Establishment and characterization of a new human hepatocellular carcinoma cell line

Summary A human hepatocellular carcinoma cell line (FOCUS—Friendship of China and United States) was derived from a patient with primary hepatocellular carcinoma. This cell line has been in continuous culture over an 18-mo period. The morphological and ultrastructural features of FOCUS are consistent with its neoplastic hepatocellular orgin. FOCUS cells contain aspartate aminotransferase and glucose-6-phosphatase activity. In addition, α₁-antitrypsin, fibrinogen, alpha fetoprotein, and carcinoembryonic antigens were detectble in the cytoplasm of the cultured cells by immunochemical staining techniques. The karyotype of the FOCUS cell is human in origin and it contains human DNA sequences as detected by molecular hybridization analysis. The FOCUS cells do not show evidence of density-dependent inhibition of growth under confluent conditions. Repeated growth curves over an 18-mo period were identical, revealing a doubling time of 42 to 48 h. The malignant potential of FOCUS cells was further demonstrated by their ability to lead to gross tumor formation after subcutaneous infection into nude mice. From one of the solid tumors grown in nude mice, recultured cell lines have been established and found to have properties identical to the original FOCUS cell line. This FOCUS cell line represents an additional model for further investigation of tumor specific antigens and the relationship between hepatitis B virus (HBV) and hepatocellular carcinoma. Preliminary molecular characterization has indicated the existence of integrated HBV sequences within the FOCUS genome.     

Malignant transformation of immortalized transgenic hepatocytes after transfection with hepatitis B virus DNA

Persistent infection by hepatitis B virus (HBV) is epidemiologically correlated with the prevalence of hepatocellular carcinoma, but its role in tumor development is not yet understood. To study the putative oncogenic potential of HBV, a non-malignant immortal mouse hepatocyte line FMH202 harboring metallothionein promoter-driven simian virus 40 large tumor antigen was transfected with HBV DNA. All stably transfected clones which replicated HBV displayed malignant growth characteristics in soft agar and were tumorigenic upon inoculation in nude mice. The nude mice tumors were histologically classified as differentiated or anaplastic hepatocellular carcinomas. As with human liver carcinomas, rearrangements of in vitro integrated HBV sequences were observed in the nude mouse tumors, and in tumor-derived cell lines. In one case, expression of viral core and surface antigens was blocked in the tumors, correlating with hypermethylation of the HBV genome. However, the expression of X gene was maintained in most tumors and tumor-derived cell lines. X protein was detected in nuclei by immune fluorescence and by immune blot. These results provide the first demonstration that HBV displays oncogenic potential in an experimental system. This system could be useful to functionally identify HBV genes which convey a tumorigenic phenotype.     

人肝癌细胞系HC-108的建立及其生物学特征

HC108细胞系为贴壁生长具有上皮细胞形态特征的分化程度较高的人肝癌细胞。AFP分泌阴性,细胞平均倍增时间为32.21小时,细胞分裂指数为27.75‰,软琼脂克隆形成率为5.0％,能形成裸鼠移植瘤,支原体为阴性。染色体和流式细胞术分析为超二倍体核型,17号染色体改变最为明显。     

Tissue-specific expression of Na,K-ATPase beta-subunit. Does beta 2 expression correlate with tumorigenesis?

We have found a substantial decrease in the level of Na,K-ATPase beta 2-subunit mRNA in xenografts of human renal, lung hepatocellular carcinomas in nude mice as compared with corresponding normal tissues, as well as in the neuroblastoma cell line as compared with the neuron primary cell culture. The level of beta 1 mRNA is decreased in kidney and lung tumor cells, but is unchanged in hepatocellular carcinoma. In the neuroblastoma cell line the level of beta 1 subunit mRNA was found to be higher then in neuron primary cell culture. The level of alpha 1 mRNA in investigated tumors was the same as in normal tissues. These results may give evidence of the involvement of beta 2-subunit in the process of tumorigenesis as was shown for some other adhesion molecules.     

Origin of laminin in the extracellular matrix of human tumor xenografts in nude mice

Monoclonal antibodies reacting exclusively with laminin of human origin and a polyclonal antibody reacting with both murine and human laminin were used to immunohistochemically study the extracellular matrix of four human tumors grown as xenografts in nude mice: a lung carcinoma and a yolk sac carcinoma because they produced cell associated laminin in vitro; and two hepatocellular carcinomas which did not produce cell associated laminin in vitro. The extracellular matrix of the xenografts of the lung carcinoma and the yolk sac carcinoma contained laminin of both human and murine origin. Xenografts of liver carcinoma contained only laminin of mouse origin. This shows that the malignant cells capable of laminin production in vitro contribute this glycoprotein to the extracellular matrix of the solid tumor formed by them in vivo.     

Establishment and characterization of a human ureteral cancer cell line producing carbohydrate antigen 19-9 and carcinoembryonic antigen

We established a new cell line (FU-UrC-1) derived from a human primary ureteral carcinoma xenografted in a nude mouse. This cell line exhibited epithelial characteristics and formed clusters in monolayer cultures. The cells were subcultured in vitro for more than 20 passages and had a doubling time of 53 hours. The modal number of chromosomes was 66. The cell line, which was xenografted again to nude mice, produced tumors essentially identical to the original tumor. Furthermore, the cultured cells expressed carbohydrate antigen 19-9 (CA19-9) and carcinoembryonic antigen (CEA) that were secreted in the culture media. This cell line appears to provide a useful system for studying ureteral carcinoma in vivo and in vitro.     

FXR激动剂GW4064对裸鼠肝癌细胞移植瘤增殖及血管生成的影响

目的:探讨研究法尼酯x受体(FXR)激动剂GW4064对裸鼠肝癌细胞移植瘤增殖及血管生成的影响。方法:选取人肝癌细胞系Hep G2进行体外培养,将细胞悬液接种于BALB/c裸鼠皮下。裸鼠成瘤后,随机分为两组,分别腹腔注射DMSO和GW4064。一周后,处死动物取肿瘤组织,通过免疫组织化学法检测肿瘤组织中Ki-67和CD31的表达,同时计数肿瘤组织中的微血管密度(CD31-MVD);Western blot法检测其FXR和白介素-8(IL-8)的蛋白表达。结果:与对照组相比,FXR激动剂GW4064处理组的肿瘤组织中FXR的蛋白表达量明显增高,微血管密度CD31-MVD值显著降低,同时Ki-67、IL-8及CD31的表达水平均显著降低。结论:FXR激动剂GW4064能显著增加FXR的表达,抑制裸鼠肝癌细胞移植瘤的增殖及新生血管的形成。     

Establishment and characterization of a human hepatocellular carcinoma cell line JHH-4

A human hepatocellular carcinoma cell line, JHH-4 was established from resected liver tumor. Morphological diagnosis of the original tumor was hepatocellular carcinoma, Edmondson type III. This cell line was composed of polygonal shaped cells. Subcellular organelle were observed in cytoplasm. Furthermore, bile canaliculi adhering junction was also remained at the cell surface. The growth rate of JHH-4 cell is slow, peaks of the chromosome number was 75 and 79, and plating efficiency was 3.0%. JHH-4 cell is transplantable to nude mouse. Furthermore, this cell line functionally synthesized and secreted human albumin, AFP and other proteins in vitro.     

Induction of lymphomas on implantation of human oral squamous cell carcinomas in nude mice

Cancer cells from five oral cancer patients and pleomorphic adenoma cells from one individual were inoculated as single cell suspension into subcutis of 30 Swiss nude mice and tail vein of additional 30 mice. Further, tumor tissue pieces from three oral cancer patients were xenografted s.c. in 18 nude mice, and 10 mice were kept as controls. In animals implanted with tumor pieces, 7/18 (39%) mice, developed squamous cell carcinoma at the site of inoculation within 8-15 days, while tumors were not observed in mice inoculated with single cell suspension, up to 60/90 days. In 8/68 (12%) mice, white foci were observed in several tissues, with hepatomegaly and splenomegaly noted in 27/68 (39%) mice. Histopathological examination of various tissues revealed presence of large cell lymphoma in several organs in 14/68 (21%) mice. No regional or distant metastasis of the implanted oral tumor cells was detected. Mice injected with cells from pleomorphic adenoma, also demonstrated large cell lymphoma in 2/10 (20%) mice, whereas none of the 10 control animals showed any gross abnormalities or microscopic abnormalities in several organs. 2/16 (12%) lymphomas exhibited positive reaction with mouse B cell antibodies illustrating the murine origin of the lymphomas, and these were immunophenotyed as B cell lymphomas. The lymphomas were also examined with mouse T cell antibodies and none reacted positively with the mouse T cell antibodies. The lymphomas also failed to react with human T cell, B cell and human Leucocyte common antigen (LCA) antibodies, indicating that the induced lymphomas were not of human origin. The tumor specimens from seven of eight oral cancer patients and the pleomorphic adenoma patient induced lymphomas in nude mice. Thus it appears that xenografting oral tumor cells into nude mice may cause induction of the murine lymphomas, and this needs further investigation.     

Pleiotrophin stimulates fibroblasts and endothelial and epithelial cells and is expressed in human cancer.

Previously we reported the purification of the heparin-binding growth factor pleiotrophin (PTN) from supernatants of the human breast cancer cell line MDA-MB-231. To investigate further the biological activities of PTN and its potential role in cancer, we cloned a PTN cDNA and expressed the gene in a human kidney and in a human adrenal carcinoma cell line (SW-13). The supernatants harvested from cells transfected with PTN contained a heparin-binding specific protein of an apparent molecular mass of 18 kDa. These supernatants stimulated the proliferation of endothelial cells as well as the anchorage-independent growth of SW-13 cells and of normal rat kidney fibroblasts. Furthermore, SW-13 cells transfected with PTN acquired autonomous growth in soft agar and were tumorigenic in athymic nude mice. In contrast to these results with PTN from human cells, PTN obtained from insect cells (Sf9) using recombinant baculovirus as a vector was biologically inactive. We detected high levels of PTN mRNA in 16 of 27 primary human breast cancer samples (62%) as well as in 8 of 8 carcinogen-induced rat mammary tumors. Furthermore, 9 of 34 human tumor cell lines of different origin showed detectable PTN mRNA. We conclude that PTN may function as a tumor growth and angiogenesis factor in addition to its role during embryonic development.     

Isolation and characterization of the major form of human MUC18 cDNA gene and correlation of MUC18 over-expression in prostate cancer cell lines and tissues with malignant progression.

Ectopical expression of huMUC18, a cell adhesion molecule in the immunoglobulin gene superfamily, causes a non-metastatic human melanoma cell line to become metastatic in a nude mouse system. To determine if MUC18 expression correlates with the development and malignant progression of prostate cancer, we investigated differential expression of human MUC18 (huMUC18) in normal prostate epithelial cells, prostate cancer cell lines, and prostatic normal and cancer tissues. We cloned and characterized the human MUC18 (huMUC18) cDNA gene from three human prostate cancer cell lines and three human melanoma cell lines. The cDNA sequences from the six human cancer cell lines were identical except differences in one to five nucleotides. The deduced amino acid sequences of the longest ORF were 646 amino acids that were identical in these cDNAs except for one to three amino acid residues. The amino acid sequences of all our huMUC18 cDNA genes are similar to that cloned by other group (GenBank access #M28882) except differences in the same seven amino acids. We conclude that huMUC18 cDNA gene reported here represents the gene product from a major allele. The MUC18 mRNA and protein was expressed in three metastatic prostate cancer cell lines (TSU-PR1, DU145, and PC-3), but not in one non-metastatic prostate cancer cell line (LNCaP.FGC). The expression of huMUC18 in these four cell lines is positively related to their extent of in vitro motility and invasiveness and in vivo metastasis in nude mice. HuMUC18 protein was also expressed at high levels in extracts prepared from tissue sample sections containing high grade prostatic intraepithelial neoplasia (PIN), but weakly expressed in extracts prepared from cultured primary normal prostatic epithelial cells and the normal prostate gland. Immunohistochemical analysis showed that huMUC18 was expressed at higher levels in the epithelial cells of high-grade PIN and prostatic carcinomas, and in cells of a perineural invasion, a lymph node, and a lung metastases compared to that in normal or benign hyperplastic epithelium (BPH). We therefore conclude that MUC18 expression is increased during prostate cancer initiation (high grade PIN) and progression to carcinoma, and in metastatic cell lines and metastatic carcinoma. Increased expression of MUC18 is implicated to play an important role in developing and malignant progression of human prostate cancer. Furthermore, the lacking of predominant cytoplasmic membrane expression of MUC18 appeared to correlate with malignant progression of prostate cancer.     

Activated Human Hepatic Stellate Cells Promote Growth of Human Hepatocellular Carcinoma in a Subcutaneous Xenograft Nude Mouse Model

Tumor cell microenvironment defines cancer development, also in hepatocellular carcinoma (HCC). Hepatic stellate cells (HSCs) are believed to be the key contributors to tumor microenvironment in HCC, yet their precise role in cancer progression is still unclear. The aim of this study was to determine the effect of human HSCs on progression of HCC using a subcutaneous xenograft nude mouse model. Nude mice were stratified to receive subcutaneous injections of human HCC cell line HepG2 and human HSC line LX-2 (HepG2 + LX-2), HepG2 alone, LX-2 alone, or phosphate-buffered saline. Tumor growth was assessed by measuring tumor size. After 30 days, final tumor size, weight, and histology were assessed. Compared with mice that were only injected HepG2 cells, mice injected with HepG2 + LX-2 exhibited more rapid tumor growth, increased tumor size and weight, higher tumor cell numbers due to increased proliferation and reduced apoptosis, increased fibrotic bands containing LX-2 cells, and increased tumor angiogenesis. In conclusion, HSCs play a significant role in promotion of HCC growth.     

Induction of stromal cell transformation in xenografts of human colonic cancer]

Two transformed stromal cell lines FM-7 and FR-7 were obtained from human xenografts of colon tumor (HCT-7), propagated in nude mice and nude rats, respectively. These two cell lines were confirmed to be murine and rat's by a cytogenetic study and were tumourigenic in BALB/c nude mice with the morphology of fibrosarcoma. Human DNA sequences were not detected by hybridization with pBlur8 probe in DNA of FR-7 cell line grown in vitro. These results indicate that human cancer cells from xenograft HCT-7 can induce malignancy in adjacent normal cells of nude mice and nude rats in the absence of DNA transfer.     

Tissue culture course of a human hepatoma cell line

A cell line, HuH-33 was cultured in vitro from a patient with hepatocellular carcinoma. This cell line has been in continuous culture over 12 month period with slow growth potential. HuH-33 was composed spindle-or polygonal-shaped cells as a major population. Chromosome number of the cells were widely distributed even in the primary culture. HuH-33 was transplantable into nude mice and secreted alpha-fetoprotein, albumin, beta 2 microglobulin, ferritin and tissue polypeptide antigen.     

Role of the alpha(v)beta6 integrin in human oral squamous cell carcinoma growth in vivo and in vitro.

Expression of the alpha(v)beta6 integrin is strikingly upregulated in several types of carcinoma, including human oral squamous cell carcinoma (SCC). Employing a neutralizing monoclonal antibody to alpha(v)beta6, we investigated its role in cell adhesion, proliferation, migration, and in vivo growth of an invasive human SCC line, termed HSC-3. We found that alpha(v)beta6 is strictly required for HSC-3 cell growth in a three-dimensional collagen gel and also prominently contributes to cell migration in two different assay systems. In addition, the anti-alpha(v)beta6 antibody inhibited the invasive growth of HSC-3 cells transorally injected into nude mice. In the presence of the coinjected antibody, the average tumor size at 10 days was reduced by 59%. Histologically, antibody-treated tumors appeared less invasive than control tumors. Furthermore, intravenous application of a neutralizing antibody to the alpha(v) integrin subunit retarded HSC-3 tumor growth. These results point to a possible critical role of the alpha(v)beta6 integrin in controlling growth and invasion of human oral cancer cells.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

New acceptor cell for transfected genomic DNA: oncogene transfer into a mouse mammary epithelial cell line.

A line of mouse mammary epithelial cells (NMuMG) has been characterized for its ability to be stably transfected with exogenous DNA. A transfection frequency of at least 1 cell per 1,000 was obtained with the pSV2neo plasmid. Several thousand G418-resistant NMuMG cell clones can easily be generated in cotransfection of genomic DNA and pSV2neo. The NMuMG cells were isolated from normal mammary glands and do not form malignant lesions when injected into nude mice. We have cotransfected NMuMG cells with pSV2neo and genomic DNA from the human EJ bladder carcinoma line, a cell line which contains an activated c-rasH oncogene. When a pool of 4,700 G418-resistant colonies was injected into nude mice, tumors were obtained. These tumors contain a transfected human rasH gene. Genomic DNA transfection into a line of mouse epithelial cells, in combination with the selection of stable transfectants and tumor induction in nude mice, can be used to screen human tumor DNA for the presence of activated oncogenes.     

利用萤光素酶标记的肿瘤细胞建立肝癌原位移植模型及其活体成像

目的：利用生物发光成像技术非侵入性地监测活体裸鼠原位肝癌发展过程。方法：将包含有萤火虫萤光素酶基因的pCI-neo-Luc载体转染人肝癌HepG2细胞系,筛选获得具有高萤光素酶活性的细胞克隆;利用流式细胞仪对萤光素酶表达的稳定性进行初步研究,并分析细胞的生物发光情况;持续表达萤光素酶的肿瘤细胞培养扩增后被植入裸鼠皮下,2周后以形成的异体瘤作为供体瘤,进行肝脏原位移植手术;对建立的肝癌原位移植模型,用影像学资料显示肿瘤部位,用IVIS成像系统动态监测肿瘤生长情况。结果：体外影像的结果显示,表达萤光素酶细胞的数量与发光强度呈正相关;活体成像的结果显示。成功地建立了萤光素酶标记的原位肝癌动物模型。结论：生物发光成像可以监测活体内肝癌演进过程,为抗肿瘤药物的筛选和评价提供了新的手段和工具。     

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors.

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. We have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possibly related to HBV integration.     

Characterization of murine cell lines from Diethylstilbestrol-Induced uterine endometrial Adenocarcinomas

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormonedependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:ytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three-to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.