Methods and compositions targeting viral and cellular ITAM motifs, and use of same in identifying compounds with therapeutic activity
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example 1
Expression of ITAM-Containing MMTV ENV Leads to Depolarization of Mammary Epithelial Acinar Structures
Materials and Experimental Methods (Examples 1-4)
Cell Lines
[0121] NMuMG and MCF-10F cell lines were obtained from the American Type Culture Collection. MMTV-transfected clones of the NMuMG cell line were generated by transfecting NMuMG cells with MMTV. Clones expressing high levels of MMTV virions were selected. All NMuMG cell lines were maintained in DMEM containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / ml penicillin-streptomycin, and 10 μg / ml insulin at 37° C. and 5% CO2.
[0122] Cell transfections were accomplished using the GenePorter® system (Gene Therapy Systems). The Q61 plasmid was used for complete envelope expression. Y>F mutations in the MMTV SU tyrosine residues were introduced using the Quickchange® XL kit (Stratagene). Stable pools were generated and maintained by sorting for SUhi expressing cells every 5-10 passages.
[0123] Cells grown i...
example 2
SRC and SYK Tyrosine Kinases Contribute to MMTV ENV-Induced Acinar Depolarization
[0134] In lymphocytes, ITAM signaling is dependent, under the conditions utilized herein, on activity of two tyrosine kinase families, the Src family kinases and the smaller Syk / Zap-70 family kinases. To directly show the role of the SU ITAM domain in signaling, direct interaction between the MMTV SU and Syk kinase was measured in the presence of tyrosine phosphatase inhibitors. Up to 34% SU protein co-immunoprecipitated with Syk (three independent experiments; FIG. 2 A). To confirm this finding, pharmacologic inhibitors of either Src (PP2) or Syk / ZAP70 (piceatannol) were shown to be sufficient to block morphological changes associated with Env transformation (FIG. 2B; quantification for one representative experiment depicted in FIG. 2C).
[0135] These findings provide further evidence that the ITAM was responsible for the observed cell transformation.
example 3
MMTV ENV-Expressing Cells Exhibit a Transformed Mammary Epithelial Phenotype
[0136] NMuMG cells stably expressing infectious MMTV virus were also evaluated. These cells (MMTV+ cells) exhibited morphological features resembling mesenchymal cells in three-dimensional cultures and a greater degree of depolarization in comparison with NMuMG.Q4 cells (FIG. 3A). A higher level of surface MMTV SU expression (FIG. 1C) or positional effects due to virus integration and long-term culture likely accounts for the differences between the MMTV+ cells and the NMuMG.Q4 cells.
[0137] NMuMG.Q4 and MMTV+ cells both exhibited down-regulation of Keratin-18 and E-cadherin expression, indicators of epithelial-mesenchymal transition, (FIG. 3 B, left panel), while expression of these markers in NMuMG.F6 cells closely resembled wild-type and mock-transfected cells. Down-regulation of E-cadherin surface expression could also be detected in Env-expressing cells maintained in two-dimensional cultures (FIG. 3 B,...
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