The metastatic tumor samples comprised brain metastases formed by breast cancer cells (231-Luc, HER2-60, and HER2-90) or melanoma cells (MeWo, WM3734), whereas the control samples were from brains injected with PBS.
The primary tumor samples thus included fat pad tumors formed by 231-Luc or HCC1937 cells, whereas the metastatic tumor samples comprised brain metastases formed by breast cancer cells (231-Luc, HER2-60, and HER2-90) or melanoma cells (MeWo, WM3734) as well as bone metastases formed bybreast cancer cells (231-Luc, HER2-60, and HER2-90) or melanoma cells (MeWo).
(a) For models of brain metastasis, human breast cancer (231-Luc, HER2-60, and HER2-90) or melanoma (MeWo, WM3734) cell lines (or PBS as a control) were injected into the left ventricle of female immunodeficient mice.
RNA-seq analysis was performed with control brain tissue or brain tissue harboring metastases formed by breast cancer cells (231-Luc, HER2-60, and HER2-90) or melanoma cells (MeWo, WM3734).
MeWo cells and RhoA mutant clones (3.5 x [10.sup.4]) were plated on 35 mm plates at 24 h before treatment.
Parental MeWo cells and MeWo-RhoA-N19 and MeWo-RhoA-V14 mutant cells were plated at a density of 2 x [10.sup.5] cells/plate on 35 mm plates 24 h before UVA, UVB, or UVC irradiation.
In the present study, we used the MeWo cell line, an adherent cell line with fibroblastic morphology derived from the lymph nodes of patients with malignant melanoma [29-32].
The MeWo cell line and the constitutively active RhoA-expressing MeWo-RhoA-V14 clones displayed cytoskeletal features characteristic of physiologic actin function and exhibited normal RhoA levels and stress fiber integrity regardless of the UV treatment applied.
To determine whether differences in viral lytic replication in MeWo and Mel1700 cells (Figure 2) also reflected patterns of viral gene expression in these cells, we analyzed the transcriptional profile of selected viral genes belonging to each of the three previously described classes of viral transcription in KSHV-infected cells [46-48].
In light of our finding that RTA is robustly expressed inMel1700-KSHV cells even in the absence of drug induction, we speculated that deregulated expression of LANA might relieve RTA repression, resulting in the relatively higher level of virus reactivation in Mel1700, but not in MeWo cells.
Although the [beta]-actin control band suggests that less total protein may have been loaded for the MeWo samples, additional repeats and longer exposures confirmed that the MeWo samples expressed the ~180, 160, or 90 kDa bands only after NaB treatment (Figure 5(b), compare lanes 1 and 2), in contrast to Mel1700-KSHV cells in which they were clearly present even in absence of NaB induction, giving us reason to believe that the ~180, 160, and 90 kDa LANA bands may be uniquely associated with lytic replication.
KSHV Blocks Expression of Anti-Inflammatory Response Proteins to Maintain a Latent State in MeWo Cells, but Fails to Do so in Mel1700 Cells That Support Robust Lytic Replication.