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. 2003 Mar 7;129(2):100–106. doi: 10.1007/s00432-002-0410-x

The alteration of PTEN tumor suppressor expression and its association with the histopathological features of human primary hepatocellular carcinoma

X-W Wan 1, M Jiang 1, H-F Cao 1, Y-Q He 1, S-Q Liu 1, X-H Qiu 1, M-C Wu 1, H-Y Wang 1,✉
PMCID: PMC12161930  PMID: 12669234

Abstract

Purpose

Although deletions or inactivating mutations of the tumor suppressor gene PTEN (phosphatase and tensin homolog deleted on chromosome 10) are involved in the development of a variety of tumors including glioblastoma, melanoma, prostate cancer, breast cancer, endometrial cancers etc., the role of PTEN expression in human primary hepatocellular carcinoma (HCC) has not yet been clarified. The aim of this study is to investigate the involvement of PTEN mRNA and protein expression in HCC.

Methods

The level of PTEN mRNA expression in HCC specimens was analyzed by Northern blot. PTEN poly-clonal antibody was raised by immunizing New Zealand white rabbit with (His)6-tagged PTEN fusion protein and characterized by Western blot. The level of PTEN protein expression was determined by immunohistochemistry. The significance of PTEN in HCC was analyzed by comparing its expression level with the clinicopathological parameters of HCC patients.

Results

Four transcripts of PTEN mRNA at 5.5 kb, 4.4 kb, 2.4 kb, and 1.8 kb were detected in most para-carcinoma liver tissues, and the expression level of PTEN mRNA in carcinoma liver tissues was found to decrease significantly. The poly-clonal antibody raised against histidine-tagged fusion PTEN protein showed specific immuno-reactivity to PTEN protein. Using the specific poly-clonal antibody prepared and characterized by ourselves, we found that PTEN protein was significantly down-regulated in HCC tissues compared with paired para-carcinoma tissues. The protein expression of PTEN is negatively associated with the pathological grading and presence of cancer thrombus of HCC.

Conclusions

Down-regulation of PTEN expression may play an important role in the development of HCC and the level of PTEN expression may be a potential adjuvant parameter in forecasting the progression and prognosis of HCC patients.

Keywords: PTEN, Hepatocellular carcinoma, Northern blot, Immunohistochemistry

Introduction

Human hepatocellular carcinoma (HCC) is one of the most common human malignancies in South-East Asia and sub-Saharan Africa (Tang 2000; Wang et al. 2001). Although the precise etiology of HCC has not yet been clarified, HCC frequently occurs in patients who have a background of chronic liver diseases, especially with cirrhosis, induced by hepatitis B (HBV) and/or C (HCV) viral infections (Rabe et al. 2001; Guo et al. 2001), excessive intake of contaminated aflatoxins (Monto et al. 2001) or alcoholic abuse (Yamanaka et al. 2001). It was believed (Munger et al. 2002) that HCC cancer cells escape the normal growth control mechanism as a consequence of activating (i.e., gain of function) mutations, and/or increased expression of one or more cellular proto-oncogenes, and/or inactivating (i.e., loss of function) mutations and/or decreased expression of one or more tumor suppressor genes. The balance of functional oncogenes and tumor suppressor products level are components of signal transduction pathways that control cell cycle entry or exit, promote differentiation, sense DNA damage, and initiate repair damage mechanisms, and/or regulate cell death programs. However, up to the present, the described genes (Reeves et al. 2000) have not yet fully disclosed the molecular mechanisms of HCC.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) (Li et al. 1997), also called MMAC1 (Steck et al. 1997) or TEP1 (Li et al. 1997), is a tumor suppressor gene originally identified by homozygous deletion mapping of 10q23. Deletions or inactivating mutations of this gene have been detected in a variety of tumor tissues and cell lines including glioblastoma (Sasaki et al. 2001), melanoma (Celebi et al. 2000), prostate cancer (Elo et al. 2001), breast cancer (De Vivo et al. 2000), and endometrial cancers (Latta et al. 2002). The identification of PTEN as a tumor suppressor gene has been supported by various reports (Weng et al. 2001) demonstrating that ectopic expression of PTEN in glioblastoma cell lines harboring PTEN mutations resulted in inhibition of cellular proliferation and suppression of both soft agar colony formation and tumorigenicity in nude mice. PTEN negatively regulates intracellular levels of PI-(3,4,5)P3, most likely via direct dephosphorylation, suggesting that it exerts its role as a tumor suppressor by negatively regulating the PI-3 K/PKB/Akt signaling pathway (Leslie et al. 2002). However, up to now, the role of PTEN in HCC has not yet been clearly clarified.

In mainland China, the death rate of HCC patients ranks second among that of all malignancies, and first even in some rural regions (Tang 2000; Wang et al. 2001). However, the studies on the involvement of PTEN in the development of HCC have exclusively focused on the presence of PTEN mutations or deletions in HCC specimens and the results of these studies suggested that PTEN mutation or deletion was not a common event of HCC (Yao et al. 1999; Yeh et al. 2000). Therefore, the correlation of PTEN mRNA and protein expression with HCC should be investigated.

To address this issue, we, for the first time, examined PTEN mRNA expression in the HCC tissues. We then expressed (His)6-tagged PTEN fusion protein and raised specific polyclonal antibody against PTEN in New Zealand rabbit. Using the antibody, the level of PTEN protein expression in HCC specimens and its clinical significance were analyzed.

Materials and methods

Specimens and cells

Primary cancerous (HCC) tissues and paired surrounding non-carcinoma liver tissues were obtained from 60 patients (men 49, women 11, mean age 49.5 years) who received surgical resection at the Eastern Hepatobiliary Surgery Hospital of the Second Military Medical University, Shanghai, China from July 1999 to September 2001. The specimens for mRNA analysis were resected for HCC tissues and paired surrounding non-carcinoma liver tissues separately and quickly frozen in liquid nitrogen. The matched specimens, including both HCC tissues and paired surrounding non-carcinoma liver tissues for immunohistochemical analysis, were fixed in formalin and embedded in paraffin. All HCC and paired surrounding non-carcinoma liver tissues were histopathologically confirmed according to a criterion of the tumor staging system (TNM). Fifty (83.3%) patients had serological evidence of hepatitis B virus infection. The serum AFP level was above 25 µg·l-1 in 41 cases (68.3%). The tumor size was smaller than 5 cm (small HCC) in 19 patients and larger than 5 cm in 41. Histologically, 58 patients (96.7%) were complicated with cirrhosis. There were 25 well-differentiated (Edmondson's grades I and II) and 35 poorly differentiated (Edmondson's grades III and IV) HCCs. Macroscopic portal vein tumor spread was found in four patients, and microscopic vascular cancer thrombi in surrounding liver were found in 15 patients. Gross and microscopic intrahepatic adjacent small satellite nodules lesions were found in 37 patients. Informed consents were obtained from all patients.

Human breast cell line MCF-7, null for PTEN expression, was kindly provided by Prof. Axel Ullrich of the Department of Molecular Biochemistry in the Max Planck Institute, Germany, and grown in DMEM (Gibco BRL, Paisley, UK) containing 10% heat-inactivated fetal bovine serum and maintained in a humidified atmosphere of 5% CO2 at 37 °C. PTEN full-length cDNA was generously provided by Carolyn Worby of the University of Michigan.

RNA preparation

Total RNA was extracted from freshly frozen tissue samples according to the method of Chomczynski and Sacchi (Chomczynski et al. 1987). Briefly, the samples were homogenized in denaturing solution D (4.0 M guanidium thiocyanate, 25 mM sodium citrate pH 7.0, 0.20 M 2-mercaptoethanol, 0.5% N-laurylsarcosine), extracted with water-saturated phenol/chloroform, and precipitated with isopropanol. The purity and concentration of total RNA was measured by the value of OD260 nm/OD280 nm.

Northern blot analysis of PTEN mRNA in HCC tissues

Aliquots containing 40 µg of total RNA were denatured in 50% formamide and 6% formaldehyde for 15 min at 65 °C, chilled on ice for 5 min, and separated on a 1.2% agrose/2.2 M formaldehyde gel. Ethidium bromide staining of the gel revealed clear 18 S and 28 S bands and the amount of RNA loading. The fragments were transferred to a Nitro membrane by blotting in 20 × SSC (0.5 M NaCl, 0.15 M sodium citrate pH 7.0) overnight. The blotting membranes were dried under vacuum and stored at −20 °C. The membranes were pre-hybridized (42 °C, 5 × SSC; 50% formamide; 5 x Denhardt's solution; 1% SDS; 100 µg/ml heat-denatured sheared salmon sperm DNA) for 4 h, then hybridized in the same solution with α-32P-labelled PTEN full-length cDNA probe overnight (Wang et al. 2001). The blots were washed to a final stringency of 50 °C in 0.1 × SSPE (1.5 mM NaCl, 1 mM NaH2PO4·H2O, 0.1 mM EDTA, pH 7.4, and 0.2% SDS). Autoradiography was performed at −20 °C with an intensifying screen. The extent of each hybridization band on the X-ray film was quantified with an image densitometer (FUJIFILM FLA-2000). The image optical density of 18S RNA loading was used as an internal control. The mRNA expression levels of PTEN were calculated after normalizing against the 18S RNA sample and correlated with the clinicopathological parameters.

Preparation of rabbit poly-clonal antibody against (His)6-tagged PTEN fusion protein

(His)6-tagged PTEN fusion protein was prepared by inserting full-length PTEN cDNA into pPROEX HTb (Gibco BRL, EcoRI and XhoI) vector and then inducing PTEN protein expression by 0.6 mM IPTG. PTEN polyclonal antibody was raised by immunizing New Zealand rabbit with (His)6-tagged fusion protein at a schedule of first immunizing with s.c. injection of 2 mg protein in complete adjuvant and rebooting with s.c. 0.5 mg protein in incomplete adjuvant 2 weeks later three times (Dunbar et al. 1990). To characterize the specificity of PTEN polyclonal antibody prepared, we selected a PTEN-deficient breast cancer cell MCF-7 for transient transfection of pcDNA3.0 and pcDNA3.0-PTEN plasmids and then detected the expression of PTEN protein in transfected MCF-7 cell (Wang et al. 2001). Briefly, MCF-7/pcDNA3.0 and MCF-7/pcDNA3.0-PTEN cells were harvested, washed in pre-chilled PBS and lysed at a density of 5 × 107 cells/ml in cold lysis buffer (50 mM Tris, pH7.5, 150 mM NaCl, 0.5% Triton X-100, 10 mM NaF, 1 mM EDTA, 1 mM phenyl-methyl-sulfonyl fluoride, 1 mM NaVO3, 1 µg/ml each leupeptin and aprotinin) at 4 °C for 30 min. Cells were further disrupted and homogenized by passage through 21 gauge needles. Lysates were clarified by centrifugation at 13,000 ×g for 20 min at 4 °C and the supernatants containing cell extract were recovered. The protein concentration was determined by Bradford assay, and 100 µg of protein from each sample were loaded onto a 12.0% SDS-PAGE gel, electrophoresed, and transferred onto a poly-vinylidene difluoride membranes (Millipore). Equal loading and transfer were verified by staining the blot with 0.05% Ponceaus (SIGMA Chemical, St. Louis, Mo., USA). The blot was incubated with PTEN antibody prepared as above, followed by incubation with horseradish peroxidase conjugated goat anti-rabbit secondary antibody at a dilution of 1:2,000. Antigen-antibody complexes were detected using an ECL chemiluminescence reagent Kit (Amersham Pharmacia, Biotech, Buckinghamshire, UK) according to the manufacturer's instructions, and then exposed to Kodak film to record results.

Immunohistochemical analysis of PTEN protein

Consecutive tissue sections of 2 µm were deparaffinized, rehydrated through graded alcohol, and subsequently incubated in H2O2 (0.3 %) to block endogeneous peroxidase. For microwave-based antigen retrieval, sections were microwaved (750 W for 5 min and 450 W for 5 min) in 10 mM citrate-buffer, pH 6.0. After pretreatment, the sections were incubated for 30 min in a normal rabbit serum (Dako, Hamburg, Germany). The rabbit-specific polyclonal antibodies against histidine-tagged PTEN fusion protein was used in a dilution of 1:200. Sections were incubated overnight in a humid chamber. After incubation with the secondary biotinylated goat anti-rabbit immunoglobulin (Dako) for 30 min, the horseradish peroxidase-labeled avidin-biotin complex (ABC-HRP) method (Dako) was performed according to the manufacturer's instructions (Bonkhoff et al. 2001; Hsu et al. 1981). A signal amplification method based on the deposition of biotinylated tyramine was used to enhance the immunodetection. Preparation of the biotinylated tyramine reagent was recently described in greater detail. After precipitation of the biotinylated tyramine (10 min at room temperature) through the enzymatic action of HRP and H2O2 (0.1 %), the biotin precipitate was detected with an additional application of the HRP-labeled avidin-biotin complex (Dako) for 30 min in a humid chamber. The peroxidase reaction was developed by 3', 3'-diaminobenzidine (Sigma) leaving a brown-colored end-product. Negative controls were performed on consecutive sections by replacing the primary antibody with a non-immune rabbit serum. Cells were considered PTEN-positive when a distinct plasma staining was identified. Positive staining was evaluated by: (1) proportion index: the proportion of PTEN protein positive cells (× 400) was scored by 0:0 %–5 %; 1:6 %–25%; 2:26%–75%; 3: >75%; (2) intensity index: the staining intensity of small cytoplasmic particles in PTEN positive cells was scored by 0: none or faint; 1: fawn; 2: brown; 3: saturated brown; and (3) reaction index = proportion index × intensity index. The level of PTEN protein expression was determined by reaction index (−): 0; (+): 1–4; (++): 5–9.

Statistical analyses

The correlation between the expression levels of PTEN mRNA and protein and clinicopathological parameters of patient was analyzed with χ2-square test. A P <0.05 was considered to be statistically significant.

Results

PTEN mRNA expression in HCC and its correlation with clinicopathological parameters

PTEN mRNA expression was investigated in 60 cases of HCC patients by Northern blot. Four major transcripts of PTEN migrating at 5.5 kb, 4.4 kb, 2.4 kb, and 1.8 kb were detected in most non-carcinoma liver tissues (Fig. 1). The expression levels of all four PTEN transcripts expression were much lower in most HCC tissues than in paired surrounding non-carcinoma liver tissues (P <0.05). The surrounding non-carcinoma liver tissues of 54 cases showed PTEN mRNA strongly positive. Among them, the lowered levels of the 5.5 kb and 4.4 kb transcripts expression were significantly correlated with serum AFP value, presence of tumor thrombus, state of satellite lesions, and histological grading (P <0.05), while no relationship with age, serum HBsAg status, tumor size, histological pattern, hepatocirrhosis, fibrocapsule, paracarcinoma hepatocyte degeneration, and microbileduct hyperplasia was found (P >0.05). The reduced level of the 2.4 kb transcript expression in HCC were significantly correlated with presence of tumor thrombus and satellite lesions in HCC patients (P <0.05), and also, no relationship with age, serum AFP, tumor size, histological pattern, histological grading, hepatocirrhosis, fibrocapsule, para-carcinoma hepatocyte degeneration, and microbileduct hyperplasia was detected (P >0.05). However, no significant relationship between the lowered expression level of 1.8 kb PTEN mRNA transcript with age, serum HBsAg status, serum AFP value, presence of tumor thrombus, satellite lesions, tumor size, histological pattern, histological grading, hepatocirrhosis, fibrocapsule, paracarcinoma hepatocyte degeneration, and microbileduct hyperplasia was found (P >0.05) (Table 1).

Fig. 1A,B.

Fig. 1A,B.

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Northern blot analysis of PTEN mRNA in HCC specimens by α-32P-labelled PTEN full-length cDNA probe. A Specimen No.1–6; B Specimen No.13–18. K HCC carcinoma tissue, N paired para-carcinoma liver tissue, upper part of A and B PTEN transcripts, lower part of A and B equivalent total RNA loaded of each sample

Table 1.

The correlation between the clinicopathological parameters of HCC patients and the number of patients whose levels of PTEN transcripts of 5.5 kb, 4.4 kb, 2.4 kb, and 1.8 kb in HCC tissues are lower than in paired surrounding non-carcinoma liver tissues

Clinicopathological parameters Case (60) Number of patients whose levels of PTEN transcripts in HCC tissues are lower than in paired para-carcinoma liver tissues (%) and its corresponding χ2 value
5.5 kb 4.4 kb 2.4 kb
AFP (µg/l)
 <400 19 7(36.8%) 19.32 P<0.01 8(42.1%)

19.98

P<0.01

 15(78.9%) 0.24 P>0.05
 401–1000 19 18(94.7%) 16(84.2%) 14(73.7%)
 >1000 22 19(86.4%) 22(100%) 16(72.7%)
Presence of tumor thrombus
 Negative 45 29(64.4%) 7.27 P<0.01 31(68.9%) 6.09 P<0.05 30(66.7%) 6.67 P<0.01
 Positive 15 15(100%) 15(100%) 15(100%)
Satelite lesion
 Negative 23 12(52.2%) 8.54 P<0.01 14(60.9%) 5.20 P<0.05 13(56.5%) 8.74 P<0.01
 Positive 37 32(86.5%) 32(86.5%) 32(86.5%)
Pathological grading
 I–II 25 12(48.0%) 14.94 P<0.01 14(56.0%) 11.16 P<0.01 18(72.0%) 0.23 P>0.05
 III 21 18(85.7%) 18(85.7%) 16(76.2%)
 IV 14 14(100%) 14(100%) 11(78.6%)
HbsAg
 Negative 11 8(72.7%) 0.0025 P<0.05 4(36.4%) 12.23 P<0.01 8(72.7%) 0.037 P>0.05
 Positive 49 36(73.5%) 42(85.7%) 37(75.5%)
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Preparation of rabbit poly-clonal antibody against (His)6-tagged PTEN fusion protein

Recombinant (His)6-PTEN fusion protein of about 48 KDa was prepared by IPTG induction and purified by (His)6-affinity column (Fig. 2A). A poly-clonal antibody against (His)6-PTEN fusion protein was obtained. The result of Western blot demonstrated that this antibody (1:200) reacted specifically with a protein molecule of about 48 KDa in MCF-7 cells transiently transfected with pcDNA3.0-wt-PTEN. While this band was not detected in MCF-7 cells transfected with an empty expression vector (Fig. 2C).

Fig. 2. A.

Fig. 2. A

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Characterization and B purification of (His)6-tagged PTEN fusion protein from XL-1 blue bacteria and characterization of rabbit anti-(His)6-PTEN fusion protein antibody by Western blot (C 1:200). A (His)6-tagged PTEN fusion protein was induced by 0.6 mM IPTG; B (His)6-tagged PTEN fusion protein was purified by Ni-affinity column; C Vector plasmid pcDNA3.0 and PTEN eukaryotic recombinant plasmid pcDNA3.0-wt-PTEN were transiently transfected into PTEN expression- deficient MCF-7 cells. The proteins in cells lysates were transferred to PVDF membrane and blot with rabbit PTEN poly-clonal antibody

PTEN protein expression and its correlation with clinicopathological parameters

The level of mRNA expression does not necessarily reflect the level of protein expression, and knowledge of the cellular localization of signal transduction protein can provide valuable insight into their functions and substrate preference. We therefore, using immunohistochemical staining, investigated the cellular localization and the level of PTEN expression in all 60 cases of paraffin-embedded HCC sections and analyzed the correlation of PTEN protein expression levels with clinicopathological parameters of patients. PTEN protein was clearly localized in the cytoplasm of hepatocytes and the expression levels were much lower or even absent in carcinoma tissues when compared with paired non-carcinoma liver tissues (Fig. 3). The reduced PTEN protein expression levels in 60 HCC samples were significantly correlated with histological grading and presence of tumor thrombus (P <0.05), but no correlation was found with age, serum AFP, serum HBsAg, tumor size, histological pattern, hepatocirrhosis, fibrocapsule, paracarcinoma hepatocyte degeneration, and microbileduct hyperplasia (Table 2, P >0.05).

Fig. 3.

Fig. 3.

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Immunohistochemical analysis of PTEN protein in HCC samples (PTEN antibody dilution 1:100, amplitude ×400, K carcinoma tissue, N non-carcinoma liver tissue)

Table 2.

Correlation between the level of PTEN protein expression in HCC tissues and the clinicopathological parameters of HCC patients

Clinicopathological parameters Cases (60) Level of PTEN protein expression (percent of patient PTEN positive)and its corresponding χ2 value
−(%) +(%) ++(%)
Presence of tumor thrombus
 Negative 45 20(44.44) 3.86 P<0.05 17(37.78) 3.11 P>0.05 8(17.78) 0.16 P>0.05
 Positive 15 11(73.33) 2(31.25) 2(13.33)
Pathological grading
 I–II 25 4(16.00) 21.85 P<0.01 12(48.00) 5.31 P>0.05 9(36.00) 11.67 P<0.01
 III 21 16(76.20) 4(19.00) 1(4.80)
 IV 14 11(78.60) 3(21.40) 0(0.0)
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Discussion

Tyrosine phosphorylation, controlled by the coordinated actions of protein tyrosine phosphatases (PTPs) and kinases (PTKS), is a critical control mechanism for numerous physiological processes, including growth, differentiation, metabolism, cell cycle regulation, and cytoskeletal function (Cheng et al. 2002; Ostman et al. 2001). PTEN, a candidate tumor suppressor involved in a variety of human cancers including glioblastoma (Li et al. 1997; Li et al. 1997; Steck et al. 1997), melanoma (Celebi et al. 2000; Poetsch et al. 2001), prostate cancer (Elo et al. 2001; Murillo et al. 2001), breast cancer (De Vivo et al. 2000), endometrial cancer (Latta et al. 2002), and hepatocellular carcinoma (Fujiwara et al. 2001; Yao et al. 1999; Suzuki et al. 2000; Yeh et al. 2000), encodes a dual specificity phosphatase (Li et al. 1997) with homology to the cytoplasmic protein tensin and auxilin. Recently, it was demonstrated that PTEN plays a role in both tyrosine and serine/threonine phosphatase-mediated signal transduction. PTEN inhibits Shc (Src-homology collagen) phosphorylation following epidermal growth factor (EGF) stimulation and therefore blocks the activation of the Ras/MAP-kinase pathway. This has been proposed as an explanation for the tumor-suppressive effect of PTEN (Gu et al. 1998). Another mechanism that involves the protein phosphatase activity of PTEN is dephosphorylation and inactivation of focal adhesion kinase (FAK), thus implying a crucial role of PTEN for the interaction between extracellular matrix and the cytoskeleton. This mechanism may lead to negative regulation of integrin-mediated cell spreading, migration, and invasion (Tamura et al. 1999a and 1999b). Besides its function as a protein phosphatase, PTEN acts as a phospholipid phosphatase with phosphatidylinositol 3,4,5-triphosphate (PIP3) as a substrate (Cantley et al. 1999). One of the direct downstream targets of PIP3, protein kinase B (Akt/PKB), is continually activated by phosphorylation in cells lacking functional PTEN and the elevated level of activation can, in turn, be reduced to normal levels by expression of wild-type PTEN (Stambolic et al. 1998). These data suggest that the tumor suppressive properties of PTEN relate in part to its ability to down-regulate the Akt/PKB pathway and hence inhibit cell proliferation and facilitate apoptosis.

Vlietstra et al. demonstrated (Vlietstra et al. 1998) that down-regulation of PTEN expression is not a common mechanism of PTEN inactivation in prostate cancer, although their results showed that down-regulation of PTEN expression by promotor methylation, mutation or other processes can cause gene inactivation. In contrast, as mentioned in the introduction, mutation of PTEN seems to play a minor role in the pathogenesis of hepatocellular carcinomas, and our results revealed that down-regulation of 5.5 kb and 4.4 kb transcripts of PTEN expression played an important role in the inactivation of PTEN activities in patients with hepatocellular carcinoma, which showed a consistent lower PTEN mRNA expression level than other transcripts in liver carcinoma tissues. We also found a complex pattern of PTEN mRNA of 5.5 kb, 4.4 kb, 2.4 kb, 1.8 kb transcripts, which resembled the profiles previously reported by Steck (Steck et al. 1997) and Gray (Gray et al. 1998). Although some of the transcripts profiles may be accounted for by alternative poly-adenylation sites, other differences are also evident, suggesting alternative PTEN splicing but related sequence, raising the possibility that PTEN may be a member of a wider gene family. Gray (Gray et al. 1998) also reported that the hybridization with PTEN cDNA probe consisting of PTEN exon 1–7 plus 5'-UTR DNA gives the same expression pattern as this study; however, a probe derived from the PTEN 5'-untranslated region (UTR) detects a single 5.5 kb transcript in all tissues examined. The probe derived from PTEN exon 1, 6 or 8 gives a similar profile displaying the strongest signal at 2.4 kb transcript. A probe from the PTEN 3'-UTR downstream of the first polyadenylation signal does not hybridize to the 2.4 kb transcript but detects the others. All these results suggested that the 2.4 kb mRNA uses this proximal polyadenylation site, whereas the others do not. Our results demonstrated that the expression level of the 2.4 kb PTEN transcript was significantly associated with the presence of tumor thrombus and satellite lesion and histological grading in 60 cases of HCC (P < 0.05). We believe that a primary mode of PTEN inactivation may be at the level of transcription. There are several potential mechanisms that might explain the reduction or loss of PTEN mRNA expression. Whang's (Whang et al. 1998) results, that demethylating agent 5-azadeoxycytidine could restore PTEN mRNA expression, argued that the most likely possibility is methylation of CpG islands in the PTEN genomic locus. Direct proof of this hypothesis in HCC requires demonstration of methylated DNA in PTEN gene, but interpretation of these experiments is difficult because the promoter and enhancer regions have not yet been precisely defined. Our findings suggested that the levels of PTEN mRNA expression may serve as an adjuvant tool for diagnosis and prediction of prognosis of HCC.

Using the specific polyclonal antibody against PTEN, we examined the PTEN protein expression levels by immunohistochemistry analysis. It was demonstrated that the expression level of PTEN protein is much lower or absolutely absent in HCC carcinoma tissues than paired surrounding non-carcinoma liver tissues. The reduced expression level of PTEN protein was significantly associated with histological grading and presence of tumor thrombus in 60 cases of HCC patients (P < 0.05), which provides further evidence to the hypothesis that PTEN might serve as a tumor progression marker in HCC and is consistent with the speculation of Vlietstra (Vlietstra et al. 1998) that complete absence and aberrant cellular distribution of PTEN protein can be expected in some malignant tumors.

Taken together, we found that the levels of PTEN mRNA and protein expression decreased significantly in HCC tissues and were closely correlated with presence of tumor thrombus and histological grading of patients. Our results suggest that PTEN might play an important role in the development and prognosis of HCC. However, whether reduced expression of PTEN mRNA and protein is the dominant factor of HCC progression remains to be further investigated.

Acknowledgements

We are grateful to Dr. Carolyn Worby of the University of Michigan for generously providing full-length wild type PTEN cDNA, and to Prof. Axel Ullrich of Department of Molecular Biochemistry in the Max Planck Institute for his generous gift of MCF-7 cells.

Footnotes

Supported by Chinese National Distinguished Young Scholar Awards (No.39825114), Chinese National Key Project of Basic Research (No.G1998051210 and No.01CB510205) and the Key Project of the Chinese National Natural Science Foundation (No. 30000159 and No.39830080).

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