A, Total RNA was extracted and reverse-transcribed from prostate lobes microdissected from three nontransgenic mice at 18, 24, and 36 weeks of age. The bars indicate the average transcript levels of ALDH1A1, ALDH1A2, and ALDH1A3 in prostate tissue from age-matched nontransgenic controls (WT, black bars) and TRAMP (white bars) normalized to 36B4 mRNA levels in each sample. B, Relative mRNA levels of RARβ₂, CYP26A1, and LRAT measured by quantitative RT-PCR in WT (black bars) and TRAMP mice (white bars) at 36 weeks of age. All samples were normalized to 36B4. VP, ventral prostate; LP, lateral prostate; DP, dorsal prostate; and AP, anterior prostate. Relative expression was calculated using the Bio-Rad Genex software, where all prostate lobes from a given age group were processed independently. Error bars= standard error. Comparisons for statistical analysis were made for each lobe between the Wt and the TRAMP mice at each of the time points. *, p ≤ 0.05 as determined by two-tailed Student's t test, comparing TRAMP mice to their age-matched littermate controls. (TRAMP = TRAMP+).

Tissue extracts were obtained from microdissected dorsal prostate lobes (30 μg protein loaded/lane) from nontransgenic (labeled as WT) littermate controls and TRAMP mice at 18, 24, and 36 weeks of age (3 mice per condition). A, Western blot analysis performed with the polyclonal rabbit anti-mouse ALDH1A2 antibody. B, Western blot analysis performed with the polyclonal rabbit anti-human S100A4 antibody. For a loading control, these blots were stripped and reblotted with a polyclonal goat anti-human actin antibody. Positive controls: ALDH1A2-wild type testis extract (15 μg protein loaded/lane); S100A4-PC3 cell extract (30 μg protein loaded/lane). This experiment was performed three times with similar results; one blot is shown. The upper arrow at the right in panel A shows ALDH1A2; the lower arrow points to the non-specific protein band at ∼30 kd. C, Densitometric quantitation of similar ALDH1A2 and S100A4 Western Blots. Band density was measured using the Fluor Chem 8800 software (Alpha Innotech) for the bands from ALDH1A2 (left) and S100A4 (right). Western blots normalized to actin for all prostate samples. Error bars = standard error. *, p ≤ 0.05 as determined by two-tailed Student's t test. TRAMP mice were compared to age-matched littermate nontransgenic controls (WT). DP, dorsal prostate.

A-J, representative slides of a human prostate tumor tissue. A-B and E-F, ALDH1A2 staining at 200× and 600× (boxed areas of A and E) magnification. B and F, ALDH1A2 staining located in the cytoplasmic compartment of basal and luminal cells in a normal prostate gland with weak staining in adjacent cancer cells. C-D and G-H, S100A4 staining at 200× and 600× (boxed areas of C and G) magnification. S100A4 staining is seen in benign glands, as well as in surrounding stroma. I-J, Negative controls, prostate specimens incubated with preimmune serum instead of primary antibody, 200× magnification. (Arrows indicate areas on sections discussed in the Results section).

Retinoids were extracted from prostate tissues of TRAMP(+) and age-matched nontransgenic littermate control mice ranging from 24-36 weeks of age (6/group) and were separated by reverse-phase HPLC analysis. Individual prostate lobes from 2 mice were pooled together for the retinoid extraction, giving a total of 3 samples each for control, non-transgenic littermate (designated WT) and TRAMP. Retinol (white) and retinyl ester (black) levels (pmol/mg tissue) in prostate tissue were calculated using known standards and normalization to prostate tissue weight. Comparisons for statistical significance were made between the Wt and the TRAMP mice within each lobe for each type of retinoid. Error bars= standard error. VP, ventral prostate; LP, lateral prostate; DP, dorsal prostate; and AP, anterior prostate. ‡, retinol was not detected. §, retinyl esters were not detected. *, p ≤ 0.05 as determined by two-tailed Student's t test, comparing TRAMP mice to their age-matched littermate controls. (TRAMP = TRAMP+, ie. containing the transgene.)

A, Wild type mouse embryo at E12/13 (WT). ALDH1A2 staining located in the metanephros. B, ALDH1A2^-/- embryo at E12/13. No ALDH1A2 staining was observed in the metanephros, confirming antibody specificity. E, Wild type mouse testis at 36 weeks of age. ALDH1A2 staining located in the germ cells but not in the spermatagonia. G, Wild type mouse kidney at 36 weeks of age. ALDH1A2 staining was observed in the tubular cells, but not in the glomeruli. C-D and G-H, Negative control incubated with preimmune serum instead of primary antibody. A-D, 200× magnification. E-H, 400× magnification.

All tissue sections were stained with hematoxylin (blue). A, C, E, and G, ALDH1A2 staining. B, D, F, and H, S100A4 staining. A-H, 600× magnification. Dorsal lobe (A) and lateral lobe (C) from an 18 week old nontransgenic mouse. Strong nuclear and cytoplasmic ALDH1A2 staining (brown stain) representative of normal prostate epithelial cells in all lobes. S100A4 (brown stain) staining in fibroblast cells surrounding a normal prostate gland in dorsal lobe (B) and lateral lobe (D). E, dorsal prostate tumor tissue in a 36 week old TRAMP mouse, showing weak cytoplasmic ALDH1A2 staining in prostate cancer cells with little to no stain in the nuclei. There is no ALDH1A2 staining in the stroma of dorsal prostate tissue in the TRAMP mouse. F, S100A4 staining in the stroma of TRAMP mice and among prostate cancer cells within the gland. G, lateral prostate tumor tissue in a 36 week old TRAMP mouse. The ALDH1A2 staining pattern in the prostate cancer cells is similar to E. Positive staining of ALDH1A2 in the lateral prostate stroma, which is unique to this lobe. F, S100A4 staining in the prostate cancer and lateral prostate stroma. I-J, Negative controls incubated with preimmune serum instead of primary antibody. I, prostate tumor tissue from dorsal prostate of 30 week old TRAMP mouse, 200× magnification. J, prostate tumor tissue from lateral prostate of 30 week old TRAMP mouse, 200× magnification. K, ALDH1A2 staining of prostate tumor tissue from 30 week old TRAMP mouse, 200× magnification. L, adjacent section of ALDH1A2 staining of prostate tumor tissue from 30 week old TRAMP mouse. Tumor tissue section was simultaneously incubated with the 20× peptides to which the antibody was generated. (Arrows indicate areas of sections discussed in the Results section).