Peptide nucleic acids (PNA) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible polyamide. In this study, a PNA construct was employed as an anti-gene agent in intact cells in culture. The cell lines studied were derived from Burkitt's lymphomas (BL) that presented a translocated and hyperexpressed c-myc oncogene. A 17-mer anti-myc PNA, complementary to a unique sequence located at the beginning of the second exon of the oncogene, and was covalently linked at its N terminus to a nuclear localization signal (NLS) (PNA-myc(wt)-NLS). When BL cells were exposed to PNA-myc(wt)-NLS, the anti-gene construct was localized predominantly in the cell nuclei and a rapid consequent downregulation of c-myc expression occurred. Under these conditions, both completion of a productive cell cycle and apoptosis were inhibited.
Here we show that apoptotic cells overexpress vinculin and are ingested by dendritic cells, which subsequently cross-prime vinculin-specific cytotoxic T lymphocytes (CTLs). Successful cross-priming requires that the apoptotic cells provide maturation signals to dendritic cells through CD40-CD40 ligand (CD40L) interactions. If apoptotic cells are CD40L-, the help of a third T cell is needed for priming, indicating a regulatory role for apoptotic cells in determining priming or tolerance. Vinculin-specific CTL priming is also related to apoptosis in vivo, given that in HIV-seropositive individuals, the frequency of specific CTLs depends on the proportion of peripheral CD40L+ apoptotic cells.
To determine whether ras p21 products are necessary for signal transduction mediated by the colony stimulating factor-1 receptor (CSF-1R, the c-fins proto-oncogene product), we determined whether CSF-1R and ras activate a common nuclear target and whether the interruption of ras action affects CSF-1R signal transduction. Expression of the NVL3 retrotransposon was activated to the same extent in NIH-3T3 cells by both ras and v-fins oncogenes, and the ras-responsive element located in the long terminal repeat of NVL3 was demonstrated to be a common target for oncogene action. Human recombinant CSF-1 stimulated expression of the NVL3 element 30-fold in NIH-3T3 cells that contained human CSF-1R. Expression of the carboxy-terminal 374 amino acid residues of the human ras GTPase-activating protein (GAP) in cells containing CSF-1R was able to inhibit CSF-1 induction of NVL3 expression by 90%. Expression of the catalytic domain of GAP was also able to suppress transformation by either v-fins or ligand-activated CSF-1R. Expression of the c-jun proto-oncogene was activated by CSF-1R but was insensitive to the action of the catalytic domain of GAP. These results provide genetic evidence that in NIH-3T3 cells, ras p21 is involved in signal transduction mediated by CSF-1R.
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