FIG. 4.

A down-regulation of Maspin is correlated with the tumorigenicity of of Atf-2^−/− cells. (A) Identification of Maspin as an ATF-2 target gene in the absence of hypoxic stress. RNAs prepared from normally cultured Atf-2^−/− and wild-type MEFs were subjected to DNA array analysis. Three and 34 genes were down- and up-regulated more than threefold by the loss of Atf-2, respectively. (B) Decreased levels of Maspin mRNA in Atf-2^−/− cells. The Maspin mRNA levels in the indicated MEFs were analyzed by real-time RT-PCR, and the relative degree (mean plus SEM; n = 3) is indicated. (C) Ectopic expression of ATF-2 leads to up-regulation of Maspin expression. Atf-2^−/− cells were infected with the retrovirus encoding ATF-2 and the puromycin resistance marker or control empty virus, and the puromycin-resistant cells were selected. Maspin mRNA levels were determined by real-time RT-PCR. (D) Reexpression of Maspin in Atf-2^−/− cells. Cells were infected with the retrovirus encoding Maspin and the puromycin resistance marker, and the puromycin-resistant cells were selected. Maspin mRNA levels were determined by real-time RT-PCR. (E) Maspin overexpression suppresses the tumorigenicity of Atf-2^−/− MEFs. Atf-2^−/− MEFs expressing v-K-ras were infected with the Maspin-expressing virus or control empty virus and injected into 12 sites of six nude mice. The average weights and SEM of the 12 tumors that formed 14 days after the injection are shown (left). Note that double the number of cells were injected in these experiments as in Fig. 3E. Maspin mRNA levels in the tumors were determined by real-time RT-PCR (right). (F) Maspin does not suppress Ras-induced tumor formation. NIH 3T3 cells expressing activated K-ras were infected with the Maspin-expressing virus or control virus. Cells were injected into nude mice, and the weights of tumors formed were analyzed as described above. (G) Gadd45α overexpression suppresses the tumorigenicity of Atf-2^−/− MEFs. Atf-2^−/− MEFs expressing v-K-ras were infected with the Gadd45α-expressing virus or control empty virus and injected into nude mice. The weights of tumors formed were analyzed as described above (left). Gadd45α mRNA levels in the tumors were determined by real-time RT-PCR (right). (H) Loss of Atf-2 does not affect c-Jun levels. Whole-cell lysates were prepared from p53^−/− and p53^−/− Atf-2^−/− cells and used for Western blotting with anti-c-Jun antibody. (I) Loss of Atf-2 does not affect c-Jun-dependent trans-activation. The CAT reporter, in which the TRE-containing promoter was linked to the CAT gene, was cotransfected into p53^−/− and p53^−/− Atf-2^−/− cells with or without a c-Jun expression vector. CAT activity was measured, and the average of relative CAT activity (n = 3) is shown with SEM.