FlG. 2. p47pl"": antisense ODN treatment inhibits C>2~ production by activated human monocytes. Human monocytes were plated in 96-well tissue culture plates at a concentration of 1.0 x 105 cells/ 0.1 ml per well. Monocytes were treated with antisense or sense ODNs (5 /iM) for 3 days (with refeeding after 2 days). After incubation, the cells were washed with RPMI without phenol red and On production was determined as described in Protocols. The data represent means ¿standard deviation of triplicate determinations. The results are from a representative experiment of three performed. Cells, human monocytes; ZOP, activator; antisense, p47ph"x antisense ODNs; sense, p47phox sense ODNs. These data were analyzed using the unpaired, one-tailed Student t test. The asterisk indicates that values from cells treated with ZOP and antisense ODNs were significantly different from those obtained with cells treated with ZOP alone (p = 0.01) or as compared with those obtained with cells treated with ZOP plus sense ODNs (p = 0.003). [Adapted from E. Bey and M. K. Cathcart, J. Lipid Res. 41,489 (2000), with permission from Journal of Lipid Research.]

No oligo Sense Antisense

FIG. 3. (A) pX7phox antisense ODN treatment inhibits <p41phox protein expression in human monocytes. Human monocytes (2.5 x 106/ml) were treated with 5 ¡xM antisense or sense ODNs for 72 hr with a refeeding at 48 hr. Cells were lysed, run on SDS-10% (w/v) polyacrylamide gels, and transferred to a PVDF membrane as described in Protocols. p47'''i"x was detected with human p47ph"x polyclonal antibody (diluted 1:1000) followed by incubation with horseradish peroxidase-conjugated rabbit anti-goat IgG (diluted 1: 1000). The blot was developed by ECL. Samples were run in duplicate as indicated. The left arrow indicates the migration of p47phox (47 kDa) based on the migration of molecular weight markers that were in adjacent lanes. The samples were loaded in duplicate and the bars represent the average density of two bands per group. The bar graph depicts integrated densities of p47plwx bands in the blot as determined by analysis of lightly exposed film by the software program NIH Image. Error bars represent the data range of duplicates. (B) In a separate experiment monocytes were treated as described above with antisense or sense ODNs to p47phox (5 ¡xM) and then Western analysis of <p47phox was performed. Again, inhibition of pA7phox was observed. The blot was then stripped and reprobed with antibody to ERK1/2 kinase as an unrelated, constitutively produced protein. This blot was similarly developed. Bar graphs represent the integrated density of the relative OD curves derived from NIH Image analysis of lightly exposed films of the blots developed by ECL. [Adapted from E. Bey and M. K. Cathcart, J. Lipid Res. 41, 489 (2000), with permission from Journal of Lipid Research.]

here indicate that 5 ¡iM p47phox antisense ODNs significantly inhibited p47phox protein expression, whereas sense ODNs had no effect. The inhibition shown here is approximately 80% as determined by NIH Image densitometry analysis. These results are representative of three experiments in which inhibition by p47phox antisense ODNs ranged from 45 to 80%. Figure 3B shows a separate experiment in which a p47phox antisense/sense Western blot was stripped and reprobed with ERK1/2. This result indicates that p47ph"x antisense, although effective in inhibiting expression of p47phox protein, did not inhibit an unrelated, constitutively expressed protein. Together, these results suggest that inhibition of p47phox protein expression correlates with a downregulation of NADPH oxidase activity and that p47'''"M protein expression in human monocytes is required for O2 production.

As just demonstrated, the specificity of antisense ODN inhibition can be assessed by evaluating levels of related or unrelated cell proteins. It is essential to remember that antisense ODN effectiveness, in inhibiting the expression of a protein, depends not only on blocking new mRNA translation but on the decay of existing protein. Information regarding the half-life of the protein of interest will aid study design for the duration of antisense ODN treatment. This issue was important in these studies with p47'',,at because we discovered that this protein has a long half-life. Thus it was necessary to treat for 3 days, with two feedings, in order to observe substantial inhibition of NADPH oxidase activity and related inhibition of y41phox expression.

Indirect Assessment of NADPH Oxidase Function Using Antisense ODN as a Tool

Inhibition of02 Production Using Antisense ODN to PKCa. In other studies, we have discovered that NADPH oxidase can be regulated tightly by upstream signaling pathways required for achieving NADPH oxidase activity. Among these are studies with PKC.22'52 Selective pharmacologic inhibitors of PKC were shown to substantially inhibit the production of by activated human monocytes. To confirm that this was indeed due to inhibition of PKC, we designed an antisense ODN to a conserved region of PKC isoforms belonging to the conventional PKC (cPKC) group of PKC enzymes. Previous studies indicating requisite roles for calcium influx and release from intracellular stores influenced our choice to target the calcium-dependent group of PKC isoenzymes.59 This group is composed of PKCa, PKC/6I, PKC/JII, and PKCy, although this latter isoform is not expressed in monocytes. The cPKC antisense ODNs blocked 02~ production, on monocyte activation, whereas sense ODNs were without effect. These results confirmed the data obtained with pharmacologic inhibitors. To determine which of the isoenzymes in this cPKC group were required for O2 production through NADPH oxidase activity, we designed an antisense ODN specific for PKCa and another antisense ODN that was specific for both PKC/3I and /3II. Only the antisense for PKCa inhibited 02~ production, thus supporting the conclusion that PKCa is the only cPKC isoenzyme required for NADPH oxidase activity.22

Inhibition of02 Production Using Antisense ODN to cPLA2. Additional studies have revealed a requisite role for another upstream enzyme regulating NADPH oxidase activity, namely cPLA2.53 Studies performed in our laboratory, using a variety of pharmacologic inhibitors as well as antisense ODNs, have implicated an essential role for this enzyme in allowing the activation of NADPH

59 Q. Li, A. Tallant, and M. K. Cathcart, J. Clin. Invest. 91, 1499 (1993).

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