) Peripheric lymphoma showing pleomorphic and multinucleated neoplastic cells (inset). Magnification, ×100 for panel D, ×400 for panels E and F.
- FIG. 5.
c-myc amplification in Cul1-N252 lymphomas. (A) Representative immunoblotting of tissue extracts from control thymi (C) and tumors developed in TMTV-Nras mice (Nras) or in CD4-Cul1-N252 Tg mice (Cul1-N252). Extracts were immunoblotted with the antibodies to the indicated proteins. (B) Immunoblot analysis of c-Myc in Daudi Burkitt's lymphoma cells and in a representative Cul1-N252 lymphoma cell line after treatment with 100 μg of cycloheximide/ml for the indicated time points (in hours). (C) Southern analysis of lymphoma DNAs. EcoRI-digested DNA from a control Cul1-N252 liver (C) and TMTV-Nras (Nras) and Cul1-N252 T-cell lymphomas was probed with c-myc or β-actin (loading control). Numbers below gels are relative gene dosages based on setting of the liver control (C) to 2. (D) c-myc FISH analysis of a metaphase from a representative Cul1-N252 tumor. Four copies of the c-myc signal (green) are shown on four different chromosomes (stained blue with DAPI).
- FIG. 6.
Karyotype heterogeneity in Cul1-N252 lymphomas. (A) Chromosome counts on tumor cells derived from CD4-Cul1-N252 and CD4-NPM-ALK Tg mice after one to five generations are given. Karyotype heterogeneity in Tg-Cul1-N252 lymphomas is shown. Lymphoma cell lines independently derived from Tg-Cul1-N252 tumors (20-13, 20-28, 20-38) constitutively accumulated an increased proportion of aneuploid cells (85 to 90%) compared with that for Tg-NPM-ALK (N15) lymphoma cell lines (30%) and displayed a widespread karyotype heterogeneity within each tumor cell line. (B) Karyotype analysis in established clones (20-28).
- FIG.7.
Chromosomal instability in Cul1-N252 cells. (A) Quantitative analysis of the number of nuclei in 293T cells transfected with empty vector (Pallino), cyclin E, wt Cul1, and the Cul1-N252 mutant is shown. Data are expressed as the percentages of cells that contained the indicated number of nuclei. Data are the means ± SD from three independent experiments. ∗∗, chi-square P < 0.0001. (B) Overexpression of Skp1 overcomes the changes induced by Cul1N252. Cul1-N252 and Cul1-N252-His-Skp1-transfected 293Trex cells were cultured with doxycycline, and protein levels of p27 were analyzed by Western blotting. The expression of Skp1 was measured with anti-His antibody (left panel). Transfected cells were cultured in the presence of doxycycline for 72 h, and the number of multinucleated cells was scored as described above (right panel). (C) FISH analysis of 293T cells with centromeric probes specific for chromosomes X, 7, 12, and 18 is shown. Mean chromosome number and SD are calculated for each transfection after counting at least 100 nuclei. (D) Unequal distribution of chromosomes in a multinucleated Cul1-N252 cell (293T) is indicated. FISH signals were detected with centromeric probes specific for chromosome 7 (red) and chromosome 12 (green). Nuclear DNA was stained with DAPI (blue). (E) Percentage of multinucleated Cul1-N252 cells showing unequal distribution of the indicated chromosomes analyzed by FISH is illustrated. At least 200 cells were counted.
- FIG. 8.
Centrosome amplification and cell division defects in cells expressing Cul1-N252. (A) Quantitative analysis of centrosome number is illustrated. Data are expressed as the percentage of cells that contained the indicated number of centrosomes. Centrosomes were counted for 100 cells per sample. Data are the means ± SD from threeindependent experiments. ∗∗, P < 0.0001. (B and C) NIH 3T3 cells transfected with the indicated constructs were stained for centrosomes with anti-γ-tubulin antibody (red) and counterstained with DAPI (blue) after 5 days of growth in selective medium. (B) Representation of a Cul1-N252 cell with multiple centrosomes is shown. (C) Multinucleated Cul1-N252 cell with nonseparated centrosomes is shown. (D through I) Abnormal mitosis in cells that express Cul1-N252 is shown in the following components and stages: mitotic spindles of unipolar (D), tripolar (E), and tetrapolar (F) mitosis stained with anti-α-tubulin antibody (red) and counterstained with DAPI (blue); metaphase plate with chromosomes not congregated with the bulk of DNA (G); anaphase with lagging chromosomes (H); and defective cytokinesis with bridging chromatin (I). For panels G through I, DNA was stained with DAPI (white). (J) Aberrant mitosis was induced by Cul1-N252 mutant expression and quantified. One hundred cells during mitosis were counted per sample. Data are given as the means ± SD from three independent experiments, ∗∗, P < 0.0001.
