Activation of fetal promoters of insulinlike growth factors II gene in hepatitis C virus-related chronic hepatitis, cirrhosis, and hepatocellular carcinoma.

Increased prevalence of hepatitis C virus (HCV) infection has been found in patients with hepatocellular carcinoma (HCC). The expression of insulinlike growth factor II (IGF-II) has been linked to hepatocarcinogenesis in the experimental animal and in humans. Since reactivation of fetal IGF-II transcripts has been observed in human HCC, we have analyzed the levels of adult P1 and fetal P3 and P4 IGF-II promoter-derived transcripts in the liver of patients with HCV-related chronic active hepatitis (CAH), cirrhosis, and HCC by means of a semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) assay. Transcripts derived from adult P1 promoter were increasingly expressed from normals to patients with CAH and cirrhosis, but were undetectable in the tumorous area of 5 of 7 HCC patients and present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were expressed abundantly in most CAH and all cirrhotic patients. Transcripts from fetal P4 promoter were detected at high levels in 3 of 9 CAH patients and in the majority of cirrhotic patients. Increased expression of fetal promoter-derived transcripts was also found in the liver of HCC patients, although levels were lower than in cirrhosis. Also, the activity of fetal P3 and P4 promoters was higher in the nontumorous than in the tumorous area of the liver of HCC patients. The expression of IGF-II transcripts was correlated with the rate of cell mitotic activity by measuring the expression of the proliferating cell nuclear antigen (PCNA) gene. PCNA messenger RNA (mRNA) levels progressively increased from normals to CAH and to cirrhotic patients, and persisted at a high level in the tumorous and in the nontumorous area of HCC subjects, thus showing that the increase of IGF-II transcripts in CAH and cirrhosis is accompanied by an activation of cell mitosis in these samples. These data suggest that the activation of IGF-II gene expression from adult and fetal promoters may play a role in premalignant proliferation observed in HCV-related chronic liver disease.

sis. Also, the activity of fetal P3 and P4 promoters was Increased prevalence of hepatitis C virus (HCV) infechigher in the nontumorous than in the tumorous area tion has been found in patients with hepatocellular carof the liver of HCC patients. The expression of IGF-II cinoma (HCC). The expression of insulinlike growth factranscripts was correlated with the rate of cell mitotic tor II (IGF-II) has been linked to hepatocarcinogenesis activity by measuring the expression of the proliferating in the experimental animal and in humans. Since reacticell nuclear antigen (PCNA) gene. PCNA messenger RNA vation of fetal IGF-II transcripts has been observed in (mRNA) levels progressively increased from normals to human HCC, we have analyzed the levels of adult P1 CAH and to cirrhotic patients, and persisted at a high and fetal P3 and P4 IGF-II promoter-derived transcripts level in the tumorous and in the nontumorous area of in the liver of patients with HCV-related chronic active HCC subjects, thus showing that the increase of IGF-II hepatitis (CAH), cirrhosis, and HCC by means of a semitranscripts in CAH and cirrhosis is accompanied by an quantitative reverse-transcription polymerase chain reactivation of cell mitosis in these samples. These data action (RT-PCR) assay. Transcripts derived from adult suggest that the activation of IGF-II gene expression P1 promoter were increasingly expressed from normals from adult and fetal promoters may play a role in premato patients with CAH and cirrhosis, but were undetectlignant proliferation observed in HCV-related chronic able in the tumorous area of 5 of 7 HCC patients and liver disease. (HEPATOLOGY 1996;23:1304-1312.) present at low levels in the nontumorous area of all HCC patients. Transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were Hepatitis C virus (HCV) is a positive-stranded RNA expressed abundantly in most CAH and all cirrhotic pavirus that plays a major role in the development of tients. Transcripts from fetal P4 promoter were detected chronic liver disease (CLD). 1,2 Acute posttransfusion at high levels in 3 of 9 CAH patients and in the majority hepatitis due to HCV is followed by chronic hepatitis of cirrhotic patients. Increased expression of fetal proin more than 50% of cases, 3,4 and 20% to 50% of these moter-derived transcripts was also found in the liver of patients eventually progress to cirrhosis. 3,5 Mounting HCC patients, although levels were lower than in cirrhoevidence suggests that HCV infection may play a role in the development of hepatocellular carcinoma (HCC) in cirrhotic patients. 5-7 Chronic injury of liver cells and Abbreviations: HCV, hepatitis C virus; CLD, chronic liver disease; HCC, the associated inflammatory and regenerative rehepatocellular carcinoma; IGF-II, insulinlike growth factor II; IGF-IR, insusponse that occurs in CLD are known to represent a linlike growth factor type I receptor; RT-PCR, reverse-transcription polymerase chain reaction; CAH, chronic active hepatitis; PCNA, proliferating cell preneoplastic process that may evolve toward malignuclear antigen; PCR, polymerase chain reaction; mRNA, messenger RNA; nancy. 8 ers in the extrahepatic and hepatic tissues. 15-18 Each sion of proliferating cell nuclear antigen (PCNA), which is a known marker of cell proliferation. 30,31 promoter is followed by one or more alternative untranslated exons, which are all spliced to the last three
Patients. The biochemical and histological characteristics of our patient population are described in Table 1. The study Evidence demonstrates that the synthesis of IGF-II population consisted of 4 anti-HCV-negative, HCV-RNAand the activation of its signaling pathway through the negative control subjects (age range, 44-66 years; 1 woman, tyrosine kinase domain of the IGF-IR play important 3 men) and 25 anti-HCV-positive patients (9 with CAH [age roles in tumorigenesis. 19 The expression of IGF-II and IGF-IR genes is activated in several human and experimental tumors, and, in some of them, an autocrine/ more evident in cirrhosis than in HCC, and the activi-HAI, histological activity index. ties of fetal promoters were higher in the nontumorous * Times the upper limit of the normal range (°37 U/L). than in the tumorous area of the liver of HCC patients. † All cirrhotic and HCC patients were in Child A class. All HCCs The expression of IGF-II transcripts in patients with were well differentiated (Edmonson's class I-II) except case 25, which was poorly differentiated (Edmonson's class III-IV).
PCR products were phenol-chlorophorm-extracted, etha-CLD and from surgical specimens in control subjects. In patients with HCC, liver biopsy was performed in the tumorous nol-precipitated, and subjected to 5% polyacrylamide gel electrophoresis and autoradiography. Sizes of the amplified frag-and nontumorous areas at distance from the tumor. In each case, a portion of the liver sample was fixed in 10% buffered ments were estimated from migration of the 1-kb ladder molecular-weight marker (Gibco-BRL), and identity was as-formalin for immunohistochemistry and routine histological examination. The remaining sample was immediately sessed by restriction-enzyme digestion. PCR products were quantified by densitometric scanning of the autoradiograms washed with 0.3% NaCl, snap-frozen in liquid nitrogen, and stored at 080ЊC until assayed. Informed consent was ob-using a Howteck Scanmaster 3 densitometer with RFL Print-TM Software (Pharmacia). tained from the patients.
Histology. Diagnosis of CAH or cirrhosis was reached ac-Statistical Analysis. Correlation was evaluated by regression analysis. Significance of differences was evaluated by cording to internationally accepted criteria. 32,33 The histological activity index was assessed according to Knodell. 34 All ANOVA, followed by Wilcoxon's rank sum test. HCCs were graded histologically according to the criteria of RESULTS Edmondson and Steiner. 35 RNA Extraction. Total RNA was extracted using the gua-

Semiquantitative RT-PCR Analysis of IGF-II Promot-
nidinum thiocyanate method, and the high quality of the ers. We have studied the expression of the IGF-II gene product was assured by analysis on 1% agarose/formaldehyde from the adult (P1) and the two fetal (P3 and P4) proelectrophoresis stained with 1% ethydium bromide. 36,37 moters, which are more abundantly expressed in nor-Reverse Transcriptase. First-strand complementary DNA mal and transformed hepatocytes, 18,29 in patients with was prepared using 200 units of reverse transcriptase (Super-HCV-related CLD by using a semiquantitative RT-PCR transcript RT, Gibco BRL, Gaithersburg, MD), 1 mg of total assay. Oligonucleotides were designed from the se-RNA as template, and 10 pmol/L of random hexamers in quences of exon 3, exon 5, and exon 6, and used as 5 the presence of 0.1 mmol/L dithiothreitol, 0.5 mmol/L dNTP (Pharmacia, Milan, Italy), and 20 units of RNase inhibitor primers to specifically amplify by RT-PCR transcripts (Promega, Madison, WI), as previously described. 38 The reacoriginating from adult P1 or fetal P3 and P4 promoters, tion profile was 37ЊC 1 10 minutes, followed by 42ЊC 1 60 respectively (Fig. 1). An oligonucleotide complemenminutes. To control for contamination by genomic DNA, all tary to exon 7 sequence was used in all cases as 3 RNA samples were run in duplicate with or without addition primer (Fig. 1). To compare the relative activities of P1, of reverse transcriptase. P3, and P4 IGF-II promoters in different RNA samples, PCR Analysis. Hybridization sites of primers used for polyeach sample was simultaneously amplified with IGFmerase chain reaction (PCR) analysis of IGF-II transcripts are II promoter-and GAPDH-specific primers, and the inshown in Fig. 1. P1-, P3-, and P4-specific IGF-II transcripts 15-17 tensities of IGF-II signals were normalized to those of were analyzed using primer A: 5-AGAACTGAGGCTGGC-GAPDH. To analyze the linearity of RT-PCR coamplifi-AGCCA-3 (P1); primer B: 5-CTGTTCGGTTTGCGACACGCA-3 (P3); primer C: 5-GAGCCTTCTGCTGAGCTGTAG-3 (P4) cation of IGF-II and GAPDH transcripts, aliquots withas 5 primers; and primer D: 5-GTAGCACAGTACGTCdrawn from the reaction tube after different numbers TCCAG-3 (exon 7) as 3 primer. The GAPDH-specific primers 39 of cycles were electrophoresed and autoradiographed were 5-CACCATCTTCCAGGAGCGAG-3 (fore); 5 TCACGC- (Fig. 2A). Specific IGF-II and GAPDH transcripts were CACAGTTTCCCGGA-3 (reverse). The PCNA-specific primamplified with increasing efficiency up to 30 cycles, as ers 30 were 5-CAAGAAGGTGTTGGAGGCAC-3 (fore); 5-TACshown by densitometric analysis of PCR products (  Table 2). IGF-II transcripts derived from adult P1 Branchburg, NJ). MgCl 2 was added at the final concentration promoter were increasingly expressed from normal paof 1.5 mmol/L, except for P1-GAPDH reaction, where the contients to patients with CAH and cirrhosis. The increase centration was 1.75 mmol/L. After an initial denaturation in P1 transcripts was evident in 6 of 9 CAH patients step, 97ЊC 1 10 minutes, the PCR amplification was per- (Fig. 3, cases 6, 7, 8, and 13, and, to a lesser extent, 2.14-13.51) in cirrhotic patients (P õ .01 vs. normal patients and P õ .001 vs. CAH) (Fig. 4). Five cirrhotic IGF-II transcripts derived from fetal P3 promoter were not detectable in normal subjects, while they were patients (cases 15, 18, 19, 20, and 22) had P3 mRNA levels 10-fold higher than the median densitometric detected with progressively increasing expression in most of the patients with CAH (with the exception of scan values of CAH patients ( Fig. 4B and Table 2).
IGF-II transcripts from fetal P4 promoter were de-case 11) and in all patients with cirrhosis ( Fig. 3 and Table 2). The median densitometric scan values for tected in all subjects except case 21 ( Fig. 3 and Table  2). Increased expression of P4 transcripts was found in IGF-II transcripts derived from fetal P3 promoter were

Anti-HCV-Negative Controls and in 25 Subjects
and adult human IGF-II promoters was tested in tu-

With HCV-Related CLD
morous and nontumorous area of the liver of patients

IGF-II Promoters
with HCV-related HCC (Fig. 5 and  (Fig. 4). These values were in the range of 8 Table 2). and cannot be used for comparing the activities of the different pro-

PCNA Expression in Normal Controls and HCV-Re-
moters in the same patient.
Because of a certain degree of variability in the ex-(P õ .05 vs. normal patients), and 0.60 (range, 0.28-0.86) in cirrhotic patients (P õ .01 vs. normal patients pression of IGF-II transcripts within each group of patients (Table 2), we evaluated whether this might be and P õ .001 vs. CAH) (Fig. 4D).
The level of expression of PCNA in HCC subjects was due to a different inflammatory activity. We did not find any significant correlation between histological ac-comparable in the tumorous and nontumorous areas (median densitometric scan values, 0.59 and 0.51; tivity index (Table 1)  range, 0.36-1.25 and 0.30-1.07, respectively). These lope polypeptide and Z2 a 1 -antitrypsin transgenic mice) indicated that, independently of the causative values were higher than in normal patients (P õ .01) and CAH patients (P õ .001), but comparable with agent, liver cell injury and chronic inflammation may those found in cirrhotic patients (Figs. 4D and 6).
stimulate mediators of hepatocellular proliferation, The increase in the expression of P1, P3, and P4 IGFwhich in turn leads to the development of precursor II promoters from normal patients to CAH and cirrhotic lesions of HCC, 9,10,40 thus suggesting the existence of a patients correlated positively with PCNA transcript common endogenous pathway for liver carcinogenesis. levels in the same subjects (r Å .46, P Å .030; r Evidence supports the hypothesis that IGF-II plays Å .57, P Å .0054; r Å .49, P Å .018, for P1, P3, and P4, a role during liver carcinogenesis in rodents and hurespectively).
mans. [22][23][24][25][26][27][28] In addition, a number of studies have shown increased expression of IGF-II RNA and/or protein lev-DISCUSSION els in HBV-related CLD and HCC. 41-43, 50 The role of IGF-II in HCV-related CLD and HCC has not yet been HCV is a major causative agent of cirrhosis, which studied. is a known risk factor for the development of HCC. [1][2][3][4]8 We hypothesized that, in the course of HCV-related As well, a strong association between HCV infection CLD, there might be an increase in the expression of and HCC has been described. 5-7 However, the mecha-IGF-II, which might contribute to the proliferative hit nisms by which HCV contributes to development of ultimately leading to development of HCC. Our data HCC is unknown. Recently, two different transgenic mouse models of HCC (i.e., hepatitis B virus large enve-show a progressive increase in the expression of tran-5p0e$$0029 05-21-96 17:59:17 hepal WBS: Hepatology whereas it was activated in CAH and cirrhosis. Since P3-derived transcripts are expressed abundantly in human HCC, 28 we postulate that the activation of IGF-II fetal promoter in CLD may represent a preneoplastic lesion. The increase in IGF-II expression significantly correlated with the expression of PCNA, which is a known marker of cell mitotic activity. 30,31 Therefore, our study indicates that there is a significant relationship between liver cell proliferation and IGF-II expression in the course of HCV-related CLD. IGF-II expression during hepatitis B virus-related CLD and HCC has been localized mainly to the hepatocytes. 41-43, 50 Additionally, isolated and cultured rat hepatocytes and human hepatoma cell lines are known to express IGF-II and IGF-IR that mediates IGF-II proliferative effects. 44,45 Therefore, the increase in IGF-II-mRNA levels during HCV-related CLD suggests that IGF-II might contribute through an autocrine mechanism to the enhanced proliferative activity of liver cells that may ultimately lead to the development of HCC. One additional possibility is that the increased IGF-II expression in the cirrhotic liver is contributed to by nonparenchymal cells. In this case, IGF-II would act in a paracrine mechanism to stimulate the growth of hepatocytes and promote hepatocarcinogenesis. We also studied the expression of transcripts derived from IGF-II promoters in patients with HCV-related P3 (but not P4) promoter, though to a lesser degree when compared with cirrhosis. In the nontumorous area of the same HCC patients, transcripts from adult P1 promoter were expressed to the same extent as in scripts originating from P1 adult and P3 and P4 fetal normal subjects, while those from both P3 and P4 fetal promoters of IGF-II from normal patients (i.e., subjects promoters were expressed more abundantly. IGF-II exwith normal liver histology not infected with HCV) to pression in the nontumorous area was higher than in subjects with CAH and cirrhosis. The increase in the the tumorous area but still lower than in cirrhosis. This expression of IGF-II was evident mostly in patients data is in agreement with a recent report by Su et with cirrhosis. P3, the most active IGF-II fetal promoter, [16][17][18]29 was not expressed in normal liver tissue, al., 43 demonstrating that IGF-II immunoreactivity was