PMC

Live cultures of 184A1 colonies at p28 showing morphologic heterogeneity. (a) Small colony that did not maintain growth; (b) colony with mixed growing and nongrowing, large vacuolated cells; (c) large colony with mostly growing cells. Magnification, 32×.

184A1 growth in TGFβ at different passages. Giemsa-stained cells labeled with [³H]thymidine for 24 h as described in MATERIALS AND METHODS. (a) Small slowly growing area at p28, part of a slightly larger area, labeled after 14 d exposure to TGFβ; this represents the best growth seen at p28. (b) Representative area of a typical p44 colony with good growth (LI >50%) labeled after 13 d exposure to TGFβ. Magnification, 125×.

Mean TRF length and telomerase activity in late passage 184B5 and subclones at different passages. (A) 184B5 and subclones. (B) B5Y9H cells at different passage levels from two separate freezedowns. The first telomerase assay at p105 was from dishes containing vacuolated, nongrowing cells. The second assay for telomerase at p105 and TRF values were obtained from sister cultures that contained good growing patches. All B5Y9 clones assayed showed good growth in TGFβ. Assays were performed as described in MATERIALS AND METHODS and the Figure legend. For TRF length, lighter shaded ovals indicate a faint signal.

Telomerase activity in 184A1 and 184B5 at different passages. Telomerase activity was determined by the TRAP assay as described in MATERIALS AND METHODS. Each sample was run in duplicate or with RNase added to the reaction mix in the indicated lanes (+) as a negative control. Each lane shows signals generated from 1 μg of cell extract. (a) 184A1 at p11 with no visible telomerase products. (b) B5Y17 at p18 shows a very weak level of telomerase activity (below the limits of detection of the PhosphorImager quantitation system). Similar results were seen with B5Y16G (p23), B5Y16L (p22, p35), B5Y16E (p24), and B5Y23 (p17). (c) 184A1 at p34 showing low levels of telomerase products. (d) 184A1 at p46 showing strong telomerase activity.

Comparison of mean TRF length, telomerase activity, and growth in TGFβ in early-passage 184B5 and subclones. (A) Subclones that did not maintain growth ± TGFβ or had only rare cells maintaining growth. Since most of the cells generated from these clones were used for assays, only 2–5 × 10⁵ cells/clone were available for observation of ability to maintain growth in mass culture. Consequently, a rare cell capable of maintaining growth may not have been detected. Y17-βR represents the progeny of rare B5Y17 cells that maintained proliferation. (B) Subclones that maintained growth and showed a gradual increase in the ability of cells to maintain growth in the presence of TGFβ. (C) The heterogeneous B5Y16 clone and B5Y16 subclones. 16G-βR represents the progeny of a rare p24 colony that grew well in TGFβ and was clonally isolated. Assays were performed as described in MATERIALS AND METHODS and the Figure legend.

Heterogeneity of subclone B5Y16G p25 colony growth in TGFβ. One thousand cells were seeded into 100-mm dishes and exposed to TGFβ 15 d after seeding. Cells remained in TGFβ an additional 18 d and were labeled with [³H]thymidine for the last 24 h. The Giemsa stained, single-cell–derived colonies shown are from the same dish. (a) Colony with no growth in TGFβ. (b) Mostly flat colony with rare scattered labeled cells. (c) Colony with growing small cobblestone cells amid flatter cells with little growth. (d) Colony with larger growing areas of small cobblestone cells amid flat cells. (e) Rare large colony with uniform good growth in TGFβ. These pictures are also illustrative of B5Y7, B5Y13, and B5Y40. In general, growth of these clones in TGFβ produced colonies: mostly like panel a and a few like panel b at ∼p17–20, ranging from panels a-c at p20–24; ranging from panels a-d at p23–27. By ∼ p27 and higher, colonies like panel e could be seen. Magnification, 125×

Comparison of mean TRF length, telomerase activity, and growth in 184A1 at different passage levels. (A) TRF length: lighter shaded ovals indicate a faint signal. (B) Telomerase activity, determined semiquantitatively by comparing the levels of HMEC telomerase products generated to those generated for a constant number of 293 cells (1,000 cell equivalents). The following categories were used to designate semiquantitative values. Note that the points are presented in a semilog form: none, no detectable telomerase products by PhosphorImager analysis; weak, ∼ 5% of telomerase activity of 293 cell control; low, ∼10% of 293 control; medium, 25–50% of 293 control; strong, 75–100% of 293 control. (C) Colony-forming efficiency and labeling index (LI) in colonies. TRF length, telomerase activity, CFE, and labeling index were determined as described in MATERIALS AND METHODS. See Figure for examples of telomerase activity assay semiquantitation.