HLE Cell Research Articles

TGF-β1-induced cell growth arrest and partial differentiation is related to the suppression of Id1 in human hepatoma cells

Transforming growth factor beta 1 (TGF-beta1) is a proposed regulator of Ids (inhibitors of DNA binding/differentiation) gene expression in epithelial cells. We previously reported that Id proteins are variously expressed in human hepatocellular carcinomas (HCC). However, the mechanism of regulation of Ids in HCC remains obscure. Here, we examined the relationship between Id1 and TGF-beta1 in four HCC cell lines, and studied the changes in cell proliferation, cell cycle and differentiation. The four HCC cell lines expressed Id1, TGF-beta1 and their receptors at various levels. TGF-beta1 strongly inhibited the growth of HuH7 cells, while the growth inhibition was moderate in PLC/PRF/5, and was not observed in HLE and HLF cell lines. TGF-beta1-induced growth inhibition in HuH7 cells was associated with cell accumulation in the G1 phase and partial induction of differentiation (with reduction of AFP and AFP-L3). Induction by TGF-beta1 dose-dependently suppressed Id1 expression in HuH7 cells; 1 ng/ml TGF-beta1 inhibited Id1 by 84.0 and 78.6% that of the untreated control at transcriptional and protein levels, respectively. HLE and HLF cells, which did not exhibit a TGF-beta1 growth inhibitory effect, lacked TGF-beta receptors and Id1 expression was not altered. In PLC/PRF/5 cells, Id1 augmentation was not observed in response to TGF-beta1, indicating that TGF-beta1-induced growth inhibition was not related to Id1 in this cell line. Our results suggest that, in some HCC cells, the pathway of suppression of Id1 by TGF-beta1 may be important in TGF-beta1-induced growth inhibition and partial differentiation.

Expression of hTERT mRNA in a mortal liver cell line during S phase without detectable telomerase activity

Normal human liver cells have a limited capacity for proliferation due to telomere shortening, whereas immortalized cells prevent shortening of the 3' single strand telomeric repeat by expressing telomerases, including human telomerase reverse transcriptase (hTERT). The hTERT transcript contains three deletion sites that give rise to alternatively spliced variants (ASVs). Recently, hTERT expression was observed in cycling primary presenescent human fibroblasts, which were believed to lack hTERT expression and telomerase activity. hTERT mRNA was expressed in the synthesis (S) phase of the cell cycle. Although hTERT mRNA has eight isoforms, it is not known which of the hTERT ASVs are expressed in S phase. In order to determine the possible relationships between the cell cycle and ASV expressions, we measured the full-length isoform and ASVs of hTERT mRNA in a mortal liver cell line and immortal cell lines that were synchronized in S phase of the cell cycle. Using RT nested-PCR analysis, the full-length isoform and alpha-deletion ASV of hTERT were detected in the LI90 mortal liver cell line at points when cells in S phase represented >48% of the cell population without detectable telomerase activity. hTERT was always expressed in the HLE and Huh-7 hepatocellular carcinoma cell lines, regardless of the cell cycle. Our results suggest the possibility that telomerase is regulated in a cell cycle-dependent manner in normal liver cells.

Effects of fibril- or fixed-collagen on matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1 production in the human hepatocyte cell line HLE

Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) are central to the spontaneous resolution of liver fibrosis. The mechanisms involved have been investigated in hepatic stellate cells (HSC), but not in hepatocytes. We investigated the effects of fibril- and fixed-collagen on MMP-1 and TIMP-1 production in hepatocytes, using the HLE cell line. Fibril type I and IV collagen were prepared by HCl digestion of type I and IV collagen, respectively. For fixed-collagen, culture dishes were coated with fibril type I or IV collagen and fixed by ultraviolet. Type I collagenase activity was measured using fluorescein isothiocyanate-labeled type I collagen. MMP-1 and TIMP-1 in HLE cells were measured by a one-step sandwich enzyme immunoassay. Both fibril type I and IV collagen significantly increased type I collagenase activity about two-fold compared with no fibril collagen. The effects of the fibril collagen were not affected by the coating condition. There was no significant difference in the effects on collagenase activity between cells cultured in medium containing fibril type I collagen and those cultured in the presence of type IV collagen. Both types of fibril collagen significantly increased MMP-1 production, and showed more than 10-fold higher levels of MMP-1 than the control. The enhanced MMP-1 production by fibril collagens was unaffected by the coating condition. By contrast, TIMP-1 production was not changed by the addition of fibril type I or IV collagen, and neither was it affected by the coating conditions. Coating with type I collagen significantly suppressed MMP-1 production by almost one-tenth compared with no coating. By contrast, TIMP-1 production was not affected by either the absence of a collagen coat or by increasing the concentration of the coating collagen. These results indicated that, in HLE cells, fibril- and fixed-collagen have opposite effects on MMP-1 production without affecting TIMP production. Fibril collagen induced collagenase activity by up-regulation of MMP-1 production without affecting TIMP-1 production. By contrast, fixed collagen reduced MMP-1 production. Our results suggest that hepatocytes might also play an important role in the regulation of the hepatic fibrosis alongside HSC.

Matrix Metalloproteinases and Tissue Inhibitors of Matrix Metalloproteinases in the Human Lens: Implications for Cortical Cataract Formation

To characterize the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) in human cortical cataract and to determine whether there is a correlation with the localization of cortical cataract. To evaluate the expression and activity of MMPs and TIMPs after cytokine and UV-B exposure in a human lens epithelial cell line. Twenty-eight human donor eyes with cortical cataract and 21 normal human donor eyes were photographed. Thirteen cortical cataract and six normal lenses were immunohistochemically analyzed for MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3. Twelve fresh cortical cataract and 12 normal lenses were divided into quadrants to quantify, by ELISA, the expression of MMP-1, -2, -3, and -9 and TIMP-1. Three fresh cortical cataract and three control lenses were assessed for MMP-1, -2, and -9 activity by SDS-PAGE zymography. Human lens epithelial cells (HLE-SRA-01/04) were exposed to proinflammatory cytokines and UV-B radiation to determine the protein expression profiles of MMP-1, -2, -3, and -9 and TIMP-1 and -2. Immunohistochemical analysis revealed specific localization of MMP-1 within lens epithelium and cortical lens fibers of cortical cataract. Normal lenses had equally low MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3 immunoreactivity, expression, and activity in all lens quadrants. IL-1 and TNF-alpha upregulated the expression of MMP-2, -3, and -9, and UV-B upregulated the expression of MMP-1 in the SRA-01/04 HLE cell line. This is the first study to localize the expression of MMP-1 in cataracts with clinically observed opacification in vivo and to examine the expression induced by UV-B, in vitro.

Molecular cloning and characterization of LAPTM4B, a novel gene upregulated in hepatocellular carcinoma.

Lysosomal-associated protein transmembrane-4 beta (LAPTM4B), a novel gene upregulated in hepatocellular carcinoma (HCC), was cloned using fluorescence differential display, RACE, and RT-PCR. It contains seven exons and encodes a 35-kDa protein with four putative transmembrane regions. Both the N- and C-termini of the protein are proline-rich, and may serve as potential ligands for the SH3 domain. Immunohistochemical analysis localized the protein predominantly to intracellular membranes. Northern blot showed that the LAPTM4B mRNAs were remarkably upregulated in HCC (87.3%) and correlated inversely with differentiation status. LAPTM4B was also overexpressed in many HCC-derived cell lines. It was also highly expressed in fetal livers and certain adult normal tissues including the heart, skeletal muscle, testis, and ovary. Promoter function assays showed a distinct difference in the gene's activities between BEL7402 and HLE cell lines, suggesting that the transcription factors responsible for regulation of the gene in the two cell lines are different, and that possible negative regulatory cis-elements may exist upstream of the promoter region. It was demonstrated that the N-terminus of LAPTM4B was essential for survival of the cells. Cells harboring the full-length LAPTM4B cDNA expression clone displayed a slightly increased efficiency in colony formation. These results suggest that LAPTM4B is a potential protooncogene, whose overexpression is involved in carcinogenesis and progression of HCC. In normal cells, it may also play important roles such as regulation of cell proliferation and survival.

Hepatocyte Growth Factor Promotes Cell Survival From Fas-Mediated Cell Death in Hepatocellular Carcinoma Cells via Akt Activation and Fas-Death–Inducing Signaling Complex Suppression

The Akt/PI-3 kinase pathway is a system essential for cell survival. In the current study, we showed that hepatocyte growth factor (HGF) activates the Akt/PI-3 kinase pathway to suppress Fas-mediated cell death in human hepatocellular carcinoma (HCC; 3 lines; SK-Hep1, HLE, and Chang Liver cell lines), hepatoblastoma (1 line; HepG2), and embryonic hepatocyte (1 line; WRL). Five tested cell lines showed the resistance to Fas-mediated cell death by the pretreatment of HGF. This HGF-induced cell survival was suppressed by wortmannin (Akt/PI-3 kinase pathway inhibitor), suggesting an involvement of Akt. When cells were pretreated with HGF, Fas-mediated cell death was suppressed, followed by Akt phosphorylation at Ser473. Fas-death–inducing signaling complex (DISC) formation, especially FADD and caspase 8 interaction, was suppressed by HGF and the suppression of the Akt/PI-3 kinase pathway by transient expression of PTEN, resulting in acquisition of Fas-DISC formation and Fas-mediated cell death in HGF-treated cells. We suggest that HGF promotes cell survival in hepatocyte-derived cell lines (HCC, hepatoblastoma, and embryonic hepatocyte) from Fas-mediated cell death via Fas-DISC suppression as a result of Akt activation.(Hepatology 2000;32:796-802.)

Effects of intermittent UVA exposure on cultured lens epithelial cells

Purpose. This paper addresses the question of whether a single non-lethal dose of UVA radiation or the same dose divided three daily one-third doses have like or unlike effects on the growth and the catalase activity of cultured rabbit epithelial (RLE) and immortalized human epithelial (HLE) cells. Methods. Non-confluent cultured RLE and HLE cells were used to study the effects of UVA radiation on their growth. The effects of three doses of 3 J/cm 2 given one day apart on cell growth (i.e. live cell number) over a three day period were compared with those of a single 9 J/cm 2 exposure. Estimation of live cell numbers was done 24 hrs after each exposure by counting trypan blue exclusive cells using a hemocytometer. Confluent cultures of RLE cells were used to study the effects of UVA radiation on their catalase activity. The effects of three doses of 1.25 J/cm 2 each given one day apart on catalase activity (breakdown of H 2 O 2 measured spectro-photometrically at 240 nm) were compared with that of a single 3.75 J/cm 2 exposure. Results. A single 9 J/cm 2 dose of UVA reduced the cell growth to only 10% of controls after 24 hrs. By three days after a single 9 J/cm 2 exposure, the number of live cells was only 70% of controls. Three intermittent exposures of 3 J/cm 2 each for three consecutive days reduced the cell number to 76% of controls. Similar results were obtained for HLE cells. Little if any recovery of cell numbers occurred after three intermittent exposures. A single dose of 3.7 J/cm 2 reduced RLE catalase activity to 20% of controls by one day. By three days after exposure, catalase activity returned to 90% of controls. After three intermittent exposures to 1.25 J/cm 2 each, over three days, RLE catalase activity was reduced to 75% of the controls. There appeared to be no recovery within two additional days. Conclusions. While single below lethal doses of UVA allow no recovery of RLE or HLE cell growth, partial recovery does occur after several daily intermittent exposures. Recovery of the catalase activity by RLE cells does occur after single sub lethal exposures, but intermittent exposures allow no recovery. The recovery of lens epithelial cell growth and catalase activity from UVA damage varies with the exposure regimen.

Quantitation of α–Fetoprotein and Albumin Messenger Rna in Human Hepatocellular Carcinoma

To analyze gene expression of α–fetoprotein (AFP) and albumin in hepatocellular carcinoma (HCC), messenger RNAs (mRNAs) of these proteins in six human hepatoma cell lines and in 30 cases of HCC were quantitatively analyzed by competitive reverse transcription (RT) followed by polymerase chain reaction (PCR). The transcriptional levels of both AFP and albumin genes in HepG2 and Huh 7 cell lines were 1010 copies/μg RNA, in contrast to approximately 105 copies/μg RNA in HLE and HLF cell lines. AFP and albumin mRNA levels in three normal livers were 105 and 1010 transcripts/μg RNA, respectively. In 30 cases with HCC AFP mRNA level in neoplasm was 10 to 105–fold enhanced as compared with that of nonneoplastic portion, and correlated with serum AFP level and tumor size ( P < .01). In contrast, albumin mRNA level was not reduced in the neoplasms presenting enhanced AFP mRNA levels, indicating that AFP and albumin gene expression in situ is not necessarily mutually exclusive. Prospective analysis revealed that an increased serum AFP was shown at the time of recurrence among patients with enhanced AFP mRNA levels in neoplasm only, indicating that AFP mRNA levels in neoplasm could be a clinically predictable tool.

Quantitation of alpha-fetoprotein and albumin messenger RNA in human hepatocellular carcinoma

To analyze gene expression of alpha-fetoprotein (AFP) and albumin in hepatocellular carcinoma (HCC), messenger RNAs (mRNAs) of these proteins in six human hepatoma cell lines and in 30 cases of HCC were quantitatively analyzed by competitive reverse transcription (RT) followed by polymerase chain reaction (PCR). The transcriptional levels of both AFP and albumin genes in HepG2 and Huh 7 cell lines were 1010 copies/microgram RNA, in contrast to approximately 105 copies/microgram RNA in HLE and HLF cell lines. AFP and albumin mRNA levels in three normal livers were 105 and 1010 transcripts/microgram RNA, respectively. In 30 cases with HCC AFP mRNA level in neoplasm was 10 to 105-fold enhanced as compared with that of nonneoplastic portion, and correlated with serum AFP level and tumor size (P < .01). In contrast, albumin mRNA level was not reduced in the neoplasms presenting enhanced AFP mRNA levels, indicating that AFP and albumin gene expression in situ is not necessarily mutually exclusive. Prospective analysis revealed that an increased serum AFP was shown at the time of recurrence among patients with enhanced AFP mRNA levels in neoplasm only, indicating that AFP mRNA levels in neoplasm could be a clinically predictable tool. (Hepatology 1996 Jun;23(6):1384-92)

Gene Therapy for α–Fetoprotein–Producing Human Hepatoma Cells by Adenovirus–Mediated Transfer of the Herpes Simplex Virus Thymidine Kinase Gene

We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms.

Gene therapy for alpha-fetoprotein-producing human hepatoma cells by adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene

We have developed a recombinant replication-defective adenovirus containing human alpha-fetoprotein (AFP) promoter/enhancer to direct cell type-specific expression of the herpes simplex virus thymidine kinase (HSVtk) gene to AFP-producing hepatocellular carcinoma (HCC) cells. After an in vitro infection by a recombinant adenovirus carrying the lacZ gene under the control of human AFP promoter/enhancer (AdAFPlacZ), an expression of the lacZ gene was demonstrated efficiently in AFP-producing HuH-7 and HepG2 cell lines, but not in AFP-nonproducing HLE and HLF cell lines, although lacZ gene expression was demonstrated in all these cell lines when infected with adenovirus vector carrying lacZ gene driven by the beta-actin-based promoter. Expression of the HSVtk gene by adenovirus, from AFP promoter/enhancer (AdAFPtk) induced the cells sensitive to ganciclovir (GCV) in the AFP-producing cell line efficiently, but not in AFP-nonproducing HLF hepatoma cells. An in vitro bystander effect was observed when only 10% of the cells were infected with AdAFPtk. These findings suggest that the AFP promoter/enhancer sequence can provide the tumor-specific activity for the therapeutic gene expression, and that the AdAFPtk vector induces the selective growth inhibition by GCV in the adenovirus-infected human hepatoma cells in vitro. Recombinant adenovirus transfer of the HSVtk gene under the control of tumor-specific promoter followed by GCV may have promise as a targeted in situ treatment for solid neoplasms. (Hepatology 1996 Jun;23(6):1359-68)