Effects on preneoplastic and malignant epithelial cells. Epithelial cells were plated on WI-38 fibroblast lawns and cultured in growth factor-deficient medium for 8 days. (A) S1 (i and iv), SCp2 (ii and v), and HaCAT (iii and vi) cells, cultured on presenescent (i–iii) or senescent (iv–vi) lawns, stained with Rhodanile (240×). (B) Cocultures described in A, and coculture with MDA231 cells, stained with DAPI. Epithelial nuclei were quantified as described in Materials and Methods. The results (in arbitrary units) shown are from one of 3–5 experiments. Error bars = SEM of duplicate or triplicate wells. Gray bars, presenescent lawns; black bars, senescent lawns. (C) SCp2 cells cultured without fibroblasts (i) or with presenescent and senescent cultures at ratios 10:0 (ii), 9:1 (iii), 8:2 (iv), 5:5 (v), and 0:10 (vi), stained with Rhodanile (240×). (D) SCp2 cells cultured without fibroblasts (No Fb) or with ratios of fibroblasts described in C, quantified by DAPI fluorescence. Error bars = SEM from triplicate wells from one of two experiments.

Effects on normal epithelial cells. Normal adult (Adult) or neonatal (Neonatal) human keratinocytes were seeded onto WI-38 lawns, cultured for 8 days, and quantified by DAPI fluorescence. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent lawns; black bars, senescent lawns.

Characteristics of the growth stimulation. (A) EGFP-expressing HaCAT cells, seeded onto presenescent or senescent WI-38 lawns. EGFP fluorescence (in arbitrary units) was measured after 1–8 days, as indicated. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent lawns; black bars, senescent lawns. (B) SCp2 cells, seeded onto WI-38 fibroblasts (Cells), plated in the upper chambers of Millicells containing fibroblasts in the lower chambers (Soluble), or plated onto matrices deposited by fibroblasts (Matrix), as described in Materials and Methods. Cell number was assessed after 8 days by DAPI fluorescence. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent fibroblasts; black bars, senescent fibroblasts. (C) HaCAT cells, seeded onto lawns of control (Left) or p14^ARF-expressing (Right) presenescent 82-6 fibroblasts, stained 8 days later with Rhodanile (250×). (D) HaCAT cells, quantified by EGFP fluorescence after coculture with control (−) or p14^ARF-expressing (+) 82-6 fibroblasts (ARF); control (−) or p14^ARF-expressing (+) hTERT-immortalized 82-6 cells (TERT-ARF); control (−) or RAS-Ha/V12-expressing (+) WI-38 cells (RAS); 82-6 cells untreated (−) or treated (+) with H₂O₂; or replicatively senescent WI-38 cells (R). Growth was assessed after 8 days, except for RAS panels, where growth was assessed after 5 days. Error bars = SEM from duplicate wells from one of two experiments. Gray bars, presenescent lawns; black bars, senescent lawns.

Tumor growth stimulated by fibroblasts. Nude mice were injected with epithelial cells alone (Control) or presenescent (Presn), senescent (Sen), or hTERT-immortalized (Telom) fibroblasts. At the indicated intervals (Days), tumor size was measured as described in Materials and Methods. The number of animals per group (n) is indicated. The last point on each line indicates when tumors were excised for histology. (A) HaCAT cells (1.5 × 10⁶) alone or with 1.5 × 10⁶ WI-38 fibroblasts. Shown are results from two experiments. The incidence of tumors >10 mm³ and average tumor size were greater in the senescent group (P < 0.05). (B) SCp2 cells (1 × 10⁶) alone or with 1 × 10⁶ WI-38 cells. Tumor incidence was greater in the senescent group (P < 0.05). (C) Ha(Pk) cells (1.5 × 10⁶) alone or with 1.5 × 10⁶ WI-38 cells. (D) MDA231 cells (2.5 × 10⁵) alone or with 1 × 10⁶ WI-38 cells. Tumor size was greater in the senescent group (P < 0.01).

Characterization of tumors induced by fibroblasts. (A) Fluorescence image of a frozen section of a tumor formed by EGFP-expressing HaCAT cells (80×). Tumors formed in the presence of presenescent or senescent fibroblasts were similarly fluorescent. (B) Section shown in A, counterstained with DAPI (80×). (C) HaCAT tumor formed in the presence of presenescent WI-38 cells (fixed section, stained by hematoxylin and eosin, 160×). Arrow shows a well demarcated tumor margin. (D) HaCAT tumor formed in the presence of senescent WI-38 cells (fixed section, stained by hematoxylin and eosin) (160×). Arrow shows a poorly demarcated tumor margin. (E) Fluorescence image of a frozen section of an SCp2 tumor formed by senescent WI-38 cells, stained for cytokeratin (160×). Solid arrow shows positive staining in the host mammary duct; open arrow shows lack of staining in surrounding tumor cells. (F) Frozen section adjacent to that shown in E, stained by hematoxylin and eosin (160×). Solid arrow, host ductal epithelial cells; open arrow, surrounding anaplastic tumor cells. (G) Paraffin-embedded section of an MDA231 tumor formed in the presence of presenescent WI-38 cells, stained with hematoxylin and eosin (40×). (H) Paraffin-embedded section of an MDA231 tumor formed in the presence of senescent WI-38 cells, stained with hematoxylin and eosin (40×).