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Clinical Chemistry 48: 1551-1559, 2002;
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(Clinical Chemistry. 2002;48:1551-1559.)
© 2002 American Association for Clinical Chemistry, Inc.

Quantitative Analysis of p40/p46 and p69/p71 Forms of 2',5'-Oligoadenylate Synthetase mRNA by Competitive PCR and Its Clinical Application

Akira Takahashi1, Yoshiaki Iwasaki3a, Jiro Miyaike1, Hideaki Taniguchi1, Hiroyuki Shimomura3, Tadashi Hanafusa4, Yasuhiro Yumoto4, Akio Moriya1, Norio Koide2 and Takao Tsuji1

Departments of
1 Medicine and Medical Science and
2 Laboratory Medicine, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan.
3 First Department of Internal Medicine, Okayama University Medical School, Okayama 700-8558, Japan.

4 Radioisotope Center, Okayama University, Okayama 700-8558, Japan.

aAddress correspondence to this author at: First Department of Internal Medicine, Okayama University Medical School, 2-5-1, Shikata-cho, Okayama 700-8558, Japan. Fax 81-86-225-5991; e-mail yiwasaki{at}cc.okayama-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: 2',5'-Oligoadenylate synthetases (2-5AS) are type I interferon (IFN)-induced proteins with antiviral capacity. Three major forms of 2-5AS with distinct enzymatic activities have been described in IFN-treated human cells. We measured distinct forms of 2-5AS mRNA to analyze the relationship with its enzymatic activity and response to IFN therapy in chronic hepatitis C.

Methods: We established a method to quantify p40/p46 and p69/p71 forms of 2-5AS mRNA by use of reverse transcription followed by competitive PCR. The 2-5AS mRNA concentrations were measured in peripheral blood mononuclear cells from 40 patients with chronic hepatitis C and 28 control individuals.

Results: Reconstitution experiments and comparison with Northern blot analyses revealed that our method accurately and linearly quantified 2-5AS mRNA. 2-5AS mRNA concentrations and 2-5AS enzymatic activity were correlated (P <0.03). Our data demonstrated a correlation in 2-5AS mRNA between p40/p46 and p69/p71 (P <0.02), indicating a similar regulation of the expression of these genes. Our data also demonstrated that pretreatment concentrations of 2-5AS mRNA correlated with responses to IFN therapy in chronic hepatitis C.

Conclusions: Our method for measuring 2-5AS mRNA concentrations could provide an important marker for selecting patients for IFN therapy and may be useful for the development of more effective therapeutic strategies for chronic hepatitis C.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The interferon (IFN)1 system is activated in response to viral infection and plays an important role in the host defense response (1). Type I IFNs (IFN-{alpha}, -ß, and -{omega}) induce different proteins, encoded by the genes designated as IFN-induced genes, with antiviral properties. These proteins include 2',5'-oligoadenylate synthetases (2-5AS), double-stranded RNA-activated protein kinase, and Mx proteins. These proteins are considered indicators of IFN actions and are responsible, at least in part, for the antiviral state established in cells in response to viral infections (1).

The 2-5AS can catalyze the synthesis of 2',5'-linked oligomers of adenosine from ATP with the general formula pppA(2',5'A)n, where n is >1. This mixture of oligonucleotides is referred to as 2-5A, and the enzymes that synthesize it are thus referred to under the generic term 2-5AS (2). Currently the only known function of 2-5A is to bind and activate a latent endoribonuclease, 2-5A-dependent ribonuclease (RNase L), which is responsible for the degradation of viral and cellular RNAs (3)(4). This also leads to inhibition of cellular protein synthesis, thus impairing viral replication. The results from several laboratories have suggested that the 2-5A system (2-5AS, 2-5A, and RNase L) is involved, at least in part, in the antiviral and antiproliferative actions of IFN (1).

Three major forms of 2-5AS have been described in IFN-treated human cells, corresponding to proteins of 40/46, 69/71, and 100 kDa (p40/p46, p69/p71, and p100, respectively). The two isoforms of p40/p46 and p69/p71 are encoded, respectively, by the same gene and are generated by alternatively spliced mRNA from each gene (2). Each form of human 2-5AS is induced by IFN-{alpha}, -ß, and -{gamma}, but in some cells there might be differential expression and induction by IFN (5)(6)(7)(8)(9)(10). Interestingly, the three forms of 2-5AS have distinct enzymatic activities, thus suggesting that they might have specific functions (5)(6)(7)(8)(9)(10)(11). In this regard, p100 synthesizes almost exclusively the dimeric form of 2-5A (7)(11)(12), which can not activate RNase L (3)(13). Thus the biological function, especially the antiviral effect, of this particular 2-5AS is currently unknown.

The enzymatic activity of 2-5AS has generally been measured by RIA methods (13)(14)(15), but this does not give any information about the composition of these three different forms of 2-5AS, which might be differently regulated and contribute variably to the total enzymatic activity.

Chronic hepatitis caused by hepatitis C virus (HCV) infection is a common health problem affecting ~3% of the world’s population. The established treatment for chronic hepatitis C (CH-C) consists mainly of the use of type I IFN (IFN-{alpha} or -ß), which leads to a sustained treatment response in only 30% of patients. Furthermore, therapy is costly, parenterally administered, and frequently associated with many undesirable effects. For these reasons, efforts have been made to identify pretreatment predictive factors of IFN efficacy to identify patients with the greatest likelihood of response. However, the exact prediction of response to treatment for an individual patient is not possible (16)(17), although several viral and host factors that are to some extent predictive of response have been unraveled.

We describe the establishment of a new quantitative assay to measure mRNA concentrations of p40/p46 and p69/p71 2-5AS by reverse transcription and competitive PCR (C-PCR). The new assay was applied to the analysis of mRNA concentrations in peripheral blood mononuclear cells (PBMCs) from CH-C patients as well as control individuals.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
participants
After appropriate informed consent, blood samples were obtained from 18 healthy volunteers and 10 patients without liver diseases as controls (20 men and 8 women; age range, 26–80 years; mean age, 45 years) as well as from 40 CH-C patients (24 men and 16 women; age range, 26–67 years; mean, 50 years). All CH-C patients were admitted to our department between January 1996 and July 1998. The diagnosis of CH-C was based on the following: increased serum alanine aminotransferase for at least 6 months; the presence of serum HCV RNA by reverse transcription followed by nested PCR targeted to the 5' noncoding region (18); and the presence of chronic active hepatitis on liver biopsy obtained through peritoneoscopy. Twenty-nine CH-C patients received an intravenous injection of 3 x 106 units of human fibroblast-derived IFN-ß (Feron; Toray Industries Inc.) twice a day or an intramuscular injection of 10 x 106 units of recombinant or natural IFN [rIFN-{alpha}2b (Schering Plough) or OIF (Otsuka Pharmaceutical Inc.), respectively] for 2–4 weeks. Subsequently, the patients received the recombinant or natural IFN-{alpha} three times per week up to 6 months. Six months of therapy and 6 months of follow-up were completed by all enrolled patients. Serum alanine aminotransferase was monitored monthly, and the presence of serum HCV RNA was assayed at the end of therapy and at 6 months after the completion of therapy. The response to IFN therapy was assessed 6 months after therapy was stopped. Complete responders (CRs) were defined as patients in whom HCV RNA was absent in serum, as assessed by qualitative reverse transcription-PCR, for 6 months after the completion of therapy. Otherwise, patients were considered nonresponders (NRs). PBMCs from controls and CH-C patients were isolated from 10 mL of fresh, heparinized venous blood by Ficoll-Paque (Pharmacia Biotech) density centrifugation according to the instructions provided by the manufacturer.

measurement of serum hcv rna and determination of hcv genotype
Serum HCV RNA was quantified by a PCR assay (Amplicor HCV Monitor; Nippon Roche). HCV genotype was determined on the basis of the nonstructural 5 and core region sequences, according to the methods described by Chayama et al. (19) and Okamoto et al. (20). HCV genotypes were classified into genotypes 1 and 2.

cell culture and treatment with ifn
HepG2 human hepatoma cells (ATCC HB-8065) were maintained in minimal essential medium (Sigma) supplemented with penicillin (0.6 g/L), streptomycin (60 g/L), glutamine (2 mmol/L), and 100 mL/L fetal bovine serum (Sigma). Recombinant human fibroblast-derived IFN-ß was purchased from Toray Industries, Inc. HepG2 cells were incubated with or without 200 kilounits/L IFN-ß for 6 h and harvested for RNA extraction as described below.

rna extraction and cDNA synthesis
Total RNA was obtained from the PBMCs (1–33 x 106 cells) or hepatoma cells by use of UltraspecTM RNA (Biotecx Laboratories), according to the instructions provided by the manufacturer. The harvested total RNA was 1–23 µg from PBMCs and >200 µg from the hepatoma cells. The quality and integrity of the total RNA were checked by spectrophotometry or denaturing agarose gel electrophoresis. cDNA was then synthesized by reverse transcription using a random hexamer (Takara Shuzo) and Moloney murine leukemia virus reverse transcriptase (Invitrogen) according to the instructions provided by the latter manufacturer.

construction of competitor cDNA for use in c-pcr
Competitor cDNA was generated by deletion using exonuclease III as described previously by our laboratory (21). Briefly, cDNA fragments for 2-5AS genes and for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were amplified by PCR using the primer sets shown in Table 1 . The oligonucleotide primers were synthesized to span introns, based on available reported genomic sequences (GenBank accession numbers AC004551 and AC006064), to avoid accidental amplification of any contaminating genomic DNA in the RNA preparations. These primer sets amplify cDNAs, as an indicated PCR product size, that encode p40/p46, p69/p71, and GAPDH, respectively (Table 2 ). The cDNA was directly cloned into the T-vectors pCR2.1 or pCRII by use of a TA cloningTM reagent set (Invitrogen), and the phagemid clones were purified with QIAprep® spin miniprep reagent set (Qiagen), followed by sequencing by the fluorescence-based Taq dye deoxy terminator cycle sequencing system (Applied Biosystems). The purified phagemid (2 µg) was digested at the site for the restriction endonuclease, which is unique for the cloned cDNA fragment but not for the vector, followed by digestion with exonuclease III, polishing with S1 nuclease, and ligation with T4 DNA ligase. Appropriate clones were selected, grown in LB medium with kanamycin, purified, and sequenced as described above to serve as DNA competitors to be used in C-PCR or as cDNA probes for Northern blot analysis as described below (Table 2 ).


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  		Table 1. Oligonucleotide sequences and their locations on distinct forms of 2–5AS and GAPDH mRNAs.


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  		Table 2. Fragments deleted from target sequences and size of competitors for distinct forms of 2-5AS and GAPDH mRNAs.

quantification of distinct forms of 2-5as mRNAs
The steady-state concentrations of mRNAs for p40/p46 and p69/p71 were quantified by reverse transcription followed by C-PCR using serially diluted homologous competitor cDNAs with short deletions prepared as described above. The C-PCR reaction contained 5 µL of diluted synthesized cDNA, an equal volume (5 µL) of defined working solution containing competitor cDNA, and 20 µL of reaction solution containing, as a final concentration, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 200 µM each dNTP, 1 µM each primer listed in Table 1Up , and 1 U of AmpliTaq Gold® DNA polymerase (Roche Molecular Systems) for 30 µL of total reaction mixture. A typical competitor series consisted of 0, 102, 102.5, 103, 103.5, 104, 104.5, 105, 105.5, 106, and 106.5 copies. The GAPDH mRNA was used for normalization of expression of other mRNAs to enable cross-comparison among the individual samples and of reverse transcription efficiency. Moreover, correction by GAPDH could contribute to the equality of mRNA used in the reaction and improved quantification. Reactions were conducted in 500-µL tubes in a DNA thermal cycler (Model PJ-2000; Perkin-Elmer Cetus) with the following PCR cycle program: initial denaturation for 12 min at 95 °C; 50 cycles of 30 s at 95 °C, 1 min at 55 °C, and 2 min at 72 °C; and final extension for 10 min at 72 °C. Twelve microliters of each reaction mixture was run on a 3% NuSieve:1% Agarose (FMC BioProducts) gel containing 1 mg/L ethidium bromide in buffer containing 40 mmol/L Tris-acetate (pH 7.8) and 1 mmol/L EDTA. The gels were visualized under ultraviolet light and photographed using Polaroid 667 film (Polaroid). The photographs were used to generate digital images of the gels on an EpsonScan GT-9500 scanner (Epson), and the digital images were subsequently imported into the NIH Image computerized densitometry program (Ver. 1.61; Wayne Rasband, NIH, Bethesda, MD). To correct for differences in nucleotide length, the band densities of the competitor were multiplied by 1.17, 1.23, and 1.13 when measuring p40/p46, p69/p71, and GAPDH mRNA, respectively. The logarithm of the density ratio of the competitor band to the target cDNA band was then plotted vs the logarithm of the initial number of competitor molecules. At the competition equivalence point (log ratio = 0), the original concentration of cDNA in the C-PCR corresponds to the initial concentration of competitor cDNA used. Taking into account the dilution factor of the cDNA used in the C-PCR, the absolute amount of the corresponding mRNA was determined and expressed as the logarithm of copies per 103 copies of GAPDH mRNA. For calculation of the CV, six samples containing 2-5AS p46 mRNA ranging from 102 to 108 copies/5 µL were used. Each sample was measured three times in an independent assay on different days.

reconstitution experiment
Quantitative accuracy and dynamic range were assessed by a reconstitution experiment. We prepared pairs containing the wild type and competitor of deletion mutant phagemid DNAs. Wild-type phagemid DNAs consisting of 102, 103, 104, 105, 106, 107, and 108 copies/5 µL were prepared and measured by C-PCR using the corresponding competitor DNA as described above. The logarithm of the DNA determined by C-PCR was then plotted against the logarithm of the number of input wild-type phagemid DNA copies.

northern blot analysis
RNAs from IFN-ß-treated and untreated HepG2 cells were mixed at ratios of 8:2, 6:4, 4:6, 2:8, and 0:10. Ten micrograms of total RNA was analyzed through a 1% 3-(N-morpholino)propanesulfonic acid gel (20 mmol/L MOPS, 5 mmol/L sodium acetate, 1 mmol/L EDTA, pH 6.8), followed by transfer to BrightStar-PlusTM Positively Charged Nylon Membrane (Ambion) by use of a VacuGene (Amersham Pharmacia Biotech). For hybridization, 32P-labeled cDNA probes for p40/p46 and p69/p71, prepared using Ready-To-Go® DNA labeling beads and [{alpha}-32P]dCTP (Amersham Pharmacia Biotech), were used. Hybridization was performed in ULTRAhybTM hybridization buffer (Ambion) for 2 h. After hybridization, the membranes were rinsed under stringent conditions (65 °C for 30 min in a washing buffer of 0.1x standard saline citrate and 1 g/L sodium dodecyl sulfate) and then analyzed on a PhosphorImager (Molecular Dynamics). Equal loading of RNA was verified by stripping and reprobing the membrane with a 32P-labeled GAPDH probe as an internal control. Signal intensities were quantified by use of the NIH Image computerized densitometry program as described above. The assays were performed in duplicate.

measurement of 2-5as activity
We used 10% of the total PBMC samples for determination of 2-5AS activity. The total activity of the enzyme 2-5AS was measured in aliquots of lysed PBMCs by RIA, according to the instructions provided by the assay manufacturer (Eiken Immunochemical Laboratory).

statistical methods
Data are expressed as median values and ranges for nonparametric data. Distributions of continuous variables were analyzed by the Mann–Whitney U-test. A relationship between different variables was investigated by linear regression analysis. The Fisher exact test was used for comparisons between group frequencies. The independent prognostic value of the selected variable was analyzed by a multiple logistic regression model. P <0.05 was considered statistically significant, unless otherwise specified. All statistical calculations were performed using the StatView 5.0 program (SAS Institute Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
quantification of distinct forms of 2-5as mRNA
The mRNA concentrations for distinct forms of 2-5AS, namely p40/p46 and p69/p71, were quantified by C-PCR. A representative example of the agarose gel electrophoresis and quantification of each mRNA is shown in Fig. 1 . The quantitative ability of the C-PCR was assessed by a reconstitution experiment. Linear quantification was observed, with a dynamic range of 102–108 copies/5 µL of sample analyzed (Fig. 2 ). The CV was estimated as <=12% at 102–108 copies/5 µL. With regard to the sensitivity of the C-PCR for 2-5AS mRNA, 0.5% of total RNA extracted from PBMCs (5–115 ng of total RNA) was used for each sample and was sufficient for the quantitative range of the assay. The minimum detectable amount of total RNA for the assay was estimated as 0.5 ng.



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  		Figure 1. Quantification of distinct forms of 2-5AS mRNA by C-PCR.

(A), representative agarose gel electrophoretic analysis of C-PCR products for p40/p46 cDNA. C-PCR products were analyzed on a 3% NuSieve:1% Agarose gel containing 1 mg/L ethidium bromide. Products derived from target cDNA and competitor are indicated on the right side of the gel image. (B), quantification of p40/p46 mRNA by plotting the density ratio of the C-PCR products analyzed in A. The density of each band was measured and converted as described in Materials and Methods. The logarithm of the density ratio of the competitor band to the target cDNA band, after correction for difference in nucleotide length of the competitor, is plotted against the logarithm of the initial number of competitor molecules. At the equivalent point (log ratio = 0), the amount of competitor corresponds to the amount of target cDNA, as indicated by the arrow.



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  		Figure 2. Reconstitution experiment for the quantitative assay of distinct forms of 2-5AS mRNA by C-PCR.

Various amounts of p40/p46 cDNA-bearing phagemid were assayed by C-PCR. For each determination, five PCRs with different concentrations of competitor cDNA were performed and analyzed as described in the legend for Fig. 1Up . Each point represents the mean of three determinations. Statistical analysis was by linear regression analysis.

relationship between 2-5as mRNA quantified by c-pcr and northern blot analysis
Total RNA samples from hepatoma cell lines containing various amounts of 2-5AS mRNA were analyzed by C-PCR and Northern blot analysis, and the results were compared (Fig. 3 ). There was a significant correlation between mRNA concentrations measured by these two methods, confirming the accuracy and linearity of the C-PCR measurement (Fig. 3 and data not shown).



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  		Figure 3. Regression analysis of the correlation between 2-5AS mRNA concentrations determined by C-PCR and those obtained by Northern blot analysis.

(A), Northern blot analysis of RNA from total RNA mixtures from IFN-treated and untreated HepG2 hepatoma cells. RNA blots were probed with p40/p46 (top; 1.6/1.8 kb) or GAPDH cDNA (middle; 1.3 kb). Also shown is the ethidium bromide-stained band for 18S ribosomal RNA (18S rRNA; bottom). (B), quantitative comparison of 2-5AS p40/p46 mRNA concentrations measured by C-PCR and Northern blot analysis. The intensities of the band representing p40/p46 mRNA revealed by Northern blot analysis (ordinate) were equalized for that of GAPDH mRNA, expressed in arbitrary unit (a.u.), and compared quantitatively with the mRNA copies per 103 copies of GAPDH by C-PCR (abscissa). Statistical analysis was by linear regression analysis.

relationship between distinct forms of 2-5as mRNA
In the next step, we compared the p40/p46 and p69/p71 mRNA concentrations by linear regression analysis (Fig. 4 ). The mRNA concentrations showed weak but significant correlation in CH-C patients (r = 0.459; P = 0.003) as well as in the control group (r = 0.443; P = 0.018).



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  		Figure 4. Linear regression analysis of the correlation between p40/p46 and p69/p71 mRNA concentrations.

A weak but significant correlation was observed by the statistical analysis, as described in the legend for Fig. 2Up . Closed circles and solid line, CH-C patients (r = 0.459; P = 0.003); open circles and dashed line, controls (r = 0.443; P = 0.018).

correlation between concentrations of distinct forms of 2-5as mRNA and 2-5as enzymatic activity
We analyzed the relationship between the concentrations of the distinct forms of 2-5AS mRNA and 2-5AS enzymatic activity by linear regression analysis. As shown in Fig. 5 , these variables showed weak but significant correlation with each other in all cases studied.



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  		Figure 5. Regression analysis of the correlation between the mRNA concentrations for the distinct forms of 2-5AS, p40/p46 (A) and p69/p71 (B), and 2-5AS enzymatic activity (expressed as logarithm of the value) in PBMCs from controls ({circ}) and CH-C patients (•).

Statistical analysis was by linear regression analysis.

correlation between hcv rna concentrations and response to ifn treatment
Of 29 CH-C patients treated with IFN, 12 were CRs and 17 were NRs. The HCV RNA concentrations were compared in terms of the responses to IFN (Fig. 6 ). Although there seemed to be more cases with lower concentrations (<100 x 103 copies/mL) of HCV RNA in CRs than in NRs, there were some CRs with high concentrations of HCV RNA; thus, HCV RNA concentrations were widely distributed. HCV RNA concentrations in CRs and NRs were compared by the Mann–Whitney U-test. There was no statistically significant difference in HCV RNA concentrations between CRs and NRs (P = 0.051).



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  		Figure 6. Comparison of HCV RNA concentrations in CRs and NRs.

Horizontal line indicates detection limit of the Amplicor HCV Monitor (1.0 x 103 copies/mL). Vertical lines indicate the 90th and 10th percentiles and median for each group. HCV RNA concentrations in CRs and NRs were compared by the Mann–Whitney U-test.

correlation between hcv genotypes and response to ifn treatment
Of 16 cases with genotype 1, six were CRs, and of 13 with genotype 2, six cases were CRs. The relationship between HCV genotype and response to IFN treatment was analyzed by the Fisher exact test. No statistically significant relationship was observed (P = 0.917).

correlation between 2-5as mRNA concentrations and response to ifn treatment
The 2-5AS mRNA concentrations were compared in terms of the responses to IFN by the Mann–Whitney U-test (Fig. 7 and data not shown). p40/p46 mRNA concentrations were higher in CRs than in NRs, and the difference was statistically significant (P = 0.032). On the other hand, there were no significant differences in the p69/p71 mRNA concentration and 2-5AS activity, although they were higher in CRs than in NRs (P = 0.452 and 0.846, respectively).



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  		Figure 7. Relationship between p40/p46 mRNA concentrations and response to IFN therapy.

Vertical lines indicate the 90th and 10th percentiles and median for each group. The concentrations of p40/p46 mRNA in CRs and NRs were compared by the Mann–Whitney U-test. *, P = 0.032.

relationship between 2-5as mRNA and hcv rna concentrations
The relationship between p40/p46 mRNA and HCV RNA concentrations is shown in Fig. 8 . Of eight cases with more than log 2.5 copies of p40/p46 mRNA, only two cases, who had high titers of HCV RNA, were NRs (25.0%). On the other hand, of 20 cases with fewer than log 2.5 copies of p40/p46 mRNA, 14 were NRs (70.0%). The difference in the frequency of NRs between those with more than log 2.5 and fewer than log 2.5 copies of p40/p46 mRNA was analyzed by the Fisher exact test and was statistically significant (P = 0.044). Moreover, of 12 cases with low HCV RNA (<100 x 103 copies/mL), 3 were NRs, and all of them had fewer than log 2.5 copies of p40/p46 mRNA (Fig. 8 ).



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  		Figure 8. Relationship between p40/p46 mRNA and HCV RNA concentrations.

Vertical dotted line indicates detection limit of Amplicor HCV Monitor (1.0 x 103 copies/mL). Horizontal dotted line divides high (more than log 2.5) and low (fewer than log 2.5) copies of p40/p46 mRNA.

relationship between 2-5as mRNA concentrations and hcv genotypes
The p40/p46 mRNA concentrations were analyzed in terms of HCV genotypes and compared by the Mann–Whitney U-test (Fig. 9 ). There was no significant differences in the p40/p46 mRNA concentrations between genotypes 1 and 2 (P = 0.677).



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  		Figure 9. Comparison of p40/p46 mRNA concentrations between HCV genotypes 1 and 2.

Vertical lines indicate the 90th and 10th percentiles and median for each group. p40/p46 mRNA concentrations in HCV genotypes 1 and 2 were compared by the Mann–Whitney U-test.

analysis of factors possibly associated with response to ifn therapy for ch-c by multiple logistic regression model
To determine the independent prognostic value of the selected variables, HCV genotype, HCV RNA concentrations, and the p40/p46 mRNA concentrations were analyzed in terms of response of the CH-C patients to IFN therapy by multiple logistic regression model. Only the p40/p46 mRNA concentration was significant (P = 0.013) for desirable response to IFN therapy, whereas HCV RNA concentrations and HCV genotype were not (P = 0.146 and 0.691, respectively).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we designed a new quantitative assay system to measure the concentrations of the distinct forms of 2-5AS mRNA, p40/p46 and p69/p71, using quantitative C-PCR. Linear and reproducible quantification was confirmed by the reconstitution experiment, correlating the results with those obtained by Northern blot analysis. To the best of our knowledge, this is the first report demonstrating the quantification of distinct forms of 2-5AS mRNA by C-PCR.

2-5AS analysis has been most commonly performed by measuring the enzymatic activity (13)(14)(15). However, different forms of 2-5AS show different localization in cells (5)(7)(9)(22), differential induction in vitro and in vivo (7)(9)(10)(23)(24), and different substrate specificities and catalytic activities (7)(11)(12). Accordingly, total enzymatic activity does not provide information on the extent to which the distinct forms of 2-5AS contribute to the total activity, unless combined with immunoprecipitation with the limited available specific antibodies (5)(6)(8). Moreover, different forms of 2-5AS produce differently sized forms of 2-5A (6)(11)(12) with different capacities for RNase L activation (4)(13), suggesting specific functions in vivo. Therefore, estimation of the distinct forms of 2-5AS is useful for detailed understanding of IFN-mediated phenomena in vitro and in vivo, including antiviral action. Analysis of 2-5AS mRNA concentrations by Northern blot analysis or RNase protection assay might provide information on the distinct forms and amounts of 2-5AS to some extent. However, only relative and semiquantitative information can be obtained, and larger RNA samples are required, usually 1–10 µg of total RNA for Northern blot analysis and 0.1–1 µg for the RNase protection assay, because of their lower reproducibility, quantitative ability, and sensitivity than C-PCR.

Because our data demonstrated that the p40/p46 and p69/p71 mRNA concentrations correlated with each other, the transcription and stability of these different species of mRNAs must be similar in PBMCs from the controls and CH-C patients examined in our study. This may be explained by the fact that the promoter regions of these 2-5AS genes bear similar elements in common, such as the IFN-stimulated response element, for transcriptional regulation (25)(26). However, the 5'-flanking sequence of the p69/p71 gene does not generally resemble the p40/p46 promoter (26). Therefore, the observed variations in the amounts of each 2-5AS mRNA, while maintaining a correlation with each other (Fig. 4Up ), suggest that the major role of the IFN-stimulated response element, which resides in the promoter region of these two 2-5AS genes, lies in the regulation of transcription in PBMCs, as analyzed in our study. Further studies are required to demonstrate the correlation in various tissues from different individuals or in various cells under certain conditions and, if no such correlations are identified, to identify the transcriptional elements that are responsible for it.

Our results showed weak but significant correlation between the mRNA concentrations for the distinct forms of 2-5AS and 2-5AS activity (Fig. 5Up ). Our data suggest that the steady-state concentrations of 2-5AS mRNA could influence the enzymatic activity of 2-5AS in PBMCs. The relationship between mRNA concentration and 2-5AS enzymatic activity was maintained even in CH-C patients. The inhibition of signal transduction after type I IFN stimulation and of IFN-induced double-stranded RNA-activated protein kinase activity has been reported for various viral infections, including HCV (27)(28)(29). However, our data indicate that for the 2-5A system, posttranscriptional events are not affected by HCV infection in PBMCs. This seems, at least in part, attributable to the poor replicative nature of HCV in PBMCs (30), although there are some reports that suggest active replication of HCV in leukocytes, including PBMCs (31). Further studies are needed, including analyses of liver tissues, where active replication of HCV has been demonstrated, to clarify the effect of HCV infection on the 2-5A system.

We have demonstrated the possible correlation between the pretreatment concentrations of 2-5AS mRNA and responses to IFN therapy in CH-C patients (Figs. 7Up and 8Up ). Studies have revealed several host and viral factors that are predictive of treatment response but do not allow clear distinction between CRs and NRs before treatment is started (16)(17). As has been reported, our data also suggest that there is some association between HCV RNA concentrations and response to IFN therapy (Fig. 6Up ). However, analysis by multiple logistic regression revealed that the p40/p46 mRNA concentration was the only significant variable. Analysis of the relationship between p40/p46 mRNA and HCV RNA concentrations in terms of response to IFN therapy also supports the result (Fig. 8Up ). Further prospective studies with more patients will be needed to validate the observation made in this study.

There have been several reports analyzing the relationship between pretreatment 2-5AS activity and response to IFN therapy. However, this relationship remains controversial (32)(33). Our data suggest that CH-C patients with higher pretreatment 2-5AS mRNA concentrations tend to have a more favorable response to IFN therapy. High pretreatment concentrations of 2-5AS mRNA might be associated with enhanced IFN production, high expression of receptors for IFN, efficient transcriptional activation, or decreased degradation of the mRNA. Further investigation, including the latter points, is required to elucidate the underlying mechanisms of our findings. Moreover, analysis of the kinetics of 2-5AS mRNA concentrations in the course of IFN therapy should provide insight for the development of more efficient therapeutic strategies against chronic hepatitis C.

In conclusion, we have described a reliable, sensitive, and quantitative assay for the mRNA concentrations of distinct forms of 2-5AS. Analysis of the concentrations of distinct forms of 2-5AS mRNAs with this method in PBMCs from CH-C patients may be useful for selecting patients for IFN therapy and for the development of more effective therapeutic strategies for CH-C.


   Acknowledgments
 
This work was supported in part by grants from the Study Group for Intractable Hepatitis Research Committee, the Ministry of Health, Labor and Welfare, Japan. We thank Drs. Kazuharu Matsuura and Kazuo Tobe for helpful discussions and Dr. Kozo Fujio for technical assistance.


   Footnotes
 
1 Nonstandard abbreviations: IFN, interferon; 2-5AS, 2',5'-oligoadenylate synthetase(s); HCV, hepatitis C virus; CH-C, chronic hepatitis C; C-PCR, competitive PCR; PBMC, peripheral blood mononuclear cell; CR, complete responder; NR, nonresponder; and GAPDH, glyceraldehyde 3-phosphate dehydrogenase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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