Expression of mutant proteins in human melanocytes. (A) Experimental schematic. (B) Confirmation of protein expression; Ras=active N-Ras^G12V, CDK4=active CDK4^R24C, p53^DN=dominant-negative p53^R248W. (C) Levels of active phosphorylated and total ERK1/2 in primary human melanocytes engineered to express active N-Ras compared to SK-Mel-2 melanoma cells harboring a known activating mutation at endogenous NRAS. (D) Reporter activity from wild-type (WT, left panel) and non-functional mutant control p53 binding sites (right panel) as a function of expression of p53^DN or LacZ control; relative reporter activity is shown, with LacZ control assigned a value of 100%. (E) Telomerase activity with combinatorial expression of hTERT.

Invasive human melanocytic neoplasia. (A) Clinical appearance. Note ulceration with hemorrhagic crust in right panel. (B) Histology. Note mild junctional melanocytic hyperplasia [brackets] in tissue with melanocytes expressing active Ras and hTERT compared with invasive melanocytic neoplasia seen with Ras, hTERT, CDK4 and p53^DN. Representative histology is shown at 6–12 weeks post-grafting (E=epidermis; D=dermis; scale bars=100µm). (C) Features of melanocytic neoplasia. Note deep dermal invasion (arrows), and pagetoid spread upwards into the epidermis (arrowheads). Note also full-thickness tumor and ulceration seen in low power view of 24 week timepoint (far right panel); scale bars=50µm. (D) Immunoperoxidase staining demonstrating that melanocytic neoplasms express HMB45, S100, Melan-A and lack epithelial keratins. Large melanocytic clusters are seen replacing keratin[+] epidermal cells; “*” denotes melanocytes; control=no primary antibody; scale bars=100µm. (E) Immunofluorescence staining demonstrating retention of introduced genes in melanocytic neoplasms. Ras, p53, CDK4 (orange), collagen VII epidermal basement membrane zone marker (green), Hoechst 33342 nuclei (blue), are shown in serial tissue sections. Note retention of introduced genes in both junctional/epidermal [E] and dermally invasive [D] HMB45[+] melanocytic regions (scale bars=100µm).

Effect of disrupting function of p53 and Rb pathways and the impact of hTERT. (A) Invasive melanocytic neoplasia occurs with Ras and hTERT with CDK4 as well as p53^DN. Representative histology is shown at 6 weeks; scale bars=100µm. (B) Tumor progression at 4, 12 and 24 weeks in the presence or absence of delivered hTERT. Note progression from junctional hyperplasia to deep dermal invasion and full-thickness ulceration of skin tissue that occurs with hTERT co-expression with Ras, CDK4 and p53^DN compared to the milder, predominantly junctional hyperplasia that occurs without hTERT co-expression; scale bars=100µm.

Capacity of active PI3K and B-Raf to recapitulate Ras-driven human melanocytic neoplasia. (A) Expression and function of active PI3K p110α-CAAX (PI3K) in primary human melanocytes. Levels of phosphorylated active Akt in melanocytes expressing either active PI3K or N-Ras are compared to SK-Mel-24 (SK24) and SK-Mel-31 (SK31) melanoma cells with known activation of PI3K via PTEN deletion. (B) Expression and function of active B-Raf^V599E. Levels of phosphorylated active ERK1/2 in melanocytes expressing either active B-Raf or N-Ras are compared to WM115 and A375 melanoma cells with known mutation of endogenous BRAF leading to expression of the active B-Raf^V599E mutant. (C) Human tissue regenerated with melanocytes expressing either p110α-CAAX, B-Raf^V599E or both in combination with CDK4, p53^DN and hTERT at 12 weeks post-grafting. Note deeply invasive melanocytic neoplasia in presence of active PI3K and only mild junctional hyperplasia seen with active B-Raf. The lower left panel represents a more deeply invasive region of the tumor shown in the upper left panel; scale bars=100µm.