Initially, we induced c-MYC expression in cultured astrocytes by infecting primary cultures of Gtv-a
astrocytes with an RCAS vector carrying the human c-MYC
cDNA (RCAS-MYC). Following infection with RCAS-MYC, astrocytes adopted a smaller, more compact morphology compared with astrocytes infected with the control RCAS-LacZ vector that carries the marker gene encoding β-galactosidase (). Immunocytochemical staining of astrocytes infected with RCAS-MYC demonstrated loss of GFAP expression and increased nestin expression (). We then performed Western blot analysis of Gtv-a
-transgenic brain cultures infected with either RCAS-MYC or RCAS-LacZ and compared the concentrations of proteins such as PLP, fibronectin and vimentin, which are expressed characteristically in glial progenitors but not in astrocytes (Lee et al., 2000
). We discovered increased expression of markers for glial progenitors in RCAS-MYC infected cells, which correlated with the level of virally transduced MYC in two independently infected populations of cells (). Expression of platelet-derived growth factor receptor α (PDGFR-α) was essentially the same in both cell populations, indicating that PDGFR-α expression is relatively unaffected by MYC, unlike other markers of glial progenitors. Last, cells infected with RCAS-MYC had a growth advantage relative to cells infected with RCAS-LacZ () and RCAS-MYC-infected cells were immortalized whereas LacZ-infected Gtv-a
astrocytes did not exhibit an undifferentiated character and senesced after extended culture (data not shown). Together, these data imply that elevating the activity of MYC in astrocytes in culture results in a rapidly growing population of cells that share many gene-expression characteristics of undifferentiated glia. They also indicate that MYC maintains the undifferentiated phenotype.
Fig. 1 Cell-culture characteristics of astrocytes infected with RCAS-MYC. (A) Morphologic differences between Gtv-a astrocytes infected with RCAS-MYC and RCAS-LacZ are indicated. (B) Immunostaining for either GFAP or nestin in cultured astrocytes from Gtv-a (more ...)
Previously, we demonstrated that combined activation of Ras and Akt induced GBMs only from undifferentiated glial progenitors in Ntv-a
mice, and that differentiated astrocytes from Gtv-a
mice were insensitive to tumor formation following such stimuli (Holland et al., 2000
). The in vitro
results indicated that MYC could promote undifferentiated Ntv-a
-like characteristics in infected cells from Gtv-a
mice. By infecting both mouse strains (Gtv-a
) with Ras+Akt, either with or without MYC, we first confirmed our previous result that MYC is not necessary for tumor formation from nestin-expressing glial progenitors (Ntv-a
mice) that are naturally undifferentiated and sensitive to the tumor-inducing capacity of double infection with Ras+Akt. After achieving this goal, we then sought to demonstrate that the addition of MYC is required to promote an undifferentiated phenotype in GFAP-expressing astrocytic cells of origin (Gtv-a
mice) sensitizing them to glioma formation by Ras+Akt.
The incidence of tumors in each group is shown in . To test the hypothesis that MYC induces gliomas when combined with Ras+Akt in Gtv-a but not Ntv-a mice, we created a logistic regression model with MYC, strain (Gtv-a and Ntv-a), and MYC by strain interaction as predictors. Using exact methods, the coefficients for strain and MYC-by-strain interaction, but not MYC-independent-of-strain, were statistically significant (P < 0.001, P < 0.001 and P = 0.5, respectively). This demonstrates that the tumor-promoting effect of MYC, when combined with Ras and Akt, depends on the mouse strain. The upper bound of the 95% confidence interval for MYC was an odds ratio of 1.6, confirming that infection with MYC when combined with Ras+Akt is unlikely to have an important effect independent of mouse strain. Therefore, MYC appears to contribute to oncogenesis from GFAP-expressing astrocytes as the cell of origin but not from nestin-expressing glial progenitors.
Fig. 2 Tumor-free survival and tumor incidence in Gtv-a and Ntv-a-transgenic mice infected with RCAS vectors carrying activated Ras, Akt and human c-MYC. Two cohorts each of Gtv-a (A) and Ntv-a (B) transgenic mice were infected with either RCAS-Ras + RCAS-Akt (more ...)
The data in vivo is consistent with MYC promoting undifferentiated character in Gtv-a cells, similar to naturally undifferentiated Ntv-a cells that are sensitive to Ras + Akt. In Gtv-a mice, the addition of MYC to Ras+Akt allows tumor formation (6/27; 22%) from more differentiated, GFAP-expressing astrocytic cells of origin, and the largest such tumor is shown in . By contrast, no tumors (0/43) were observed Gtv-a mice with Ras+Akt in the absence of MYC. This difference was statistically significant (P = 0.002, Fisher's exact test). In Ntv-a mice, where nestin-expressing cells are naturally undifferentiated and sensitive to tumor formation by Ras + Akt, the addition of MYC was unnecessary. There was no significant difference in glioma incidence (P = 0.7 by χ2 test) when comparing double infection (Ras+Akt, 13/43, 30%) to triple infection (MYC + Ras+Akt, 10/38, 26%); moreover, the confidence interval from this statistical analysis further demonstrated that addition of MYC to Ras+Akt had no important effect on tumor growth in Ntv-a mice. The data is consistent with MYC promoting an undifferentiated character of GFAP-expressing astrocytes from Gtv-a mice, which is similar to the naturally (i.e. without MYC) undifferentiated, nestin-expressing tumor cells in Ntv-a mice. These results are further illustrated in the Kaplan-Meier survival curves ().
Fig. 3 Characteristics of GBMs induced from astrocytes by infection of mice with RCAS-MYC, RCAS-Ras and RCAS-Akt. Whole-mounts illustrating (A) the largest GBM arising in Gtv-a mice after infection with a combination of Ras+Akt+MYC, (B) the only GBM that arose (more ...)
To further interrogate the effect of MYC on other oncogenic combinations, we infected Gtv-a
mice with double combinations, including MYC+Ras, MYC + Akt and Ras + Akt. As reported previously (Holland et al., 2000
), Ras + Akt induced no tumors. MYC+Akt induced one tumor from 27 Gtv-a
mice (4%) at 12 weeks (), which, histologically, resembled the Ras+Akt+MYC gemistocytic astrocytomas. Immunohistochemical analysis of this particular tumor demonstrated expression of exogenous Akt and MYC and endogenous Ras (not shown). Therefore, although MYC+Akt did induce a glioma in one mouse, activation of Ras was achieved spontaneously.
Three out of 25 mice (12%) infected with the double combination of Ras+MYC developed small tumors with the histologic characteristics of lower grade astrocytomas (largest tumor shown in ). These lesions contained abundant cytoplasm when stained with H&E, expressed GFAP as shown by immunohistochemistry, and showed occasional evidence of mitotic activity. Because these lesions exhibited neither pseudopalisading necrosis nor microvascular proliferation, they were classified as anaplastic astrocytoma (grade III astrocytoma) rather than GBM (grade IV astrocytoma). It is possible that these lesions might have acquired additional oncogenic mutations and increased in either size or grade had they been allowed to progress over time. From this data it appears that postnatal gene transfer of Ras + MYC is weakly oncogenic in differentiated astrocytes, but that Ras alone is not under these experimental conditions (Holland et al., 2000
The gliomas induced by the combination of Ras+Akt+MYC in Gtv-a
mice were characterized by large, epithelioid cells that closely resemble human gemistocytic astrocytomas (Bigner et al., 1998
). illustrates the microscopic features of these tumors which include a mixture of low and high-grade elements, as seen in human gemistocytic astrocytomas undergoing progression from a low grade tumor to a GBM (). In addition, the mouse tumors contained elements of higher malignancy, comprising an overgrowth of small tumor cells and the classic findings of GBM, including pseudopalisading necrosis and microvascular proliferation.
Fig. 4 Microscopic characteristics of gliomas arising from astrocytes following infection of Gtv-a mice with RCAS vectors encoding Ras, Akt and MYC. H&E staining illustrates (A) the mixed cell type and (B) pseudopalisading necrosis (arrow). (C–F) (more ...)
We then sought to determine if the tumors that arose from the GFAP-expressing cells showed evidence of undifferentiated character. These tumors expressed nestin strongly, which is consistent with an undifferentiated phenotype and analogous to the effects of c-MYC that we observed in vitro (). This data indicates that in vivo, c-MYC might convert GFAP-expressing astrocytes in Gtv-a mice to cells that resemble more closely nestin-expressing cells from Ntv-a mice, which are sensitive to the oncogenic effects of Ras+Akt without MYC. The expression of human c-MYC was demonstrated by immunohistochemical staining of glioma sections. Antihuman c-MYC staining in tumors induced in Gtv-a mice following triple infection with Ras+Akt+MYC corresponded to regions of tumor cells that express exogenous Akt, as indicated by staining for the HA epitope tag on the Akt vector.
To characterize further the cells comprising gliomas induced by Ras+Akt+MYC in Gtv-a
mice, we performed immunohistochemical staining with antibodies to the astrocyte markers GFAP and S100 protein, and the neuronal markers synaptophysin and NeuN. The tumors expressed variable amounts of GFAP and S100 as occurs in human GBMs (Bigner et al., 1998
), which indicates limitation to glial lineage. By contrast, no tumor expressed either of the neuronal markers synaptophysin or NeuN (data not shown), indicating that Gtv-a
astrocytes do not dedifferentiate to cells that have features of neurons, such as those found in primitive neuroectodermal tumors.