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4/2009
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Hepatic mRNA expression of histone (H3): an early predictor of tumorgenic changes in chronic hepatitis C

Azza El Bassiouny
,
Tarek Abou-Shousha
,
Mona Moussa
,
Eman El-Ahwany
,
Nora El-Bassiouni
,
Kesmat Maher
,
Ayman Galli
,
Magdy Youssef
,
Suher Zada

Arch Med Sci 2009; 5, 4: 506-512
Online publish date: 2009/12/30
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Introduction
Hepatocellular carcinoma (HCC) is one of the major health problems throughout the world. It is the fifth most common cancer in the world, accounting for an estimated 500,000 deaths annually [1, 2]. Although much is known about both the cellular changes that lead to HCC and the etiological agents responsible for the majority of HCC cases (Hepatitis B virus, Hepatitis C virus, alcohol), the molecular pathogenesis of HCC is not well understood [3, 4].
Chronic infection with hepatitis C virus (HCV) is a well-established risk factor for the development of HCC. Chronic infection with HCV appears to cause HCC as a result of chronic or recurrent hepato-cellular necrosis and hepatic inflammation and regeneration [4]. The HCV itself has direct carcinogenic potential. The HCV core protein has been implicated as being possibly carcinogenic as a result of its effect on normal growth, apoptosis, repair and cellular signaling pathways [5].
Cancer is recognized as a genetic disorder at the cellular level that involves mutation of a small number of genes. Many of these genes normally act to suppress or stimulate progression through the cell cycle, and loss or inactivation of these genes causes uncontrolled cell division and tumor formation [6].
Molecular genetics are concerned with the inter-relation between the information macromolecules DNA (deoxy-ribonucleic acid) and RNA (ribonucleic acid) and how these macromolecules are used to synthesize polypeptides, the basic component of all proteins. In human cells, genetic information is stored in DNA molecules [7]. A variety of nucleotide sequences in DNA and RNA can serve as markers for the molecular genetic diagnosis of cancers. Most of these markers directly or indirectly represent alterations in DNA that distinguish malignant cells. These markers may be related to the cause of cancer (e.g mutations in oncogenes or tumor suppressor genes) or to modifications in nucleic acid sequences that are acquired during the progression of the tumor [8].
Cells protect their DNA by organizing it as a higher order nucleoprotein complex termed chromatin in which the basic unit is the nucleo-some. Each nucleosome is composed of an octamer of core histones (two each of H2A, H2B, H3 and H4), around which two super-helical turns (80 base pairs) of DNA are wrapped [9, 10]. Histones are the numerous DNA-binding proteins that control the level of DNA condensation, and post-translation modification of histone tails plays a critical role in the dynamic condensation/decomposition that occurs during the cell cycle [11, 12].
Histones have an unusual amino acid composition in that they are extremely rich in the positively charged amino acids lysine and arginine. This property enables them to bind to the negatively charged phosphates of the DNA so they have an important role in the condensation of the DNA in chromatin [11]. Elevated levels of histone H3 and phosphorylated H3 may be responsible for the less condensed chromatin structure and aberrant gene expression observed in the oncogene-transformed cells [13, 14]. Aberrant acetylation or deacetylation of histone lead to many human disorders including cancer. The various groups of histone acetyl transferases and deacetylase were regarded to be involved in cancer progression and human diseases [15-18].
Determining the proliferative activity of tumor cells is very important for diagnostic purposes and subsequent treatment decisions. Evaluation in tissue sections is generally performed by quantitation of mitotic figures, PCNA or Ki-67 immunohistochemical labeling, silver argyrophilic nuclear organizer region staining (AGNORs), thymidine (or BrdU) labeling and flow cytometry [19]. Each of these methods has its own merits and limitations [20].
Synthesis of histones was associated with DNA replication but these synthetic rates might be different in tumor cells [11, 21]. All histones were not detected in human HCC, only H3 was detected on western blot, while H1, H2A, H2B, H4 were not detected in human HCC [11].
In situ hybridization (ISH) for some histone mRNA species, including histone 3 (H3) has been introduced as an extremely accurate technique for assessment of S-phase cell proliferation indices [22]. Thus the detection of H3-mRNA-expressing cells can be used to evaluate the proportion of cells in the S-phase, because H3-mRNAs are rapidly degraded after DNA synthesis is complete [23].
The present study employing histone H3-mRNA in-situ hybridization was undertaken to assess cell kinetics and clarify the relationship between proliferation activity and the H3-mRNA expression in chronic hepatitis C and subsequent hepato-cellular carcinoma.

Material and methods
Patients and controls

This study was conducted on 50 chronic liver disease (CLD) patients with age range for 20 to 63 years (32 males and 18 females) admitted to the inpatient section of Hepato-Gastroenterology department, Theodor Bilharz Research Institute (TBRI). All patients were subjected to thorough clinical examination and laboratory investigations including urine and stool analysis, liver function tests and serological diagnosis of schistosomiasis and hepatitis (B and C). Patients were further subjected for abdominal ultrasound (Hitachi EuB-515A) and liver biopsy specimens were obtained from hepatitis patients by percutaneous needle using the Menghini-technique and from HCC patients by ultrasound-guided needle biopsy for histopathological and in-situ hybridization studies. All biopsied patients had prothrombin time ł 60% to avoid bleeding. Serum samples from all studied subjects were collected, aliquoted and kept frozen at –70°C till further use. Patients who had any history of alcohol intake or other causes of chronic liver disease than CHC and its sequelae; cirrhosis and/or HCC, were excluded from the study.
The control group included 10 wedge liver biopsy specimens taken during surgical cholecystectomy from ten healthy subjects, well matched for age and sex. They all had clinical, biochemical, serological, ultrasonographic and histological findings within the normal range. Our study followed the tenets of the Declaration of Helsinki [24].

Laboratory tests
Liver function tests including albumin, aspartate aminotransferase (AST) and alanine amino-tranferase (ALT) were carried out using commercially available kits. Hepatitis B markers including hepatitis B surface antigen and anti-HBs antibodies, total and IgM class antibodies against hepatitis B core antigen, HBe antigen and anti-HBe antibodies were tested using enzyme immunoassay kits (Abbott laboratories, North Chicago, IL). Circulating anti-HCV antibodies were detected using a third generation enzyme immunoassay kit (Murex anti-HCV, Version III, Murex Diagnostics, Dart ford, England). Serum levels of HCV RNA were detected by nested RT-PCR according to Saber et al. [25]. Serum a-fetoprotein (a-FP) was measured using enzyme immunoassay kit (Euro genetics N-V Belgium).

Histopathological procedures
Liver specimens were fixed in 10% buffered formalin and processed into paraffin blocks. Routine hematoxylin-eosin and Masson trichrome stained sections were prepared for all cases. As regard CHC, the degree of hepatic necro-inflammation and the stage of fibrosis were scored according to the METAVIR system [26]. Grade 1 inflammation as well as the first and second stages of fibrosis were considered low score, while, grades 2 and 3 inflam-mation as well as the third and fourth stages of fibrosis were considered high. Biopsies of HCC were graded according to the histological differentiation of Ishak [27].
According to the above mentioned investigations, patients were classified into three groups: group I – 30 patients diagnosed as CHC, group II – 20 pa-tients diagnosed as HCC on top of HCV-infection, group III – 10 normal controls.

In situ hybridization (ISH)
We performed H3 mRNA-ISH on hepatic tissues using the method described by Maeyama et al. [28]. All reagents, including histone H3 probe, were obtained from Dako (Glostrup, Denmark). The H3 probe is a 550-base single-stranded, antisense, fluorescein-labeled DNA fragment that hybridizes to the entire coding region of the human histone H3 mRNA. It was used for in situ hybridization in the hybridization buffer supplied by the manufac-turer. Briefly, for formalin fixed specimens, deparaffinized, rehydrated slides were treated with protinase K for 5 min and rinsed in distilled water several times. Sections were immersed in pre-warmed Target Retrieval solution for 20-40 min at 95°C. Fluorescein-labelled H3 probe was applied to the sections and incubated overnight at 55°C. The slides were then washed in pre-warmed stringent wash buffer 1 : 50 (Dako) at 55°C for 15 min and rinsed in Tris-buffered saline (TBS). Sections were then incubated with alkaline phosphatase-conjugated anti-fluorescein antibodies at room temperature for 20 min. Development was performed in BCIP/NBT substrate for 2 h. The slides were then dehydrated and coverslipped. Negative control was treated identically except that the H3 probe was replaced by TBS.

Assessment of H3-mRNA-ISH staining
Randomly selected fields of the stained slides were evaluated at high power for H3 labeling.
A minimum of 400 cells from three different high powered (40×) fields were examined for each slide. We counted the numbers of positive and negative cells in each hepatic tissue. Scores were then obtained by calculating the percentage of cells showing positive nuclear and/or cytoplasmic staining. The H3-mRNA-ISH was evaluated inde-pendently by two observers (T.A and M.M).

Statistical analysis
The data are presented as mean ± standard error of mean (X ± SEM). The means of the different groups were compared globally using the analysis of variance ANOVA test, on SPSS software version 9. The data were considered significant if p values were less than 0.05.

Results
Fifty patients, 32 males and 18 females (mean age 20 to 63 years), with circulating anti-HCV antibodies and no serological evidence of co-infection with hepatitis B virus were selected for study. All patients were sero-positive for HCV RNA as detected by nested RT-PCR.
Tables I and II showed that patients with CHC accompanied with cirrhosis or HCC were more symptomatic with more evident signs of CLD than those without cirrhosis.
There was a significant increase in liver enzymes (ALT and AST) in HCC group (p < 0.01) compared to CHC group with a significant decrease in serum albumin level (p < 0.001) in HCC than CHC group. As regard the HCV markers, both hepatitis C and HCC groups showed seropositivity for anti-HCV antibodies. However, the quantitation of the hepatitis C viral genome by PCR test showed a highly significant increase of the HCV particles in sera of HCC compared with those of CHC group (p < 0.01) (Table III).
Nuclear expression of histone H3 was found to be statistically higher in cases of CHC than in HCC, while cytoplasmic expression showed the reverse (p < 0.05) (Table IV). It was found that with increasing grade of necro-inflammatory activity and stages of fibrosis in CHC cases, the nuclear expression of H3 was decreased while cytoplasmic expression showed increasing values compared with lower scores but with no statistical difference (Table V, Figures 1A, 1B). As regards the different grades of HCC, cytoplasmic expression of H3 was found to be statistically more in high grade than in low grade (p < 0.01) (Table VI, Figures 1C, 1D).

Discussion
The expression of histone genes is a funda-mental step constituting the process of cell proliferation [29]. Evaluation of cell proliferative activity in tissue sections can be achieved by many different methods. In vivo labeling of DNA by nucleotide analogues such as [3H] thymidine or BrdU has proven useful for identifying S-phase cells in animal experiments, but this “gold standard” is not generally suitable for human studies, especially when repeated samplings are required from the same individual [30, 31]. However there are problems of epitope masking or loss, and detectable antigen levels can vary among different cell types, leading to spurious differences in the calculated proliferation rate [32, 33]. Proliferation-related functions can be regulated not only by protein levels but also by conformational changes not detectable by immunohistochemistry [34]. An alternative approach is the expression of the histone H3 gene that has shown to be tightly coupled to DNA synthesis in all cell types examined (normal and malignant) [35]. The non isotopic histone mRNA in situ hybridization was proved to be a useful method for estimating cell proliferation in clinical pathology [36].
In this study, nuclear expression of H3-mRNA was decreased with increasing grade of hepatitis activity while cytoplasmic expression showed increasing values with increased grades of hepatitis and stages of fibrosis. This could be explained by accumulation of H3 in the hepatocytic nuclei during early hepatic insult. With progression of the disease, liberation of an already formed H3 took place from the nucleus into the cytoplasm of the hepatocytes.
S-phase markers (e.g. histone mRNA) label only those cells that have passed the G1/S boundary. The expression of the histone genes (H2A, H2b, H3, H4) is closely connected to the replicative stages of the cell cycle [37]. When DNA synthesis is completed or inhibited, histone mRNAs are selectively and rapidly degraded with a half-life of 10 min [38]. Thus the visualization of histone mRNA expressing cells can be used to evaluate the proportion of cells in the S-phase. Histone H3 gene expression has been shown to correlate with the mitotic index of tumor cells [36, 38].
In general, the incidence of proliferating cells parallels that of carcinogenesis [40, 41]. Our finding that as the differentiation of HCC decreased, the H3-mRNA increased is in agreement with that of Nagoa et al. [29], who demonstrated that H3 was significantly increased in less-differentiated HCCs, implying that in HCCs, the tumor cells have accomplished cell proliferative activity during tumor progression. Our result was also in agreement with similar results that have been also reported for primary mammary [42], easophageal [43], prostate cancers [44] and Bowen’s disease [20]. It is of utmost importance in our study that the H3-mRNA which might represent the tumor-cell fraction in S-phase among a proliferating-cell population in G1, S, G2 or M phase- was significantly higher (p < 0.01) in high grade HCC than in low grade of differen-tiation. In pancreatic ductal adenocarcinoma, the S-phase fraction as defined by the H3-mRNA labeling index increased as the degree of differentiation decreased [23]. Possible alterations in tumor-suppressor genes, which have been reported to correlate with the proliferation activity in tumor cells [29, 45], might induce more efficient DNA replication in the less-differentiated carci-nomas [29].
In conclusion, H3-mRNA-ISH (which may identify S-phase cells in formalin-fixed, paraffin-embedded hepatic tissue), should promote a better under-standing of the biological and clinical behavior of HCC. Thus, Histone H3 in situ hybridization could offer broader perspectives on the biology of cell proliferation. As histone H3 mRNA expression, was correlated with histologic grades, it could be used as an early predictor for tumorgenic changes in chronic hepatitis C, as well as an indicator of a malignant potentiality of HCCs.

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