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Figure 2 Dose-response of purified IL-8 RNA. Error bars represent the standard deviation of three experiments.

the samples in an incubator to automate the assay for increased throughput of the screen.

IV. IN SITU mRNA DETECTION BY ANTI-RNA:DNA ANTIBODIES

This ISH technique was applied for direct detection of mRNA expression in per-meabilized cells. The assay was validated by measuring the expression of the IL-8 gene in uninduced and IL-1-induced endothelial cells. A schematic of the assay procedure is given in Figure 3. The assay takes about 4 hr to complete and is readily automatable for HTS. Figure 4, columns 1 and 2 show the signal obtained for uninduced and IL-1-induced cells, respectively. The luminescence signal in IL-1-treated cells was increased nearly threefold over that of uninduced cells. When IL-8 o-nts were omitted from the hybridization step of the assay (column 3), the IL-1-treated cells yielded a signal identical to that obtained for uninduced cells. Clearly, the IL-8 o-nts are required for detection of the IL-1-induced signal, indicating that formation of RNA:DNA hybrids is necessary for l>

Figure 3 Schematic diagram of the HTS cell-based mRNA detection assay. Dotted line and thin solid line, mRNA besides IL-8 mRNA; thick line, IL-8 mRNA; TX-100, triton X-100; Oligos, IL-8 cDNA o-nts.

signal production. Luciferase reporter gene assays, and Northern (RNA) blot analyses [46] typically show a tenfold induction of IL-8 mRNA after treatment with IL-1. The lower level of induction observed here with the RNA : DNA duplex assay method (3-fold induction by IL-1) is most likely a result of lower sensitivity of the assay. The observation that there is no difference between the signal obtained for uninduced cells and the background signal level (no IL-8 cDNA o-nts in the hybridization step) indicates that the assay is unable to detect any IL-8 mRNA produced by uninduced cells. Hence the fold stimulation seen in the presence of IL-1 may not be a true quantitative reflection of IL-8 RNA levels following IL-1 stimulation.

The assay was validated further with respect to the need for RNA:DNA hybrid formation (Fig. 4, column 4). When the cells were treated with RNase A prior to the addition of IL-8 o-nts, the IL-1-induced signal was at background levels. RNase A presumably degraded the single-stranded IL-8 mRNA, thus preventing the formation of RNA:DNA hybrids. The assay specifically detects the desired target mRNA, as adding a control o-nt unrelated in sequence to IL-8 mRNA (Fig. 4, column 5) to the IL-1-treated cells yielded no signal.

Figure 4 Detection of IL-8 mRNA in endothelial cells. Column 1, uninduced cells; column 2, cells treated with the cytokine IL-1; column 3, cells treated with IL-1, and no IL-8 cDNA o-nts; column 4, cells treated with IL-1 and subjected to RNAseA treatment prior to incubation with IL-8 cDNA o-nts; column 5, cells treated with IL-1 and incubated with control cDNA o-nts. Unless otherwise mentioned, all the experiments were performed with 30,000 cells. Error bars represent the standard deviation obtained from four experiments.

Figure 4 Detection of IL-8 mRNA in endothelial cells. Column 1, uninduced cells; column 2, cells treated with the cytokine IL-1; column 3, cells treated with IL-1, and no IL-8 cDNA o-nts; column 4, cells treated with IL-1 and subjected to RNAseA treatment prior to incubation with IL-8 cDNA o-nts; column 5, cells treated with IL-1 and incubated with control cDNA o-nts. Unless otherwise mentioned, all the experiments were performed with 30,000 cells. Error bars represent the standard deviation obtained from four experiments.

The assay was optimized for a number of parameters, such as cell per-meablization conditions, hybridization conditions, assay steps, and incubation times. Figure 5A shows the result of permeablizing uninduced and IL-1-induced cells by incubation in (1) 2X SSC for 10 min, (2) 100% methanol for two min, (3) 0.5% Triton X-100 in 2X SSC for 10 min. The largest IL-1-induction was observed in cells treated with Triton X-100 (0.5%). Figure 5B shows the effect of temperature (25 to 55°C) on hybridization of IL-8 o-nts to IL-8 mRNA. Because RNA is known to contain extensive secondary structure, an increase in the hybridization temperature might be expected to enhance RNA:DNA hybrid formation and hence increase the sensitivity of the assay. However, increasing the hybridization temperature > 37°C increased hybridization of induced mRNA

Figure 5 Optimization of cell permeablization conditions (panel A) and cDNA o-nt-mRNA hybridization temperature (panel B). IL-8 mRNA expression was monitored for uninduced cells (right inclined stripes), IL-1-induced cells (solid) and IL-1-induced cells that were not treated with IL-8 cDNA o-nts (left inclined stripes). (A) column 1, cells were incubated with 2X SSC for 10 min during the permeablization step; column 2, cells were incubated with 100% methanol for 2 min; column 3, cells were incubated with 0.5% (v/v) Triton X-100 in 2X SSC for 10 min. (B) The effect of variation of hybridization temperature on signal production.

Figure 5 Optimization of cell permeablization conditions (panel A) and cDNA o-nt-mRNA hybridization temperature (panel B). IL-8 mRNA expression was monitored for uninduced cells (right inclined stripes), IL-1-induced cells (solid) and IL-1-induced cells that were not treated with IL-8 cDNA o-nts (left inclined stripes). (A) column 1, cells were incubated with 2X SSC for 10 min during the permeablization step; column 2, cells were incubated with 100% methanol for 2 min; column 3, cells were incubated with 0.5% (v/v) Triton X-100 in 2X SSC for 10 min. (B) The effect of variation of hybridization temperature on signal production.

and also increased the background signal level. The maximal fold induction by IL-1 was obtained at hybridization incubation temperature of 37°C.

An increase in the cell number yielded a nearly linear increase in the IL-1-induced signal, while the uninduced signal remained unchanged (curve A, Fig. 6) at cell numbers above 5000. Below 5000 cells in the assay the increase in IL-8 mRNA upon IL-1 induction was not detectable, and optimal signal of threefold induction of the signal was observed with IL-1 stimulation with 30,000

Figure 6 Dose-response obtained with increasing numbers of cells. Curve A, unin-duced; curve B, IL-1 induced.

cells. Increasing the number of cells beyond 30,000 did not increase significantly the IL-1-induced signal.

Using a HTS luciferase reporter gene assay, a selective inhibitor (compound T339142) of IL-8 mRNA expression induced by IL-1 was identified. This inhibitor was further characterized by Northern blot analysis. The inhibition by T339142 of IL-1-induced IL-8 mRNA expression was compared with the luciferase reporter gene assay (Fig. 7, curve B) and the direct IL-8 mRNA detection method described in this review (Fig. 7, curve A). Both methods revealed similar inhibitory properties of the compound. This data further validates the technique described herein and illustrates that it is suitable for the identification of inhibitors of gene expression.

This assay measures directly endogenous gene transcription and therefore can be used for identifying drugs that regulate the expression of selected genes. In addition, the method eliminates some of the key disadvantages associated with the more commonly used HTS cell-based assay formats (reporter gene assays and knock-in assays). The assay is sensitive enough to measure moderate- to high-abundance mRNA.

1.00E-04 1.00E-03 1.00E-02 1.00E-01 1 .OOE+OO 1.00E+Q1 T339142 (uM)

Figure 7 The effect of increasing concentrations of T339142 on IL-8 mRNA expression. IL-8 mRNA expression was monitored by the luciferase gene expression assay (curve A) and the direct anti-RNA:DNA antibody method described in this chapter (curve B). T339142 was added to the reaction mixture about 1 hr prior to IL-1 treatment. The compound was added in 10 ||L aliquots containing 2% DMSO in phosphate-buffered saline to about 30,000 plated cells in 90 |L of media to yield the final desired compound concentration in 0.2% DMSO.

1.00E-04 1.00E-03 1.00E-02 1.00E-01 1 .OOE+OO 1.00E+Q1 T339142 (uM)

Figure 7 The effect of increasing concentrations of T339142 on IL-8 mRNA expression. IL-8 mRNA expression was monitored by the luciferase gene expression assay (curve A) and the direct anti-RNA:DNA antibody method described in this chapter (curve B). T339142 was added to the reaction mixture about 1 hr prior to IL-1 treatment. The compound was added in 10 ||L aliquots containing 2% DMSO in phosphate-buffered saline to about 30,000 plated cells in 90 |L of media to yield the final desired compound concentration in 0.2% DMSO.

V. HTS ASSAY FOR DETECTION OF IN VITRO TRANSCRIPTION REACTION PRODUCT BY ANTI-RNA:DNA ANTIBODIES

This RNA:DNA duplex formation assay was further developed for a HTS assay to measure the product of a eukaryotic in vitro transcription reaction. HIV-1 basal and activated transcription was used as a test system. HIV-1 and other related lentiviruses encode an essential regulatory protein called Tat [47]. Tat is a transcriptional elongation factor that activates the expression of HIV-1 genes by binding to the transactivation-responsive region (TAR), a stem-loop structure at the 5' end of all HIV-1 mRNA transcripts. Tat consists of a basic RNA binding region and a transcriptional activation domain that interacts with RNA polymerase II

(RNAPII) and possibly other cellular proteins. Small molecules that inhibit the activity of Tat would likely be effective drug candidates for the treatment of HIV infection. The complete assay procedure is shown schematically in Figure 8.

Figure 9 shows the signal obtained for RNA product generated by in vitro transcription from the HIV LTR promotor using crude HeLa cell nuclear extracts [44]. The sequence and number of steps in the assay were optimized to achieve maximum throughput. Figure 9A shows that the product transcript G-less RNA obtained from (1) basal transcription, (2) Tat-activated transcription, and (3) treatment with the RNAPII inhibitor a-amanitin. Generation of the signal was completely dependent on the presence of complementary DNA o-nts, indicating that RNA:DNA heteroduplex formation is necessary for detection of the RNA product. The signal obtained from the basal transcription reaction was ~ 1.5-fold greater than that obtained from a sample treated with the RNAPII inhibitor a-amanitin (defined as background). Addition of Tat to the in vitro transcription reaction resulted in a six-fold increase in signal over the basal level. When the

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