Fig. 4. Cytogenetic analyses of anchorage-independent colonies. Representative metaphases from RAS + hTERT-expressing Leiden HDF colonies recovered from semi-solid medium and analysed by M-FISH. Clone 5A contained additional and rearranged chromosomes (A), whereas clone 4A showed a normal diploid karyotype (B).

Fig. 6. Induction of CDKN2A gene products by RAS and E2F-1. Leiden (A) and FOO3 HDFs (B) were infected with retroviruses encoding RAS (lanes 2 and 6) or E2F-1 (lanes 4 and 8), or with appropriate vector controls. Because of the extensive apoptosis induced by E2F-1, the cells were analysed at 3 days post-infection, whereas the RAS-expressing cells were harvested at 11 days to maximize the effects (see Figure 6B). Samples (30 µg) were fractionated by SDS–PAGE and immunoblotted for the indicated proteins, using a 15% gel in the case of RAS, phospho-MEK and p14^ARF- or p16^INK4a-related proteins, and a 10% gel for E2F-1, p53 and Cdk4.

Fig. 5. Induction of p53 in Leiden and FOO3 cells by UV irradiation but not RAS. (A) Anchorage-independent colonies of Leiden cells recovered from agarose (clones 1 and 2), and pools of Leiden cells infected with empty vector controls (39 PDs), expressing hTERT alone (41 PDs) or RAS + hTERT (46 PDs), were exposed to UV radiation (15 J/m²). Samples (20 µg) of total protein were then subjected to SDS–PAGE in a 10% gel and immunoblotted with a mAb against p53 (DO-1). Cdk4 served as a loading control. (B) FOO3 cells were infected with either pBABE-RAS-puro retrovirus (+ lanes) or empty vector (– lanes) and harvested at various times post-infection, as indicated, without drug selection. Samples (30 µg) were fraction ated by SDS–PAGE on a 10% gel and immunoblotted for the indicated proteins.

Fig. 3. Phenotypic effects of RAS + hTERT in Leiden HDFs. (A) Pools of Leiden HDFs expressing RAS + hTERT or the respective empty vector controls were examined by phase microscopy ∼4–5 weeks post-selection. (B) The saturation densities of Leiden cells expressing hTERT alone (stippled bar) or RAS + hTERT (filled bar) were compared by counting the numbers of cells per 75 cm² flask at 4–7 days post-confluence. The figure presents the results of four independent comparisons, and the error bars indicate the standard deviation. (C) Leiden cells expressing RAS + hTERT formed macroscopically visible colonies in soft agar (centre) whereas Leiden cells expressing hTERT alone did not (left). No significant agar growth was observed with control fibroblasts expressing RAS + hTERT (right).

Fig. 2. Functional appraisal of the β-ARF/p16 fusion protein. (A) U20S cells were transiently transfected with plasmids encoding p53 and green fluorescent protein, with and without human MDM2. As shown in the control lanes, MDM2 causes the marked destruction of p53 in this assay. Introduction of either wild-type p14^ARF or β-ARF/p16 protected p53 from MDM2-mediated degradation. (B) NARF2 cells (U20S cells expressing p14^ARF from an IPTG-inducible promoter) were transfected with a plasmid encoding β-ARF/p16 and analysed by indirect immunofluorescence. In the upper panels, p14^ARF was detected with the mouse mAb 4C6/4 (red) whereas the β-ARF/p16 fusion protein was detected with a rabbit polyclonal antibody against p16^INK4a (green). In the lower panels, β-ARF/p16 fusion protein was detected with the mouse mAb DCS50 (red) and the nucleolar protein B23 was visualized using a goat polyclonal antibody (green). (C) Samples (20 µg) of total protein from Leiden cells infected with retroviruses encoding the E6 or E7 proteins of HPV-16 (as indicated) or control virus (C) were fractionated by SDS–PAGE and immunoblotted with the mAbs DCS50 to detect the β-ARF/p16 fusion protein (upper panel) and DO-1 to detect p53 (lower panel). A sample (500 µg) of each lysate was immunoprecipitated with polyclonal antiserum against p16^INK4a (DPAR12) and immunoblotted with mAb IF2 against human MDM2 (middle panel). (D) Control (Hs68) and Leiden HDFs expressing E2F-1 fused to the hormone-binding domain of the oestrogen receptor were treated (+) or not (–) with 4-hydroxy tamoxifen (OHT) for 24 h. Samples (20 µg) of cell lysate were fractionated by SDS–PAGE and immunoblotted with an antiserum raised against amino acids 54–75 of human p14^ARF, and with mAbs against MDM2 and p53. Cdk4 served as a loading control. Samples (500 µg) of each lysate were immunoprecipitated with the same p14^ARF antiserum and immunoblotted for MDM2 and p53.

Fig. 1. Leiden deletion and resultant CDKN2A fusion proteins. (A) Schematic representation of the CDKN2A locus with the exons shown as boxes. The sequences encoding p16^INK4a are identified by stippling, and those encoding p14^ARF by cross-hatches. The nucleotide sequence of the relevant region of exon 2 is shown, with direct repeats identified by arrows and the 19 bp Leiden deletion boxed. The encoded amino acids in the three different reading frames are shown in single letter code. The –1 (p14^ARF), 0 (p16^INK4a) and +1 frames are identified by the distinctive shading, and their contributions to the β-ARF/p16 and α-p16/X fusion proteins are indicated. Y defines the regions of p16^INK4a recognized by the DCS50 and JC8 mAbs. (B) Lysates from the Leiden and FOO3 strains of HDFs were immunoprecipitated with either non-immune serum (– lanes) or a polyclonal antibody (DPAR12) against p16^INK4a (+ lanes). After fractionation by SDS–PAGE, the samples were immunoblotted with JC8 (upper panel) and DCS50 (lower panel). The positions of wild-type p16^INK4a and the β-ARF/p16 and α-p16/X fusion proteins are shown. (C) Immunoprecipitation of Cdk4 from the same Leiden and FOO3 lysates followed by immunoblotting with JC8, DCS50 and a mAb against Cdk4. (D) In vitro binding assay using unlabelled Cdk4 (lanes 1–4) and Cdk6 (lanes 5–8) mixed with [³⁵S]methionine-labelled wild-type p16^INK4a (lanes 1 and 5), the P114L variant (lanes 2 and 6), β-ARF/p16 (lanes 3 and 7) or α-p16/X (lanes 4 and 8). The mixtures were immunoprecipitated with polyclonal antisera against Cdk4 or Cdk6 and the labelled proteins were fractionated by SDS–PAGE and visualized by autoradiography. The multiple bands reflect initiation at internal methionines. (E) BrdU incorporation assays in Hs68 and Leiden cells infected with retroviruses encoding p16^INK4a, p14^ARF, β-ARF/p16 or α-p16/X as indicated.