PMC

Representative microscopic analysis of lung metastases growing from melanoma cells producing different amounts of PTN mRNA. Hematoxylin/eosin-stained whole mount sections (a–c) and microscopic views (d–f) of representative lungs from the groups producing high (a and d), medium (b and e), and low levels (c and f) of PTN are shown. For quantitative data, see Fig. d and Results.

Tumor growth in nude mice of mixtures of melanoma cells producing high and low levels of PTN mRNA. Control cells producing high levels and ribozyme-transfected cells producing low levels of PTN (pRz66-10; see Fig. b) were mixed at different ratios. Of these mixtures, 10⁶ cells per site were injected subcutaneously into two sites per mouse (n = 5 animals per group). Tumor sizes after 3 weeks of growth are shown relative to control.

In vitro characteristics of 1205LU melanoma cells after stable transfection with PTN-targeted ribozymes. (a) Northern blot. (b) PTN mRNA levels. (c) Colony growth of melanoma cells in soft agar. (a) Representative Northern blot of some of the cell lines used. The PTN hybridization signal (Upper) and the ethidium bromide stain (Lower) of the respective gel are shown. 28S and 18S indicate the ribosomal RNA bands. (b) PTN mRNA levels of all cell lines used in the studies. After transfection of 1205LU cells with different ribozyme constructs, PTN mRNA levels of G418-resistant clonal or mass-transfected cell lines were examined by Northern blot analysis with β-actin as a loading control. Values are expressed relative to control. Clonal cell lines (pRz66-4, pRz66-6, pRz66-10, pRz261-1, and pRz261-6) and a pool of G418-resistant cells (pRz66-mass) were used. The cell lines are grouped according to their PTN mRNA levels into high (= 100%), medium (>50%), and low (<50%). (c) Soft agar colony growth of melanoma cells. The number of colonies (> 60 μm) grown after 10 days of incubation is shown.

Subcutaneous tumor growth (a and b), angiogenesis (c), and lung metastasis (d) in nude mice of 1205LU melanoma cells producing different levels of PTN mRNA. (a and b) Complete growth curves of subcutaneous tumors from representative cell lines (a) and tumor sizes of all cell lines after 3 weeks (b) are shown. The cell lines producing different levels of PTN mRNA (see Fig. b) were injected subcutaneously into nude mice at 10⁶ cells per injection site and two sites per animal (n = 12 animals for control and pRz261-6; n = 5 animals for the other groups). (c) Angiogenesis in subcutaneous tumors grown from representative groups of the melanoma cells expressing different levels of PTN mRNA was quantitated. Capillaries in tumors were highlighted by staining for PECAM (CD31). In three representative tumors from each group, the average number of capillaries in eight high-power fields (×400) was counted. The number of capillaries per field (±SD) of the counts of three investigators not informed about the origins of the samples are shown. (d) Quantitation of lung metastases. Subcutaneous tumors were resected after 3 weeks (controls) or 11 weeks (pRz66-6) or when they reached at least 50 mm². Lungs were examined macroscopically or after hematoxylin/eosin staining (see Fig. ). Animals with tumors from high-level PTN-producing cells showed macroscopically visible metastases (see Fig. a) whereas metastases in the others groups were only detectable after microscopic inspection of lung sections (see Fig. e). Animals with any detectable metastases were scored positive and the relative incidence of animals with lung metastases is shown.