Production of fibronectin by HUH6 C15 cell line established from a human hepatoblastoma

Fibronectin was detected by indirect immunofluorescence on the cell surfaces of HUH6 C15 cells, established from a human hepatoblastoma and maintained with serum-free RPMI 1640 medium. Fibronectin synthesized by HUH6 Cl5 was purified by gelatin-Sepharose affinity chromatography and compared with human plasma fibronectin in respect to molecular weight, electrophoretic mobility and antigenicity. Fibronectin synthesized by this cell line was proved to be identical with human plasma fibronectin.     

Radiosensitivity of the cells of an established human melanoma cell line and the parent melanoma xenograft

Survival curves of cells from a human melanoma xenograft (E.F.) and a cell line (FME) established from this xenograft were determined. The cells of the established line were harvested from exponentially growing cultures, plateau phase cultures or solid tumours in athymic mice (FME-X) before irradiation. During irradiation the cells were kept suspended in culture medium. The colony forming ability of the cells was assayed in soft agar. The Do-value was significantly higher for the parent xenograft than for the established line, whether grown in vitro or in vivo (p less than 0.0001). In addition, the Dq-value was significantly lower for the xenograft than for exponentially growing cultures of the established line (p less than 0.05). Thus the radiation response of the cells of the established line was not representative for that of the cells from the parent xenograft. It is concluded that survival curves for established cell lines should be used with great caution in attempts to predict the radiocurability of human tumours of corresponding histological type.     

Characterization of amylin binding sites in a human hepatoblastoma cell line

Amylin binding sites in a human hepatoblastoma cell line (HepG2) have been characterized in detail. ¹²⁵I-Amylin (rat) bound to HepG2 cells with high affinity. Binding was reversible and selective, and dependent on time and temperature. Scatchard analysis revealed the presence of high (K_d = 0.11 ± 0.04 nM) and low (K_d = 1.3 ± 0.4 μM) affinity binding sites for ¹²⁵I-amylin in HepG2 cells. The dissociation experiments also showed that ¹²⁵I-amylin dissociated from high- and low-affinity sites. The association data, however, indicated the presence of only one binding site. Rat amylin was more potent than human amylin and rat calcitonin gene-related peptide (CGRP) in displacing ¹²⁵I-amylin bound to HepG2 cells. Nonhomologous peptides did not displace ¹²⁵I-amylin. Rat amylin was, however, less potent than rat CGRP in displacing ¹²⁵I[Tyr⁰]CGRP from HepG2 cells. Pretreatment of HepG2 cells with rat amylin (10 nM) reduced the specific binding of ¹²⁵I-amylin by 75%, whereas rat CGRP (10 nM) pretreatment had no effect on amylin binding. Calcitonin gene-related peptide, as well as rat and human amylin, stimulated the adenylate cyclase activity of HepG2 cell membrane preparation in a dose-dependent manner, with an order of potency of CGRP > rat amylin > human amylin. A CGRP antagonist, CGRP(8–37), significantly attenuated the stimulatory effect of both amylin and CGRP on adenylate cyclase activity. These investigations show that distinct receptors of amylin and CGRP are present in HepG2 cells and that amylin stimulates adenylate cyclase activity through CGRP receptors. This system could now be exploited for studying amylin receptors and amylin-mediated signal transduction.     

Localization ofα 2-macroglobulin in human primary sarcomas and synthesis in established cell linesin human primary sarcomas and synthesis in established cell lines

Summary The presence ofα ₂-macroglobulin was detected with the avidin-biotin technique in more than 20-yr-old paraffin blocks from human sarcomas.α ₂M was found mainly in the cytoplasm of the tumor cells, and almost all tumor cells were positive. This serum glycoprotein, which is a major plasma proteinase inhibitor with a wide specificity, was also shown to be synthesized and secreted by all three cell lines derived from primary sarcomas but was not detected in cultures of the autologous skin fibroblasts. For the detection ofα ₂M in situ and in vitro an antiserum to tumor-associatedα ₂-macroglobulin was used. Our work was supported by grant no. 55-B86-21XB, from the Swedish Cancer Society.     

Changes in blood perfusion and hypoxia after irradiation of a human squamous cell carcinoma xenograft tumor line

The effect of irradiation depends on the oxygenation status of the tissue, while irradiation itself also changes the oxygenation and perfusion status of tissues. A better understanding of the changes in tumor oxygenation and perfusion over time after irradiation will allow a better planning of fractionated radiotherapy in combination with modifiers of blood flow and oxygenation. Vascular architecture (endothelial marker), perfusion (Hoechst 33342) and oxygenation (pimonidazole) were studied in a human laryngeal squamous cell carcinoma tumor line grown as xenografts in nude mice. The effect of a single dose of 10 Gy X rays on these parameters was evaluated from 2 h to 11 days after irradiation. Shortly after irradiation, there was an 8% increase in perfused blood vessels (from 57% to 65%) followed by a significant decrease, with a minimum value of 42% at 26 h after irradiation, and a subsequent increase to control levels at 7 to 11 days after irradiation. The hypoxic fraction showed a decrease at 7 h after treatment from 13% to 5% with an increase to 19% at 11 days after irradiation. These experiments show that irradiation causes rapid changes in oxygenation and perfusion which may have consequences for the optimal timing of radiotherapy schedules employing multiple fractions per day and the introduction of oxygenation- and perfusion-modifying drugs.     

Comparative characterization of a human large cell lung carcinoma cell line and the xenograft derived cell line.

The HK-1 cell line established from a human large cell lung carcinoma shows a high transformed phenotype and undifferentiated characteristics. This cell line grows as an adherent monolayer in fetal calf serum-supplemented medium, shows a high proliferation index, is able to grow in semi-solid agar and is tumorigenic in athymic nude rats. The cell line HK-2 derived from the HK-1 xenograft in athymic nude rats shows basically the same features found in the original HK-1 cell line, which include aneuploid nuclear DNA content, abnormal chromosomal number. rearranged marker chromosomes and abnormally localised nucleolar organizer regions. Cytokeratin and vimentin intermediary-sized filaments were found in both cell lines as well as in the original and induced tumour, while neither oncofetal antigens (alphafeto-protein, carcinoembryonic antigen, chorionic gonatropin and human placental lactogen) nor neural differentiation markers (neurofilament and neural specific enolase) were expressed. Analysis of sulphated glycosaminoglycans in the cell cultures and in the nude rat induced tumour showed high expression of chondroitin sulphate.     

Purification and characterization of Alpha-Fetoprotein from the human hepatoblastoma HepG2 cell line in serum-free medium

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function remains unclear. A complete definition of the physiological activities of this oncofetal protein has been severely limited, until now, by the lack of a purification procedure appropriate to obtain pure AFP in appreciable amount. The present report describes a purification procedure extremely rapid and simple and takes advantage of the well-known fact that AFP contains copper. We have developed a single-step purification procedure by immobilized copper-chelate affinity chromatography using the culture medium from human hepatoblastoma cell line HepG2 grown in the absence of serum. This method yields AFP at high purity and high yield. Purified AFP amino acid sequence, molecular mass, carbohydrate structure, and copper content were found to be in line with previous studies. Moreover, we found that the purified AFP has superoxide dismutase activity with efficiency similar to that of the native Cu, Zn SODs at physiological pH. This result may provide further support to the idea that AFP is a bifunctional protein, acting in cellular defence against oxidative stress both as a copper buffer and as a superoxide radical scavenger.     

Methylation of the promoter region of the RASSF1A gene, a candidate tumor suppressor, in primary epithelial tumors

Methylation of the promoter CpG-islands of the candidate tumor suppressor gene RASSF1A (3p21.31) was studied in primary tumors of kidney, breast and ovary (172 cases). Methylation-specific PCR (MSP) and methyl-sensitive restriction endonuclease digestion followed by PCR (MSRA) were applied. Statistically significant correlation (P < 10(-6)) was shown for the results of the MSP and MSRA, and the data of bisulfite sequencing reported earlier. The frequency of RASSF1A methylation according to MSP and MSRA was 86% (25/29) and 94% (50/53) in renal cell carcinoma (RCC) and 64% (18/28) and 78% (32/41)--in breast carcinoma (BC) samples, and 59% (17/29) and 73% (33/45) in ovarian epithelial tumors (OET), respectively. The use of several methyl-sensitive restriction enzymes (HpaII, HhaI, Bsh12361, AciI) enhanced the sensitivity of MSRA and allowed to analyze methylation status of 18 CpG-pairs in the RASSF1A CpG-island. Density of methylation of the RASSF1A CpG-island was 72% (644/900) in RCC, 63% (361/576) in BC, and 58% (346/594) in OET samples (18 CpG-pairs multiplied to the number of samples shown methylation were assumed as 100%). The RASSF1A gene methylation was also observed in samples of morphologically normal tissues adjacent to corresponding tumors (11-35%), but it was not detected in blood DNAs of healthy donors (0/15). The RASSF1A methylation frequency did not show significant correlation to tumor stage, grade and metastases (P = 0.3-1.0). The RASSF1A gene methylation was observed more frequently than other investigated aberrations--hemi- and homozygous deletions inside or around this gene. These observations are consistent with the hypothesis that the RASSF1A gene methylation is an early event in the carcinogenesis and one of the dominant ways of its inactivation.     

Peroxisomal enzyme activities in the human hepatoblastoma cell line HepG2 as compared to human liver.

In order to develop an in vitro model allowing investigation of the long-term effects of hormones and other agents on peroxisomes in liver cells, we measured the activity of a series of peroxisomal enzyme activities in HepG2 cells, a proliferating cell line derived from a human hepatoblastoma. The results obtained show that although in absolute terms peroxisomal enzyme activities are lower in HepG2 cells as compared to human liver, relative activities were comparable in HepG2 and human liver, respectively. Furthermore, it is shown that peroxisomes can easily be isolated from HepG2 cells using density gradient centrifugation. It is concluded that HepG2 cells represent a good model system to study the characteristic (long-term) regulation and control of metabolism of human liver peroxisomes.     

omega-hydroxylation activity toward leukotriene B(4) and polyunsaturated fatty acids in the human hepatoblastoma cell line, HepG2, and human lung adenocarcinoma cell line, A549

The addition of glucose to the culture medium of HepG2 or A549 cells for 22 h caused a dose-dependent increase in leukotriene B(4) omega-hydroxylation activity in the homogenate. The addition of genistein to the culture medium of HepG2 or A549 cells for 22 h caused a dose-dependent decrease in the activity, although the number of living cells was not influenced by the addition of genistein. The inhibition by genistein was reversed by removal of genistein from the culture medium in 22 h. The specific leukotriene B(4) omega-hydroxylation activity was high in the nuclear envelope fraction of HepG2 or A549 cells, and a large portion of the activity was concentrated in the nuclear envelope fraction. In the nuclear envelope fraction, leukotriene B(4) omega-hydroxylation activity was accompanied by high polyunsaturated fatty acid omega-hydroxylation activity. The apparent K(m) values for arachidonic acid and leukotriene B(4) in the fractions of HepG2 or A549 cells were 25 and 50 microM, or 22 and 66 microM, respectively. The V(max) values were 222 and 104 pmol/min/mg protein, or 175 and 370 pmol/min/mg protein, respectively. NADPH-dependent omega-hydroxylation of LTB(4) in the nuclear envelope fraction of HepG2 or A549 cells was strongly inhibited by metyrapone and CO. The expression of cytochrome P450 4F2 mRNAs was detected in HepG2 and A549 cells, and thus the arachidonic acid and leukotriene B(4) omega-hydroxylation activities in the nuclear envelope fractions of HepG2 and A549 cells are likely due to cytochrome P450 4F2.     

Distinct population of highly malignant cells in a head and neck squamous cell carcinoma cell line established by xenograft model

The progression and metastasis of solid tumors, including head and neck squamous cell carcinoma (HNSCC), have been related to the behavior of a small subpopulation of cancer stem cells. Here, we have established a highly malignant HNSCC cell line, SASVO3, from primary tumors using three sequential rounds of xenotransplantation. SASVO3 possesses enhanced tumorigenic ability both in vitro and in vivo. Moreover, SASVO3 exhibits properties of cancer stem cells, including that increased the abilities of sphere-forming, the number of side population cells, the potential of transplanted tumor growth and elevated expression of the stem cell marker Bmi1. Injection of SASVO3 into the tail vein of nude mice resulted in lung metastases. These results are consistent with the postulate that the malignant and/or metastasis potential of HNSCC cells may reside in a stem-like subpopulation.     

Insulin-like growth factor (IGF)-II regulates CCAAT/enhancer binding protein alpha expression via phosphatidyl-inositol 3 kinase in human hepatoblastoma cell lines

To reveal growth factor and its signal pathway to CCAAT/enhancer binding protein alpha (C/EBPalpha) in hepatocyte differentiation, we used Huh-6 and HepG2, human hepatoblastoma (HBL) cell lines that maintain the expression of genes in hepatoblasts and remain at that stage of differentiation. Insulin-like growth factor (IGF)-II, hepatocyte growth factor (HGF), and dexamethasone (Dex) stimulated HBL cells for Northern blot analysis. Bromodeoxyuridine (BrdU) up-take assay and Western blot analysis on albumin was performed to unveil proliferation and differentiation activity of IGF-II. C/EBPalpha and phosphorylation of Akt were analyzed by Western blot analysis. LY294002 and wortmannin, specific inhibitors of PI3 kinase, and PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase, were used to examine the signaling pathway of C/EBPalpha upregulated by IGF-II. Luciferase assay was performed to study the promoter activity of C/EBPalpha. Actinomycin D was used to analyze half-life of C/EBPalpha mRNA. IGF-II up-regualted C/EBPalpha by Northern blot and Western blot while HGF and Dex did not by Northern blot. IGF-II promoted proliferation and differentiation by BrdU up-take assay and Western blot analysis on albumin. Akt phosphorylated by IGF-II, suggested that phosphatidyl-inositol (PI) 3 kinase mediated the signaling pathway of IGF-II. LY294002 and wortmannin suppressed expression of C/EBPalpha. IGF-II activated the promoter activity and prolonged half-life of mRNA, suggesting that IGF-II activated promoter and stabilized mRNA. LY294002 and wortmannin suppressed the promoter activity of C/EBPalpha while PD98059 did not, suggesting that activation of the promoter was mediated by PI3 kinase.