t10,c12-CLA accelerates mammary tumor development in ErbB2 transgenic mice irrespective of the time of its administration. (A, B) Tumorigenesis study. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets as follows. Group 1, mice were fed the control diet continuously throughout the experiment; group 2, mice were fed a 0.5% t10,c12-CLA-supplemented diet from 10 to 14 weeks of age, then returned to the control diet until the end of the experiment; group 3, mice were fed a 0.5% t10,c12-CLA diet continuously from 10 weeks of age; group 4, mice were fed the control diet until 21 weeks of age, the 0.5% t10,c12-CLA-supplemented diet from 21 to 25 weeks of age and then returned to the control diet for the duration of the experiment; group 5, mice were fed the control diet until 21 weeks of age, then switched to the 0.5% t10,c12-CLA-supplemented diet for the duration of the experiment; group 6, mice were fed a 0.5% c9,t11-CLA-supplemented diet continuously from 10 weeks of age (data from this latter group are reported in Table I). (A) and (B) are Kaplan–Meier plots showing percentage of tumor-free mice at various ages in mice fed control or 0.5% t10,c12-CLA-supplemented diets. In (A) mice were fed a t10,c12-CLA-supplemented diet continuously from 10 weeks of age until killing, or from 10 to 14 weeks of age, followed by a return to the control diet until killing. The control group was fed the control diet continuously. In (B) mice were fed a t10,c12-CLA-supplemented diet continuously from 21 weeks of age until killing, or from 21 to 25 weeks of age, followed by a return to the control diet until killing. The same control group as in (A) is shown in this panel. Latency was significantly decreased in mice fed the t10,c12-CLA-supplemented diet continuously from 10 weeks of age (P < 0.001), but did not quite achieve statistical significance in the mice fed the t10,c12-CLA-supplemented diet continuously from 21 weeks of age (P = 0.068). The apparent accelerated tumor development in mice fed the t10,c12-CLA diet from 10 to 14 (A) or 21 to 25 (B) weeks of age was not statistically different than control. (C). Established tumor study. ErbB2 transgenic mice with palpable mammary tumors were fed control (9 mice, 12 tumors total) or 0.5% t10,c12-CLA-supplemented diets (11 mice, 12 tumors total) for 4 weeks, and tumor diameter measured weekly. Each point represents the mean ± standard error of the mean of the diameter of the 12 tumors per group that were present at time zero. *, Statistically different from control (P = 0.008 and 0.018 at 3 and 4 weeks, respectively). Inset: the percentage of mice in each group developing a new tumor during treatment (1 of 9 mice in the control group; 9 of 11 mice in the CLA group).

Four weeks of t10,c12-CLA supplementation is sufficient to induce marked changes in the mammary epithelium and stroma. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets for 4 weeks between the ages of 10 and 14 weeks (A, C), or in the tumorigenesis study (D) as described in Figure 1. The mice were killed at the end of 4 weeks (A, C), or when a tumor reached an average diameter of 18–20 mm (D), and mammary whole mounts and paraffin sections prepared. (A). Left panel: mean ± standard error of the mean of mammary gland developmental scores of mice fed the control (n = 6), c9,t11-CLA-supplemented (n = 7) and t10,c12-CLA-supplemented (n = 7) diets, respectively. Since the average score of the FVB/ErbB2 transgenic mice in this study was 2 or greater, the developmental score graphs are plotted between 2 and 4 to better illustrate the dramatic differences that were visually observed. Right panel: mammary lymph node (LN) length in the same mice. (B) The developmental status of the gland was scored using an arbitrary score of 1–4 based on the examples shown in (B) and in (12). Score 1, whole mount of a 11.5-week-old wild-type FVB mouse fed a control diet (12); score 2, whole mount of a 14-week-old ErbB2 transgenic mouse fed a control diet for 4 weeks; scores 3 and 4, whole mounts of mice fed a diet supplemented with 0.5% t10,c12-CLA continuously from 10 weeks of age until a tumor in each mouse reached 18–20 mm in the larger diameter. (C) H&E-stained sections of mammary glands from erbB2 transgenic mice fed control, c9,t11-CLA and t10,c12-CLA-supplemented diets for 4 weeks between 10 and 14 weeks of age. The top panel (i, iii and v) was photographed using the ×4 objective. The bottom panel (ii, iv and vi) was photographed using the ×20 objective. The arrow in vi points to areas of PMN infiltration; the asterisks indicate the lymph node. (D) Mean ± standard error of the mean of mammary development scores (left) and mammary lymph node length (right panel) of the groups described in the above tumorigenesis study. Bars without a common letter are statistically different; N = 14–16 per group.

Effect of CLA feeding for different times on proliferation and apoptosis of mammary epithelial cells. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets for 10 days (n = 5 per group), 4 weeks (n = 7 per group) or until killing because of tumor size (‘lifetime’) (n = 7 per group), starting at 70 days of age. These three experiments were performed independently. The data for the 10 days feeding have been reported previously (12) and are presented here for comparison purposes. Left panels: Ki67 immunohistochemistry. For the control, c9,t11-CLA and t10,c12-CLA groups, respectively, the number of mammary structures counted were as follows. Ten days: ducts 62, 60, 66; lobules 50, 53, 57 and TEBs 60, 58, 57. Four weeks: ducts 72, 84, 84; lobules 66, 77, 84 and TEBs/lesions 45, 77, 79. Lifetime: ducts 84, 87, 70; lobules 83, 79, 82 and TEBs/lesions 86, 79, 56. Right panels: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) immunohistochemistry. For the control, c9,t11-CLA and t10,c12-CLA groups, respectively, the number of mammary structures counted were as follows. Ten days: ducts 66, 71, 72; lobules 52, 43, 59 and TEBs/lesions 86, 79, 56. Four weeks: ducts 77, 86, 85; lobules 72, 79, 84 and TEBs/lesions 59, 75, 81. Lifetime: ducts 87, 84, 70; lobules 85, 84, 78 and TEBs/lesions 91, 87, 79. Within a specific time period, bars without a common letter are statistically different.

t10,c12-CLA activates the PI3K/Akt and MEK/ERK pathways. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets continuously from 24 or 70 days of age, and protein expression evaluated in mammary tumors. (A) Phospho-PI3K p85 (Tyr458)/p55 (Tyr199), total PI3K p85, phospho-Akt (Ser473), total Akt, phospho-MEK (Ser217/221), total MEK, phospho-ERK1/2(Thr202/Tyr204) and total ERK1/2 were measured by western blot in eight mammary tumors per group. Hsc70 was used as a loading control. (B) The western blots were quantified by densitometry. Mean ± standard error of the mean of the ratio of phospho-PI3K p85 to PI3K p85, phospho-Akt (Ser473) to Akt, phospho-MEK to MEK and phospho-ERK1/2 to ERK1/2. Bars without a common letter are statistically different.

t10,c12-CLA activates IGF-IR/IR, but not ErbB2. ErbB2 transgenic mice were fed control or 0.5% CLA-supplemented diets continuously from 24 days or 70 days of age and protein expression evaluated in mammary tumors. (A) Phospho-IGF-IRβ (Tyr1135/1136)/insulin receptor (Tyr1150/1151), total IGF-IRβ, phospho-ErbB2 (Tyr877), total ErbB2 and phosphotyrosine were measured by western blot in eight mammary tumors per group. Hsc70 was used as a loading control. (B) The western blots were quantified by densitometry. Mean ± standard error of the mean of the ratio of phospho-ErbB2 to ErbB2, phospho-tyrosine to Hsc70 and phospho-IGF-IRβ/insulin receptor to IGF-IRβ. One control sample (right panel, lane 3) was not included in the calculation of mean ± standard error of the mean of the ratio of phospho-IGF-IRβ/insulin receptor to IGF-IRβ since its ratio was 25-fold higher than the average of the other samples in the group. Bars without a common letter are statistically different. Note that in this figure, the label ‘P-IGF-IR’ refers to the phospho form of both the IGF-I and insulin receptors, since the phospho-specific antibody recognizes both species.