Osteopontin expression correlates with adhesive and metastatic potential in metastasis-inducing DNA-transfected rat mammary cell lines

Abstract

A metastatic phenotype can be induced in benign rat mammary cells (Rama 37 cells) by transfecting them with metastasis-inducing DNAs (Met-DNAs). Stable transfection of Met-DNAs increases the level of the metastasis-associated protein, osteopontin. Randomly picked clonal cell lines have been established from the pool of Rama 37 cells transfected with one metastasis-inducing DNA, C9-Met-DNA. In these cell lines, moderate correlation is observed between the copy number of C9-Met-DNA and their metastatic potential (linear regression coefficient, R(2)=0.48). A very close correlation is observed between the cell lines' metastatic potential in vivo and the osteopontin mRNA levels in vitro (R(2)=0.74), but not with another metastasis-associated protein in this system, S100A4 (R(2)=0.21). A close correlation is also observed between osteopontin mRNA levels and the adhesive potential (R(2)=0.91) of the cells, but not with their growth rate in vitro (R(2)=0.03). These observations support the previous suggestion that osteopontin is the direct effector of C9-Met-DNA and that the presence of C9-Met-DNA is necessary, if not sufficient, for the induction of metastasis in vivo in this system. Additionally, these results suggest that Rama 37 cells with increased osteopontin mRNA levels become metastatic not through an increased growth rate, but through an increase in cellular adhesiveness.

Figure 1

An example of a Northern blot for OPN and S100A4 mRNA levels in the clonal cell lines. The following samples were electrophoresed through 0.8% (w v⁻¹) formaldehyde agarose gels and blotted onto Hybond-N membrane: Rama 800 (lane 1), R37C9Pooled (lane 2), R37C9VM13 (lane 3), R37C9VM16 (lane 4), R37C9VM8 (lane 5), R37C9VM6 (lane 6), R37C9VM25 (lane 7), Rama 37 (lane 8), R37C9VM29 (lane 9), R37C9VM14 (lane 10), R37C9VM3 (lane 11), R37C9VM18 (lane 12). In panel (A) the membrane was incubated with α³²P-labelled OPN cDNA and subjected to autoradiography. In panel (B) the membrane was incubated with α³²P-labelled S100A4 cDNA and subjected to autoradiography. In panel (C) the ethidium bromide-stained 18S and 28S ribosomal RNA bands on the membrane before hybridisation were photographed. The 1.6 kb OPN mRNA, 0.8 kb S100A4 mRNA and 18S and 28S ribosomal RNAs are shown. (D) Graph of OPN mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative OPN mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). (E) S100A4 mRNA levels of the cloned cell lines in culture relative to that in Rama 37 cells (Relative S100A4 mRNA level) plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). (D,E) Contain results for 9 C9-Met-DNA-transfected Rama 37 cell lines plus the uncloned C9-Met-DNA-transfected cells and the parental Rama 37 cells.

Figure 2

An example of Southern hybridisation of C9-Met-DNA to DNA obtained from the clonal cell lines. (A) DNA from each of the cell lines was digested with EcoR1, electrophoresed on agarose gels, and blotted onto Hybond-N membrane. The samples loaded on the gel were 500 copies C9-Met-DNA (lane 1), 50 copies C9-Met-DNA (lane 2), Rama 37 DNA (lane 3), R37C9VM18 (lane 4), R37C9VM13 (lane 5), R37C9VM6 (lane 6), R37C9VM29 (lane 7), R37C9VM14 (lane 8), R37C9VM3 (lane 9), R37C9VM8 (lane 10), R37C9VM16 (lane 11) and R37C9VM25 (lane 12). The membrane was incubated with α³²P-labelled C9-Met-DNA under hybridising conditions and subjected to autoradiography. The position of the authentic C9-Met-DNA is shown by the arrow. (B) Graph of C9-Met-DNA copy number (copy no) of the cell lines plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods). Results for the nine cloned C9-Met-DNA-transfected cell lines plus Rama 37 parental cells are shown.

Figure 3

(A) Adhesive potential of the transfectant cell lines and the pool of Rama 37 cells transfected with C9-Met-DNA. Plating efficiency defined as percentage of cells attached to plastic substratum after 15 min incubation in self-conditioned medium at 37°C is shown for the parental Rama 37 (R37), the C9-Met-DNA-transfected cell lines (R37C9VM3 etc) and the C9-Met-DNA pooled transfectants (R37C9Pooled). ^* Statistically different plating efficiency from the Rama 37 cell line (P<0.05; Student's t-test). (B) Graph of the mean plating efficiency of the cell lines plotted against their metastatic potential in vivo (% Metastasis) (Materials and Methods).