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Gp120 Inhibitor

Casarotto, PC;

cell lines. To further confirm the functional link between anticancer effects of Efavirenz, and LINE-1 inhibition, a LINE-1 RT functional activity assay was performed in MCF10A, a non-cancerous and low-LINE-1-expressing cell line. The plasmid pBS-L1PA1-CH-mneo (Fig. 5a) encodes a LINE-1 sequence with a CMV promoter and in the opposite orientation, an intron disrupted Neomycin resistance gene with an SV40 promoter for expression in mammalian cells [27]. The encoded LINE-1 amino acid sequence has been optimized and is 74% consistent with the original LINE-1 sequence ensuring its escape from the cells’ self-defence mechanisms while maintaining most of the LINE-1 char- acteristics. It has previously been shown to be involved in reverse transcription in mammalian cells [27]. LINE-1 reverse transcriptase activity removes the intron and allows the expression of the Neo gene to produce Geneticin (G418) resistance in the host cells. In this experiment, only the MCF10A cells successfully transfected with pBS-L1PA1- CH-mneo survived in culture medium containing Geneticin (G418) (Fig. 5bIII, control), which selects against normal untransfected cells (Fig. 5bI). This Geneticin resistance was suppressed by Efavirenz treatment with fewer drug-treated transfected cells surviving (Fig. 5bIII) (compare Fig. 5bIII, control to Fig. 5bIII, EFV). To quantify the numbers of via- ble cells in each condition, the cells were stained with crystal violet solution and de-stained with methanol. The de-stained crystal violet-methanol solution was collected and the opti- cal densities of each flask measured at 570 nm. A signifi- cant decrease was shown in Efavirenz treated-transfected cells compared with their control (Fig. 5c). The LINE-1 RT functional assay confirmed the effectiveness of Efavirenz in inhibiting LINE-1 reverse transcriptase in MCF10A breast cells over-expressing LINE-1. Efavirenz anticancer effects are associated with downregulation of fatty acid metabolism