Transduction of human hepatocellular carcinoma cells with human γ-interferon gene via retroviral vector

AIM To investigate the therapeutic potential of gamma interferon (IFN γ) gene modified human hepatocellular carcinoma (HCC) cells. METHODS The IFN γ gene was introduced retrovirally into four HCC cell lines. Secreted IFN γ activity was assessed using bioassay. The expression of MHC molecules was detected by FACS. Tumorigenicity was analysed by tumor formation in nude mice. RESULTS Four IFN γ gene transduced HCC cell lines secreted different amounts of IFN γ, as in the same case of five clones derived from one HCC cell line. Transduction with IFN γ caused significant increase in the expression of major histocompatibility complex (MHC) antigens on HCC cells. The expression of HLA class Ⅰ was increased by 2-3 times in terms of mean fluorescence intensities, while for class Ⅱ expression, the percentage of positive cells augmented from <10% to >50%. When equal amount of tumor cells were injected into nude mice, the tumorigenicity of some transduced cells decreased dramatically. CONCLUSION IFN γ gene transduction can convert weakly immunogenic HCC cells to activate antitumor immune response, and further pave the way for the future use of such gene modified tumor cells as a modality for the cancer immunotherapy.

P21^(WAF1/Cip1)Gene Expression in Primary Human Hepatocellular Carcinoma and its Relationship with P53 Gene Mutation

Summary: P21 WAF1/Cip1 , an inhibitor of cyclin dependent kinases, is a critical downstream effector in the P53 specfic pathway of growth control. Increased expression of P21 WAF1/Cip1 has been found to reflect the status of the P53 tumor suppressor pathway. We investigated the expression of P21 WAF1/Cip1 in a relatively small, but well characterized group consisting of 28 hepatocellular carcinomas. The samples were previously studied for P53 gene mutation. P21 WAF1/Cip1 expression were identified by in situ hybridization and immunohistochemistry. Positive ISH for P21 WAF1/Cip1 transcripts was found in 18 of 28 cases (64.3 %). All positive cases by ISH showed detectable P21 WAF1/Cip1 protein reactivity by IHC. No relationship was found between P21 WAF1/Cip1 staining and P53 mutational status. No associations were seen with tumor metastasis, size and tumor grade, except for tumor differentiation status which showed higher frequency of P21 WAF1/Cip1 expression in moderate well differentiated HCCs than poorly differentiated tumors ( P <0.05). It is concluded that expression of P21 WAF1/Cip1 is common in HCCs, but does not correlate with P53 mutational status or pathological parameters investigated except for tumor differentiation. Also, there may be other factors beside P53 that regulate P21 WAF1/Cip1 gene expression in HCCs.

Clinicopathologic risk factors and prognostic evaluation in hepatocellular carcinoma recurrence after surgery

AIM To analyze the clinicopathologic risk factors in hepatocellular carcinoma recurrence after surgery. METHODS Significance test (χ 2 and Student t test) of the single and multiple factors, and Wilcoxon Cox tropic examination were used, a retrospective clinicopathologic analysis was made in 156 cases of hepatocellular carcinoma after hepatectomy. RESULTS Of the 156 cases, 68 4%, 57 3%, 46 7%, 31 5% and 28 6% had 1, 2, 3, 4 and 5 postoperative tumor free years respectively with a total recurrence rate of 53 2% (83/156). In the 83 recurrent cases, 65 were of intrahepatic sabclinical type, with a re resection rate of 78 3% (65/83). The relevant factors involved in recurrence were: males, tumor number and size, capsule infiltration, portal veins involvement, etc. Those factors obviously influenced the prognosis of the patients with postoperative hepatocellular carcinoma ( P <0 05). 63 1% tumor nodes (41/65) of recurrent liver cancinomas were located at the ipsilateral segment of the primary ones. CONCLUSION Males, tumor number and size, capsule infiltration and portal veins involvement are the factors for postoperative hepatocellular carcinoma recurrence after surgery. The recurrence is mainly unicentral. Right front lobe is the liver segment with a high recurrence rate.

Segmental transcatheter arterial embolization for primary hepatocellular carcinoma

AIM To evaluate the therapeutic effects of segmental transcatheter arterial embolization for primary hepatocellular carcinoma, and to recognize the menifestation and clinical value of lipiodol overflow into portal veins surrounding the tumors. METHODS A total of 50 cases of nonresectable primary hepatocellular carcinoma underwent segmental transcatheter arterial embolization. Two methods of superselective segmental catheterization were used, one was the method of wire guiding, and the other the technic of co axial infusion catheter. RESULTS The 1 , 2 , 3 and 4 year cumulative survival rates of 50 cases with segmental transcatheter arterial embolization for primary hepatocellular carcinoma were 83 8%, 65 4%, 42 9% and 24 5% respectively. The incidence of the lipiodol overflow into portal veins was 64%. The overflow of lipiodol into portal veins, represented as 3-5 grade branches of portal veins visualized by lipiodol, was “star like” or “tree like”, and there was a relatively large vessel in the center surrounded with radicalized small branches of vessels. CONCLUSION The lipiodol overflow into portal veins was one of the signs of complete embolization for tumors, and may play a partial role in embolizating the portal venous supply for hepatocellular carcinoma.

Long-term survival for spontaneous rupture of hepatocellular carcinoma treated with hepatectomy

Objective- To determine the prognostic factors of ruptured hepatocellular carcinoma （HCC） and report the management of patients with spontaneous rupture of HCC in a single center during a 5-year period and to evaluate one-stage hepatectomy Methods- A series of 4,209 patients with HCC were collected at Eastern Hepatobiliary Surgery Hospital from April 2002 to November 2006, of whom 200 patients （4.8%） with ruptured HCC were studied retrospectively regarding their clinical characteristics and prognostic factors. The one-stage therapeutic approach to manage ruptured HCC consisted of initial management by conservative method, transarterial embolization （TAE） or surgical hepatectomy. Results of various treatments were evaluated and compared in the randomly selected 202 patients with no history of rupture during the same study period. Results： A total of 200 patients with spontaneous rupture of HCC were studied who underwent surgical treatment （n=105）, TAE 33 and conservative treatment （ConT 62）. A multivariate analysis using the Cox hazard regression model （including all the patients n=200） identified surgical hepatectomy as the only independent factor determining a relatively long survival period （P〈0.0001） On the other hand, in a further analysis of the patients in whom surgical hepatectomy was successfully performed （n=105）, which identified a maximum tumor size exceeding 6 cm as significant determinants of a poor 12-month （P=0.036）, and a multivariate analysis did not identify as any inverse independent factor determining relatively long-term survival, only a maximum tumor size exceeding 6 cm exhibited a tendency toward being a determinant factor （P=0.083）. Conelusionz Considering the high propensity to spontaneous rupture, as long as preoperatively clinical evaluation meet surgery requirements, elective one-stage hepatectomy for patients with ruptured HCC is the first treatment option. Prolonged survival could be achieved in selected patients with hepatic resection, although the survival results were inferior to those of the patients who did not have the complication of rupture

Establishment and characterization of four human hepatocellular carcinoma cell lines containing hepatitis B virus DNA

AIM To investigate the characteristics of newly established four hepatocellular carcinoma cell lines (SNU 739, SNU 761, SNU 878 and SNU 886) from Korean hepatocellular cancer patients. METHODS Morphologic and genetic studies were done. RESULTS All four lines grew as a monolayer with an adherent pattern, and their doubling times ranged from 20 to 29 hours. The viability rate was relatively high (88%-94%). Neither mycoplasmal nor bacterial contamination was present. The lines showed different patterns in fingerprinting analysis. The hepatitis B virus (HBV) DNA was integrated in the genomes of all four lines, and in all of them HBx, HBc and HBs transcripts were detected by reverse transcriptase PCR methods. Among the three cell lines used as control (Hep 3B, SK Hep1 and Hep G2), only Hep 3B showed HBx expression, and this line was used as a HBV integrated control. The RNA of albumin was detected in three lines (SNU 761, SNU 878 and SNU 886), that of transferrin in two lines (SNU 878, SNU 886), and that of IGF Ⅱ was detected in none of the cell lines. CONCLUSION These well characterized cell lines may be very useful for studying the biology of hepatocellular carcinoma in association with the hepatitis B virus.

Stage Ⅱ surgical resection of hepatocellular carcinoma after TAE:a report of 38 cases

AIM To evaluate the curative effect of stage Ⅱ surgical resection of hepatocellular carcinoma after TAE. METHODS Thirty eight patients with unresectable hepatocellular carcinoma were treated by transcatheter arterial embolization (TAE). When the sizes of tumors were markedly reduced after TAE, stage Ⅱ surgical resections were performed. RESULTS Before TAE, the diameters of tumors were 12 84cm±4 87cm ( ±s ), but reduced to 5 12cm±1 82cm ( ±s ) after TAE ( P <0 001). Pathologic examination of the resected specimens revealed obvious necrosis in most cases. After surgery, 26 patients were alive, with the longest survival of 96 months, twelve died and 10 had tumor recurrence. CONCLUSION Patients in moderate and advanced stages of hepatocellular carcinoma after TAE should be treated surgically, but the indication must be controlled strictly.

Expression of DNA Repair Enzyme hMTH1 mRNA and Protein in Hepatocellular Carcinoma

Summary： To study the expression of DNA repair enzyme hMTH1 mRNA and protein in hepatocellular carcinoma （HCC） tissues, tissues adjacent to the cancers, normal liver cells and hepatoma cell lines, and to investigate their function in the progress of HCC, semi-quantitative reverse-transcription polymerase chain reaction （RT-PCR） was employed to examine the expression of hMTH1 mRNA in matched HCC tissues （HT）/surrounding tissues （HST） of HCC, normal liver cell L02 and hepatoma cell lines SMMC7721, HepG2. hMTH1 protein was detected in corresponding HT as well as their HST by immunohistochemistry. Our results showed that the expression level of hMTH1 mRNA in HT was higher than that in HST （t=2. 424, P 〈0.05）. The expression level of hMTH1 mRNA in two hepatoma cell lines was higher than that in normal liver cell line （F=6. 810, P〈0.01）. The expression of hMTH1 mRNA inSMMC7721 was similar to that in HepG2. hMTH1 protein was 88.2%（15 of 17） positive in HT and 82.4%（14 of 17） in HST. The protein level of hMTH1 in HT was correspondingly higher than in their HST （t=2. 618,P〈0.05）. It is concluded that hMTH1 mRNA and protein were over-expressed in HCC and hepatoma cell lines. It may be one of the key events during the carcinogenesis, progression of HCC and may promote the malignant growth. These results suggest that hMTH1 plays a role in HCC and may be a candidate marker for the diagnosis of HCC.

Clinical significance of CD44v mRNA detection in peripheral blood of patients with hepatocellular carcinoma by RCR

IM To study the clinical significance of detecting the CD44v mRNA expression in the blood of patients with hepatocellular carcinoma (HCC).METHODS The expression of CD44v mRAN was detected in blood with RT and diploid PCR and the clinical significance was discussed based on the result of pathological examination and followup.RESULTS CD44v mRNA was detected in the blood of 10/15 patients, with a positive rate of 6667%. In 13 patients who responded to the followup, CD44v mRNA expression was positive in 9 cases and negative in 4 cases. Recurrence rate in the patients with positive expression of CD44v mRNA was higher than in those with negative CD44v mRNA expression, and the clinical pathological indexes were also higher in the former than in the latter.CONCLUSION Detection of the CD44v mRNA in blood of the patients with HCC can be used as an adjuvant means for differential diagnosis, prediction and monitoring of the recurrence of HCC.

Sequencing of p53 mutation in established human hepatocellular carcinoma cell line of HHC4 and HHC15 in nude mice

AIM To set up cell lines of human hepatocellular carcinoma in nude mice for the research of cell biology and gene therapy. METHODS Xenotransplantation of human hepatoma into nude mice was carried out and the growth rate, histopathology and immunology of the nude mice were studied. The DNA from xenografts were analyzed by HBV gene and PCR amplification of a fragment of p 53 gene exon 7, which were identified by dot blot hybridization, restriction fragments length polymorphism and DNA sequencing. RESULTS hHCC4 and hHCC415 cell lines could be successively transplanted in nude mice and the population doubling time was 7 and 5 days respectively. These strains retained the original characteristics of histopathology, secreting AFP and heteroploid karyotypes in human hepatocellular carcinoma. The fragment of HBV gene was detected in the genomic DNA of both hHCC4 and hHCC15, however only hHCC4 secreted HBsAg. The mutation at 250 code (C→A) and 249 code (G→T) were detected respectively in the genomic DNA of hHCC4 and hHCC15. CONCLUSION The two cell lines are useful material for studying cell biology and gene therapy in human hepatocellular carcinoma and provide molecular biological trace of the relationship between high mortality of hepatoma and AFB1 severe pollution of the daily common foods in this district.

Effect of hepatitis C virus infection on expression of several cancer-associated gene products in hepatocellular carcinoma

AIM To study hepatocarcinogenesis of hepatitis C virus (HCV). METHODS Expression of HCV antigens (CP10, NS3 and NS5) and several cancer associated gene products (ras p21, c myc, c erbB 2, mutated p53 and p16 protein) in the tissues of hepatocellular carcinoma (HCC, n =46) and its surrounding liver tissue were studied by the ABC (avidin biotin complex) immunohistochemical method. The effect of HCV infection on expression of those gene products in HCC was analyzed by comparing HCV antigen positive group with HCV antigen negative group. RESULTS Positive immunostaining with one, two or three HCV antigens was found in 20 (43 5%) cases, with either of two or three HCV antigens in 16 (34 8%) cases, and with three HCV antigens in 9 (19 6%) cases. Deletion rate of p16 protein expression in HCC with positive HCV antigen (80%, 16/20) was significantly higher than that in HCC with negative HCV antigen. Whereas no significant difference of the other gene product expression was observed between the two groups. CONCLUSION HCV appears related to about one third of cases of HCC in Chongqing, the southwest of China, and it may be involved in hepatocarcinogenesis by inhibiting the function of p16 gene, which acts as a negative regulator of cell cycle.

Expression of nm23 gene in hepatocellular carcinoma tissue and its relation with metastasis

Ｅｘｐｒｅｓｓｉｏｎｏｆｎｍ２３ｇｅｎｅｉｎｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａｔｉｓｓｕｅａｎｄｉｔｓｒｅｌａｔｉｏｎｗｉｔｈｍｅｔａｓｔａｓｉｓＨＵＡＮＧＢｅｉ，ＷＵＺｈｏｎｇＢｉａｎｄＲＵＡＮＹｏｕＢｉｎｇＳｕｂｊｅｃｔｈｅａｄｉ...

Construction of retroviral vectors to induce a strong expression of human class Ⅰ interferon gene in human hepatocellular carcinoma cells in vitro

AIM To establish the hepatoma cell specific expression of human interferon gene mediated by retroviral vectors. METHODS Human interferon α and interforon β complementary DNA (IFNs cDNA) were cloned into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNA and pMNSIFNB, where the transcription of IFN gene was driven by SV40 early region promoter, and pMNAIFNA, pAMNSIFNA and pMNAIFNB, where the transcription of IFN gene was driven by SV40 early region promoter regulated by α fetoprotein enhancer. The retroviral constructs were respectively introduced into retroviral amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection rate was (4～40)×10 3 colonies/μg DNA/10 6 PA317 cells. The retrovirus infection rate was (5～500)×10 4 colony forming units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal carcinoma cells and melanoma cell lines in the presence of 4mg/L polybrene. RESULTS Northern and Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α fetoprotein enhancer induced efficient and apecific transcription and expression of IFNs gene driven by the promoter of different origin in human hepatoma cells by which α fetoprotein was highly produced. CONCLUSION Cis active element of α fetoprotein gene can drive specific expression of IFNs gene in human hepatoma cells, which provides some valuable data for the hepatoma specific immune gene therapy.

Codon 249 mutations of p53 gene in development of hepatocellular carcinoma

AIM To investigate the mechanisms of codon 249 mutation of p53 gene in the formation of hepatocellular carcinoma (HCC). METHODS Codon 249 mutation accompanied by loss of heterozygosity (LOH) and its effect on translation and transcription were studied using SSCP, IHC and RT PCR/slot hybridization. RESULTS Codon 249 mutations were detected in 32 9%, LOH detected in 68 4% among the HCC patients. Mutations of condon 249 were accompanied by LOH in 90%. The positive rates of p53 protein and mRNA were 91 3% and 95 7%, in mutational group, both were significantly higher than those in the non mutational group (91 3% vs 19 1% and 95 7% vs 40 4%, respectively, both P <0 01). The translation of p53 gene was strongly related to its transcription by correlation analysis ( r =0 8208). CONCLUSIONS LOH might play an important role in hepatocarcinogenesis of codon 249 mutation, which could increase both transcription and translation of p53 gene. The increased expression of p53 protein mainly depend on the increased transcription of p53 gene.

UROKINASE-TYPE PLASMINOGEN ACTIVATOR,ITS RECEPTOR AND INHIBITOR EXPRESSION IN HEPATOCELLULAR CARCINOMA RELATION TO CANCER INVASIVENESS AND PROGNOSIS

Objective: To study the relevance of uPA, uPAR and PAI 1 to hepatocellular carcinoma (HCC) Methods: The expression at protein level of uPA, uPAR and PAI 1 was determined in 48 cases of HCC and 12 cases of benign tumors of liver (as control) by immunohistochemistry Results: When compared to cancer adjacent liver tissue and the control, positive rate of immune staining for uPA, uPAR and PAI 1 on cell membrane were significantly higher in HCC cells ( P <0 05) Positive staining of uPA and uPAR was seen in 16 of 22 and 19 of 22 cases of HCC with invasion, respectively ( P <0 01 and P <0 001) In 8 of 8 cases with cancer embolus, and in 6 of 6 cases with lymph node metastasis was the expression of positive uPAR Compared with 2 of 17 cases without recurrence, uPAR was positive in 15 of 17 recurrent cases ( P <0 01) In 36 cases who survived, 17 was positive uPAR and 15 positive PAI 1, while in 12 cases who died 2 years after surgery, 12 were positive for uPAR and 9 positive PAI 1, respectively ( P <0 01 and P <0 05) In 15 positive cases for all three parameters, 11 had cancer invasion and 7 died within 2 years, while in negative cases, 2 had invasion and none died within 2 years ( P <0 05) Conclusion: Expression of uPA, uPAR and PAI 1 is increased in HCC, uPA and uPAR may contribute significantly to HCC invasion and metastasis uPAR and PAI 1 are associated with poor prognosis of HCC

^(99m)Tc-labeled HAb18 McAb Fab fragment for radioimmunoimaging in nude mice bearing human hepatocellular carcinoma

AIM To establish a method of labeling anti hepatoma McAb (HAb18) Fab fragment modifier with 99m Tc. METHODS HAb18 Fab was modified with 2 iminotholane and labeled with 99m Tc by transchelation from 99m Tc GH. Labeling yield, radiochemical purity and immunoreactivity were determined by thin layer chromatography (TLC SG), paper chromatography (PC), gel chromatography (GC) and cell binding assay, respectively. The nude mice bearing human hepatoma were used for radioimmunoimaging (RII). RESULTS A radiolabeling yield of 50%-80% was obtained, and immunoreactivity (IR) was 30%-40%. Radioimaging results showed that 99m Tc HAb18 McAb Fab fragment was concentrated in the tumor 4-8 hours after injection, and the maximum concentration was seen in 12-24 hours, and the T/NT value was 5 18 and 7 48 at 6h and 8h after the injection. CONCLUSION 99m Tc HAb18 McAb Fab fragment could be specifically localized in the tumor of nude mice bearing human hepatocellular carcinoma within 24 hours and this method might be effectively used for labeling McAb Fab fragment with

Analysis of N-ras gene mutation and p53 gene expression in human hepatocellular carcinomas

IM To study the relationship between Nras gene mutation and p53 gene expression in the carcinogenesis and the development of human hepatocellular carcinomas (HCC).METHODS The Nras gene mutation and the p53 gene expression were analyzed in 29 cases of HCC by polymerase chain reactionsingle strand conformation polymorphism (PCRSSCP) and immunohistochemistry.RESULTS Thirteen cases of HCCs were p53 positive (448%), which showed a rather high percentage of p53 gene mutation in Guangxi. The aberrations at Nras codon 2-37 were found in 7931% of HCCs and 8077% of adjacent nontumorous liver tissues. More than 2 point mutations of Nras gene were observed in 22 cases (7586%). Twelve cases (4137%) of HCCs showed both Nras gene mutation and p53 gene expression.CONCLUSIONS Nras gene and p53 gene may be involved in the carcinogenesis and the development of HCC. That 38% of HCCs with Nras gene mutation did not express p53 protein indicates that some other genes or factors may participate in the carcinogenesis and the development of HCC.

Construction of cell lines with CD44 cDNA and its application in hepatocellular carcinoma

ＣｏｎｓｔｒｕｃｔｉｏｎｏｆｃｅｌｌｉｎｅｓｗｉｔｈＣＤ４４ｃＤＮＡａｎｄｉｔｓａｐｐｌｉｃａｔｉｏｎｉｎｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａＸＩＡＯＣｈｅｎｇＺｈｉ，ＤＡＩＹｉＭｉｎ，ＹＵＨｏｎｇＹｕ，ＷＡＮＧＪｉａｎＪｕｎ，ＮＩＣ...

A MOLECULAR EPIDEMIOLOGIC MARKER OF HEPATOCELLULAR CARCINOMA FROM AFLATOXIN B1 CONTAMINATED AREA IN THE SOUTHWEST OF GUANGXI

HCC specimens from high and low AFB1 risk areas in Guangxi showed different frequency of p53 mutational hot spot, which were 20/35 (57%) and 1/10 by DNA sequencing and 36/52 (69%) and 2/10 by RFLP analysis respectively. Their differences were significant (P<0.01). Mutational points of p53 gene induced by AFB1 mutagen almost exclusively clustered at codon 249 third nucleotide and by the form of G to T transversion only. We call it 'AFB1 mutational hot spot'. It turns out to be a significant marker for molecular epidemio logic survey to decide how many HCC and which individuals are induced by AFB1 mutagen, and if emergence of this marker in HCC is frequent in certain region it indicated that there is heavy contamination by AFB1.

Combined measurement of serum tumor markers in patients with hepatocellular carcinoma

ＣｏｍｂｉｎｅｄｍｅａｓｕｒｅｍｅｎｔｏｆｓｅｒｕｍｔｕｍｏｒｍａｒｋｅｒｓｉｎｐａｔｉｅｎｔｓｗｉｔｈｈｅｐａｔｏｃｅｌｕｌａｒｃａｒｃｉｎｏｍａＣＡＩＷｅｎＸｉｕ，ＺＨＥＮＧＨｕｉ，ＳＨＥＮＧＪｉａｎａｎｄＹＥＱｉｎｇＬｉｎＳｕｂｊｅｃｔｈ...